PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation
Open accessAbstract
Background: DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved. Results: Here we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation. Conclusions: The work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process.
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TL;DR
In matched human monocyte-to-osteoclast differentiation samples, DNA methylation is extensively remodeled, with thousands of genes gaining or losing methylation. PU.1 binds both hypo- and hypermethylated targets and recruits TET2 or DNMT3b, and PU.1 knockdown impairs these methylation and expression changes.
Key findings
- In three matched CD14+ monocyte/osteoclast pairs, 3,515 genes (8,028 CpGs) were differentially methylated (FC ≥2 or ≤0.5; FDR ≤0.05): 1,895 hypomethylated genes (3,597 CpGs) and 2,054 hypermethylated genes (4,429 CpGs).
- Hypomethylation occurred early (about 60% of the range by days 0–4), without replication (BrdU-positive cells <9.8% between days 1 and 4; 5azadC treatment had no significant effect on osteoclastogenesis), and was associated with 5hmC changes at genes such as ACP5 and TM4SF19.
- Motif analysis found PU.1 enrichment in both directions: 15% of hypomethylated genes had PU.1/ETS motifs (PU.1 alone 9%) and 15% of hypermethylated genes had PU.1 motifs; ChIP-seq showed PU.1 peaks within 500 bp of 10.7% of promoter hypomethylated CpGs and 25.1% of promoter hypermethylated CpGs.
- PU.1 co-immunoprecipitated with DNMT3b and TET2; siRNA PU.1 knockdown (~60%) impaired acquisition of DNA methylation and expression changes at PU.1 targets and reduced TET2 and DNMT3b association at those promoters.
Methods
- Primary human CD14+ monocytes from buffy coats were differentiated to osteoclasts with M-CSF and RANKL for 21 days; three matched monocyte/osteoclast pairs were profiled.
- Genome-wide DNA methylation used Illumina HumanMethylation450 arrays, with differential methylation by paired limma (FC ≥2 or ≤0.5; P<0.01; FDR<0.05), validated by bisulfite pyrosequencing; expression data came from ArrayExpress E-MEXP-2019.
- Mechanistic assays included TRANSFAC/STORM motif analysis, PU.1 ChIP-seq (GSE31621), ChIP, immunoprecipitation, siRNA knockdown, 5hmC detection, BrdU, and 5azadC treatment.
Limitations
- Stated: PU.1 knockdown only partially impaired DNA methylation/expression changes and only partially reduced TET2/DNMT3b binding, indicating additional transcription factors participate; TM7SF4 showed no effect, possibly because it demethylates before PU.1 knockdown is effective.
- Stated: the detailed mechanisms coupling 5mC hydroxylation and demethylation remain debated, and the study did not address whether PU.1 interactions involve different subpopulations or post-translational modifications.
- Evident: array analyses used three matched pairs, and some ChIP experiments are described as three biological triplicates but only one experiment is shown; TRANSFAC predictions had limited overlap with ChIP-seq (20.6% hypomethylated, 46.9% hypermethylated), which the authors note reveals weakness in predictive motif searches.
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- Findingown data
DNA methylation changes also occurred at a similar scale in gene bodies (51% for hypomethylated CpGs, 40% for hypermethylated CpGs).
“although DNA methylation changes also occurred at a similar scale in gene bodies (51% for hypomethylated CpGs, 40% for hypermethylated CpGs)”
Quote in the paperNot a substantial claim - Findingown data
CX3CR1 displayed an increase in methylation of over 28%.
“Conversely, CX3CR1, an important factor for MO adhesion to blood vessels that is downregulated during osteoclastogenesis, displayed an increase in methylation of over 28%”
Quote in the paperNot a substantial claim - Findingown data
TM7SF4 undergoes 59% hypomethylation.
“TM7SF4, which encodes for DC-STAMP, a seven-pass transmembrane protein involved in signal transduction in OCs and dendritic cells, undergoes 59% hypomethylation.”
Quote in the paperNot a substantial claim - Findingown data
Hypermethylation and hypomethylation occur in many relevant functional categories and key genes, including CTSK, ACP5, and DC-STAMP.
“Hypermethylation and hypomethylation occur in many relevant functional categories and key genes, including those whose functions are crucial to OC biology, like CTSK, ACP5, and DC-STAMP.”
Quote in the paperSubstantial claim - Findingown data
Hypomethylation during osteoclastogenesis was associated with a change in 5-hydroxymethylcytosine.
“associated with a change in 5-hydroxymethylcytosine”
Quote in the paperSubstantial claim
- Findingown data
ChIP-seq data analysis confirmed PU.1 association to both hypermethylated and hypomethylated gene sets.
“ChIP-seq data analysis confirmed its association to both gene sets.”
Quote in the paperSubstantial claim - Findingown data
PU.1 interacts with both DNMT3b and TET2.
“PU.1 interacts with both DNMT3b and TET2”
Quote in the paperSubstantial claim - Findingown data
Statistical analysis of combined data from three paired monocyte/osteoclast samples revealed 3,515 genes (8,028 CpGs) displayed differential methylation (FC ≥2 or FC ≤0.5; FDR ≤0.05).
“Statistical analysis of the combined data from the three pairs of samples revealed that 3,515 genes (8,028 CpGs) displayed differential methylation (FC ≥2 or FC ≤0.5; false discovery rate (FDR) ≤0.05).”
Quote in the paperSubstantial claim - Findingown data
There was a very close correlation between array and pyrosequencing data (R2 = 0.9707).
“This analysis showed a very close correlation between the array and the pyrosequencing data (R2 = 0.9707)”
Quote in the paperSubstantial claim - Findingown data
Gene ontology analysis of hypomethylated CpGs revealed significant enrichment for functional categories of relevance in osteoclast differentiation and function.
“Gene ontology analysis of hypomethylated CpGs revealed significant enrichment (FDR ≤0.05) for a variety of functional categories of relevance in OC differentiation and function”
Quote in the paperSubstantial claim - Findingown data
Transcription factor binding motif analysis revealed over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes.
“Inspection of transcription factor binding motif over-representation in genes undergoing DNA methylation changes revealed the enrichment of the PU.1 binding motif in hypermethylated genes and AP-1, NF-kB, and also PU.1 motifs among hypomethylated genes.”
Quote in the paperSubstantial claim - Findingown data
Most expression changes occurred within the first 5 days of osteoclastogenesis.
“Our analysis showed that most changes occurred within the first 5 days”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [32] Gallois A, Lachuer J, Yvert G, Wierinckx A, Brunet F, Rabourdin-Combe C, Delprat C, Jurdic P, Mazzorana M: Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes. J Bone Miner Res. 2010, 25: 661-672. 10.1359/jbmr.090829. source
Not checked in the free run.
- Findingown data
Osteoclastogenesis was associated with drastic reshaping of the DNA methylation landscape.
“We found that osteoclastogenesis was associated with the drastic reshaping of the DNA methylation landscape.”
Quote in the paperSubstantial claim - Findingown data
ACP5/TRACP gene is hypomethylated around 47%.
“The ACP5/TRACP gene is hypomethylated around 47%.”
Quote in the paperNot a substantial claim - Findingown data
NFATC1 shows significant hypomethylation at CpG sites in its gene body region.
“We also observed significant hypomethylation at the osteoclast-specific transcription factor gene NFATC1, although in this case hypomethylation occurred at CpG sites located in its gene body region.”
Quote in the paperNot a substantial claim - Findingown data
Changes corresponding to the average of three monocyte/osteoclast pairs were almost identical to the pattern obtained for each individual pair of samples.
“Changes corresponding to the average three pairs of monocytes/osteoclasts (Figure 1B) were almost identical to the pattern obtained for each individual pair of samples”
Quote in the paperNot a substantial claim - Findingown data
Only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes.
“Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes”
Quote in the paperSubstantial claim - Findingown data
Hypomethylation during osteoclastogenesis was DNA replication-independent.
“was DNA replication-independent”
Quote in the paperSubstantial claim - Findingown data
Over one-third of differentially methylated CpG-containing probes (33% for hypomethylated CpGs, 45% for hypermethylated CpGs) mapped to gene promoters.
“Over one-third of the differentially methylated CpG-containing probes (33% for hypomethylated CpGs, 45% for hypermethylated CpGs) mapped to gene promoters”
Quote in the paperNot a substantial claim - Findingown data
CTSK is hypomethylated more than 60% in osteoclastogenesis.
“CTSK, the lysosomal cysteine proteinase involved in bone remodeling and resorption, is hypomethylated more than 60%.”
Quote in the paperNot a substantial claim - Findingown data
No significant DNA methylation changes were observed in repetitive elements (Sat2, D4Z4, NBL2, Alu) or ribosomal RNA genes.
“These experiments showed no significant DNA methylation changes in any of these repetitive elements”
Quote in the paperSubstantial claim - Findingown data
Hypomethylation occurred early and concomitantly with transcription changes during osteoclastogenesis.
“Hypomethylation occurred early, concomitantly with transcription changes”
Quote in the paperSubstantial claim - Findingown data
During osteoclastogenesis, PU.1 mRNA levels increase.
“PU.1 revealed an increase at the mRNA levels, although there was no change at the protein level.”
Quote in the paperNot a substantial claim - Findingown data
In hypomethylated genes, there was highly significant enrichment of binding motifs of AP-1 family and NF-kB subunits.
“In the case of hypomethylated genes, we observed highly significant enrichment of binding motifs of the AP-1 family and NF-kB subunits”
Quote in the paperSubstantial claim - Findingown data
For genes that become hypomethylated, DNMT3b is initially enriched at their promoters but its association is lost after M-CSF and RANKL stimulation.
“whereas DNMT3b was initially enriched but its association with these promoters was lost after M-CSF and RANKL stimulation (Figure 4D).”
Quote in the paperSubstantial claim - Findingown data
PU.1 binds to promoters of both hypomethylated and hypermethylated genes at 0 and 2 days after M-CSF/RANKL stimulation.
“Our analysis showed binding of PU.1 at both 0 and 2 days (Figure 4D).”
Quote in the paperSubstantial claim - Findingown data
For genes that become hypomethylated, TET2 recruitment to their promoters increases after 2 days of M-CSF/RANKL stimulation.
“For genes that become hypomethylated, we observed an increased recruitment of TET2 at these promoters after 2 days”
Quote in the paperSubstantial claim - Findingown data
The overlap between ChIPseq PU.1 targets and TRANSFAC predictions is around 46.9% for hypermethylated genes.
“observed that the overlap between the two sets of list was around 20.6% for hypomethylated genes and 46.9% for hypermethylated genes, again using the same 500 bp for both datasets (Figure 4F).”
Quote in the paperNot a substantial claim - Findingown data
Fewer than 9.8% of cells were BrdU-positive between 1 and 4 days, confirming virtual absence of replication.
“Consistent with previous observations, fewer than 9.8% were found to be BrdU-positive between 1 and 4 days, confirming the virtual absence of replication”
Quote in the paperSubstantial claim - Findingown data
Promoters of genes like ACP5, CTSK, TM7SF4, and TM4SF19 rapidly became hypomethylated following RANKL and M-CSF stimulation.
“We found that the promoters of genes like ACP5, CTSK, TM7SF4, and TM4SF19 rapidly became hypomethylated following RANKL and M-CSF stimulation”
Quote in the paperSubstantial claim - Findingown data
PU.1 downregulation partially impairs the upregulation of ACP5 and CTSK during osteoclastogenesis.
“We then observed that the upregulation of genes like ACP5 and CTSK (both PU.1-direct targets) was partially impaired (Figure 5D).”
Quote in the paperSubstantial claim - Findingown data
For hypermethylated genes, DNMT3b recruitment increases at 2 days after M-CSF/RANKL stimulation.
“We also observed increased recruitment of DNMT3b at 2 days after M-CSF/ RANKL stimulation.”
Quote in the paperSubstantial claim - Findingown data
PU.1 downregulation does not detectably affect DNA methylation dynamics of TM7SF4.
“In the case of TM7SF4, one of the key genes undergoing hypomethylation, we did not detect an effect of PU.1 downregulation on its DNA methylation dynamics (Additional file 10).”
Quote in the paperNot a substantial claim - Findingown data
ChIP assays demonstrated interaction of PU.1, p65 NF-kB subunit, and c-Fos with most of the selected promoters, even before stimulation with M-CSF and RANKL.
“ChIP assays demonstrated the interaction of these factors with most of the aforementioned promoters, even before the stimulation with M-CSF and RANKL”
Quote in the paperSubstantial claim - Findingown data
The proportions of hypomethylated and hypermethylated CpGs with PU.1 peaks within 500 bp are similar in distal regions.
“These numbers were similar when focusing on CpGs located in distal regions (Figure 4F).”
Quote in the paperNot a substantial claim - Findingreasoning
Hypermethylation does not seem to be predominant over hypomethylation.
“However, hypermethylation does not seem to be predominant over hypomethylation.”
Quote in the paperNot a substantial claim - Findingown data
During osteoclastogenesis, PU.1 protein levels do not change.
“PU.1 revealed an increase at the mRNA levels, although there was no change at the protein level.”
Quote in the paperNot a substantial claim - Findingown data
PU.1 downregulation reduces the association of DNMT3b and TET2 with their target promoters.
“Most importantly, it also reduced the association of DNMT3b and TET2 reinforcing the notion of the role of these factors and their association with PU.1 in the DNA methylation changes occurring at these CpG sites (Figure 5E).”
Quote in the paperSubstantial claim - Findingown data
For several genes that become hypomethylated, like ACP5 and TM4SF19, there was an initial increase in 5hmC levels followed by a slight but significant decrease.
“For several genes that become hypomethylated, like ACP5 and TM4SF19, we observed an initial increase in 5hmC levels followed by a slight but significant decrease”
Quote in the paperSubstantial claim - Findingown data
PU.1 downregulation in MOs using siRNAs reduces PU.1 levels close to 60%.
“confirmed the PU.1 downregulation close to 60% (Figure 5B and 5C).”
Quote in the paperSubstantial claim - Findingown data
The PU.1 binding motif is present in 15% of all hypermethylated genes.
“In fact, the PU.1 binding motif is present in 15% of all hypermethylated genes”
Quote in the paperNot a substantial claim - Findingown data
In hypermethylated genes, there was even greater enrichment of binding motifs of ETS-related transcription factors, especially PU.1.
“In the case of hypermethylated genes, we identified even greater enrichment of the binding motifs of ETS-related transcription factors, especially PU.1”
Quote in the paperSubstantial claim - Findingown data
10.7% of all hypomethylated CpGs located in promoter regions have PU.1 peaks within a 500-bp window centered on the CpG.
“we found that 10.7% of all hypomethylated CpGs located in promoter regions genes and 25.1% of all hypermethylated CpGs located in promoter regions had PU.1 peaks within this 500-bp window (Figure 4F).”
Quote in the paperSubstantial claim - Findingown data
PU.1, DNMT3b, and TET2 do not associate with the MyoD promoter negative control.
“We did not observe association of PU.1, DNMT3b, and TET2 in the negative control for PU.1 binding, the MyoD promoter.”
Quote in the paperSubstantial claim - Findingown data
The overlap between ChIPseq PU.1 targets and TRANSFAC predictions is around 20.6% for hypomethylated genes.
“observed that the overlap between the two sets of list was around 20.6% for hypomethylated genes and 46.9% for hypermethylated genes, again using the same 500 bp for both datasets (Figure 4F).”
Quote in the paperNot a substantial claim - Findingown data
Changes in mRNA levels occurred at a similar pace or slightly delayed with respect to changes in DNA methylation.
“Changes in mRNA levels occurred at a similar pace or, in some cases, in an even more gradual manner and were slightly delayed with respect to changes in DNA methylation.”
Quote in the paperNot a substantial claim - Findingown data
Much lower or insignificant levels of enrichment were obtained for AP-1 family members and NF-kB subunits among hypermethylated genes.
“Much lower or insignificant levels of enrichment were obtained for AP-1 family members and NF-kB subunits among the hypermethylated genes.”
Quote in the paperNot a substantial claim - Findingown data
No binding was observed in control sequences like Sat2 repeats and the MYOD1 and TDRD1 promoters.
“No binding was observed in control sequences like Sat2 repeats and the MYOD1 and TDRD1 promoters.”
Quote in the paperNot a substantial claim - Findingown data
Detailed analysis of sequences undergoing DNA methylation changes produced evidence of participation of several transcription factors, given over-representation of certain motifs in hypo- and hypermethylated genes.
“First, a detailed analysis of the sequences undergoing DNA methylation changes produced evidences of the participation of several transcription factors, given the specific over-representation of certain motifs in hypo- and hypermethylated genes.”
Quote in the paperSubstantial claim - Findingown data
Hypermethylated genes like PPP1R16B, CD6, and NR4A2 displayed loss of expression before experiencing an increase in DNA methylation.
“In contrast, hypermethylated genes like PPP1R16B, CD6, and NR4A2 (Figure 2D, bottom) displayed loss of expression before experiencing an increase in DNA methylation”
Quote in the paperSubstantial claim - Findingown data
PU.1 downregulation impairs the downregulation of CX3CR1 and NR4A2 during osteoclastogenesis.
“In the case of genes like CX3CR1 and NR4A2 we determined that downregulation was also impaired in PU.1-siRNA treated MOs.”
Quote in the paperSubstantial claim - Findingown data
25.1% of all hypermethylated CpGs located in promoter regions have PU.1 peaks within a 500-bp window centered on the CpG.
“we found that 10.7% of all hypomethylated CpGs located in promoter regions genes and 25.1% of all hypermethylated CpGs located in promoter regions had PU.1 peaks within this 500-bp window (Figure 4F).”
Quote in the paperSubstantial claim - Findingown data
During osteoclastogenesis, p65 NF-kB protein levels clearly increase.
“In the case of p65 NF-kB we only observed a clear increase at the protein level (Figure 4B).”
Quote in the paperNot a substantial claim - Findingown data
An inverse relationship between DNA methylation and gene expression was mainly observed for changes in CpGs in proximity of the TSS and within the first exon, and less frequent in gene bodies and 3′UTR.
“An inverse relationship between DNA methylation and gene expression was mainly observed for changes occurring in CpGs in the proximity of the TSS and within the first exon (Figure 2C) and it was less frequent in those at gene bodies and 3′UTR”
Quote in the paperSubstantial claim - Findingown data
In other genes, like TM7SF4 and CD59, there were high levels of 5hmC before addition of RANKL/M-CSF.
“In other genes, like TM7SF4 and CD59, of there were high levels of 5hmC before the addition of RANKL/M-CSF as if these genes were already primed for demethylation.”
Quote in the paperNot a substantial claim - Findingown data
Treatment with 5-Aza-2′-deoxycytidine (5azadC) had no significant effect on osteoclastogenesis.
“treatment with 5-Aza-2′-deoxycytidine (5azadC), a pharmacological compound that results in replication-coupled DNA demethylation [33], had no significant effect on osteoclastogenesis”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [33] Momparler RL: Pharmacology of 5-Aza-2′-deoxycytidine (decitabine). Semin Hematol. 2005, 42: S9-S16. source
Not checked in the free run.
- Findingown data
Over-representation of transcription factor binding motifs among sequences undergoing DNA methylation changes was very specific to the direction of the DNA methylation change.
“We observed that the over-representation of binding motifs was very specific to the direction of the DNA methylation change (hypomethylation or hypermethylation).”
Quote in the paperSubstantial claim - Findingown data
In hypomethylated genes, there was enrichment of PU.1 (FDR 1.07E-12).
“We also observed enrichment of PU.1 (FDR 1.07E-12).”
Quote in the paperSubstantial claim - Findingown data
Around 60% of the entire range of hypomethylation occurred between days 0 and 4.
“In fact, around 60% of the entire range of hypomethylation occurred between days 0 and 4.”
Quote in the paperNot a substantial claim - Findingown data
Hypermethylation affects genes that are specific to other cellular types.
“In fact, we found that hypermethylation affects genes that are specific to other cellular types.”
Quote in the paperNot a substantial claim - Findingown data
PU.1 downregulation decreases PU.1 association with promoters of both hypo- and hypermethylated genes.
“we observed that PU.1 dowregulation resulted in a decrease of the levels of PU.1 associated with the promoters of both hypo- and hypermethylated genes.”
Quote in the paperSubstantial claim - Findingown data
PU.1-siRNA treatment has only small effects on expression changes of non-PU.1 target genes PLA2G4E and FSCN3 during osteoclast differentiation.
“PU.1-siRNA treatment had only small effects on gene expression changes during osteoclast differentiation (perhaps due to indirect effects) when compared to control siRNA, confirming the specificity of the changes observed for the other genes (Figure 5D, bottom).”
Quote in the paperSubstantial claim - Findingown data
Downregulation of PU.1 impairs the acquisition of DNA methylation changes in both PU.1-target genes that become hypomethylated and those that become hypermethylated.
“In both cases, we observed that downregulation of PU.1 impaired the acquisition of DNA methylation changes, in contrast with the changes observed for control siRNA-treated MOs (Figure 5D).”
Quote in the paperSubstantial claim - Findingown data
NF-kB p65 does not bind DNMT3b or TET2 in osteoclastogenesis samples.
“In the case of NF-kB, we did not observe binding with either of these factors (Figure 4C).”
Quote in the paperNot a substantial claim - Findingown data
PU.1 and other ETS-related binding motifs are highly enriched in both hypomethylated and hypermethylated genes.
“Among these, PU.1 and other ETS-related binding motifs are highly enriched in both hypomethylated and hypermethylated genes.”
Quote in the paperSubstantial claim - Findingown data
Analysis of over-representation of transcription factor binding motifs reveals enrichment of specific motifs for hypomethylated and hypermethylated genes.
“The analysis of over-representation of transcription factor binding motifs reveals the enrichment of specific motifs for hypomethylated and hypermethylated genes.”
Quote in the paperSubstantial claim - Findingown data
Hypomethylation occurs concomitant with or even earlier than expression changes.
“Also, when comparing the dynamics of DNA methylation and expression changes, hypomethylation occurs concomitant or even earlier than expression changes.”
Quote in the paperSubstantial claim - Findingown data
For the majority of genes becoming hypermethylated, hypermethylation follows expression changes.
“In contrast, for the majority of genes becoming hypermethylated, hypermethylation follows expression changes.”
Quote in the paperSubstantial claim - Findingown data
In monocyte-to-osteoclast differentiation, both hypomethylation and hypermethylation changes occur.
“Our study of the DNA methylation changes in monocyte-to-osteoclast differentiation reveals the occurrence of both hypomethylation and hypermethylation changes.”
Quote in the paperSubstantial claim - Study design and conductown data
Bisulfite pyrosequencing confirmed the results of the beadchip array for the selected genes.
“In all cases, bisulfite pyrosequencing confirmed the results of the beadchip array”
Quote in the paperNot a substantial claim - Study design and conductown data
At 21 days, over 84% of the nuclei detected in these preparations could be considered osteoclastic nuclei.
“At 21 days, over 84% of the nuclei detected in these preparations could be considered to be osteoclastic nuclei”
Quote in the paperSubstantial claim - Interpretationown data
PU.1 may act as a dual coupling transcription factor that can interact with DNA methyltransferases and enzymes participating in or leading to demethylation.
“Our results suggested that PU.1 may play a dual coupling transcription factor that can interact with the DNA methyltransferases and enzymes perhaps participating in, or leading to, demethylation.”
- Also stated in: § Discussion · hedged
“Our study shows that PU.1 may act as a dual adaptor during osteoclastogenesis, in the directions of hypomethylation and hypermethylation.”
- Also stated in: § Discussion · plain
“Therefore, our data reveal a novel role of PU.1 as a dual adaptor with the ability to bind both epigenetically repressive and epigenetically activating events and targeting DNA methylation changes in both directions (Figure 6).”
Quote in the paperSubstantial claim - Also stated in: § Discussion · hedged
- Interpretationown data
Our results provide evidence that transcription factors, focusing on PU.1, participate in determining DNA methylation changes during monocyte-to-osteoclast differentiation.
“Our results provide evidence of the participation of transcription factors, focusing on PU.1, in determining changes in DNA methylation during monocyte-to-osteoclast differentiation.”
Quote in the paperSubstantial claim - Interpretationown data
The large DNA methylation changes observed in this time period are independent of DNA replication.
“This implies that the large DNA methylation changes observed in this time period are independent of DNA replication.”
Quote in the paperSubstantial claim - Interpretationreasoning
Both hypomethylation and hypermethylation events are equally important.
“Our study suggests that both hypomethylation and hypermethylation events are equally important.”
Quote in the paperSubstantial claim - Interpretationown data
DNA methylation changes are involved in the differentiation dynamics and stabilization of the OC phenotype because they are concomitant with, or precede, expression changes.
“our data show that DNA methylation changes are involved in the differentiation dynamics and stabilization of the OC phenotype since they are concomitant with, or even precede, expression changes.”
Quote in the paperSubstantial claim - Interpretationown data
In relation with methylation changes, PU.1 appears to be better correlated with changes in the direction of repression.
“However, in relation with methylation changes, PU.1 appears to be better correlated with those changes in the direction of repression.”
Quote in the paperSubstantial claim - Interpretationown data
PU.1 is associated with a high number of genes undergoing DNA methylation changes.
“These analyses reinforced the notion of PU.1 associated with a high number of genes undergoing DNA methylation changes”
Quote in the paperSubstantial claim - Interpretationown data
PU.1 has a novel role in mediating DNA methylation changes during osteoclastogenesis by direct binding of both DNMT3B and TET2.
“Second, further analyses on one these candidate transcription factors, PU.1, and manipulation of its levels revealed a novel role for this factor in mediating DNA methylation changes during osteoclastogenesis, by direct binding of both DNMT3B and TET2.”
Quote in the paperSubstantial claim - Premisecitation
TET2 has high levels in hematopoietic cells of myeloid origin.
“We focused on TET2 given their high levels in hematopoietic cells of myeloid origin [44,45].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [44] Kallin EM, Rodriguez-Ubreva J, Christensen J, Cimmino L, Aifantis I, Helin K, Ballestar E, Graf T: Tet2 facilitates the derepression of myeloid target genes during CEBPalpha-induced transdifferentiation of pre-B cells. Mol Cell. 2012, 48: 266-276. 10.1016/j.molcel.2012.08.007. source
Not checked in the free run.
- cites [45] Klug M, Schmidhofer S, Gebhard C, Andreesen R, Rehli M: 5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes. Genome Biol. 2013, 14: R46-10.1186/gb-2013-14-5-r46. source
Not checked in the free run.
- Interpretationown data
Hypomethylation events affect genes specific to osteoclast differentiation or related to the function of differentiated OCs.
“Hypomethylation events, in many cases associated with gene activation, affect genes that are specific to this differentiation process or are related with the function of differentiated OCs.”
Quote in the paperSubstantial claim - Interpretationown data
Hydroxymethylation, and therefore activity of Tet proteins, participates in genes that undergo a reduction in DNA methylation.
“our results suggested the participation of hydroxymethylation, and therefore the activity of Tet proteins, in genes that undergo a reduction in DNA methylation.”
Quote in the paperSubstantial claim - Interpretationown data
Genes that become hypomethylated exchange PU.1-DNMT3b by PU.1-TET2 during osteoclastogenesis.
“Genes that become hypomethylated exchange PU.1-DNMT3b by PU.1-TET2 (although whether pre-existing subpopulations of these associations may exist or, alternatively, post-translational or another mechanisms may mediate exchange of TET2 and DNMT3b; this is not elucidated at present).”
Quote in the paperSubstantial claim - Interpretationown data
This is the first report where the association between PU.1 and DNMTs in association with gene repression is shown in this context.
“However, this is the first report where the association between PU.1 and DNMTs in association with gene repression is shown in this context.”
Quote in the paperSubstantial claim - Interpretationown data
Hypermethylated genes experience an increase in the binding of DNMT3b as differentiation to OCs is triggered.
“Hypermethylated genes experience an increase in the binding of DNMT3b as differentiation to OCs is triggered.”
Quote in the paperSubstantial claim - Interpretationown data
Manipulation of PU.1 levels by siRNAs shows that PU.1 has a direct role in recruiting DNMT3b and TET2 to its target promoters.
“The manipulation of PU.1 levels by using siRNAs has shown that PU.1 has a direct role in recruiting DNMT3b and TET2 to its target promoters”
Quote in the paperSubstantial claim - Interpretationown data
The incomplete impairment of DNA methylation and expression changes, as well as partial loss of Tet2 and DNMT3b following PU.1 knock-down, indicates that additional transcription factors also participate in this process.
“The incomplete impairment of DNA methylation and expression changes, as well as partial loss of Tet2 and DNMT3b following PU.1 knock-down indicates that additional transcription factors are also participating in this process.”
Quote in the paperNot a substantial claim - Study design and conductown data
On day 21, almost 85% of the nuclei detected in the cultures were osteoclastic nuclei.
“We used several methods to determine that on day 21 almost 85% of the nuclei detected were ‘osteoclastic nuclei’ (in polykaryons, nuclei and not cells were quantified).”
Quote in the paperNot a substantial claim - Interpretationown data
Our findings constitute the first report where the binding of PU.1 to TET2 has been described.
“Moreover, our findings constitute the first report where the binding of PU.1 to TET2 has been described.”
Quote in the paperSubstantial claim
- May combine two claims. 31
- The quote only partly states this claim. 1
- Classified as probably not a substantial claim. 21
May combine two claims.
Findingown data1,895 hypomethylated genes (3,597 CpG sites) and 2,054 hypermethylated genes (4,429 CpGs) were identified.
“Specifically, we identified 1,895 hypomethylated genes (3,597 CpG sites) and 2,054 hypermethylated genes (4,429 CpGs)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown datasiRNA-mediated PU.1 knockdown in primary monocytes impaired acquisition of DNA methylation and expression changes, and reduced association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation.
“siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation.”
Quote in the paperSubstantial claimMay combine two claims. The quote only partly states this claim.
Findingown dataHypomethylated CpGs showed very high significance for immune response (FDR = 4.25E-25) and signal transduction (FDR = 1.45E-21), and specific categories such as ruffle organization, calcium ion transport, and OC differentiation.
“We observed very high significance for terms like immune response (FDR = 4.25E-25) and signal transduction (FDR = 1.45E-21), but also more specific categories such as ruffle organization (FDR = 9.91E-2), calcium ion transport (FDR = 4.6E-2), and OC differentiation (FDR = 1.94E-1).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe 0-to-20-day comparison showed 2,895 genes upregulated (FC >2; FDR <0.05) and 1,858 downregulated (FC <0.5; FDR <0.05).
“The 0-to-20-day comparison showed that 2,895 genes were upregulated (FC >2; FDR <0.05) and 1,858 were downregulated (FC <0.5; FDR <0.05).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [32] Gallois A, Lachuer J, Yvert G, Wierinckx A, Brunet F, Rabourdin-Combe C, Delprat C, Jurdic P, Mazzorana M: Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes. J Bone Miner Res. 2010, 25: 661-672. 10.1359/jbmr.090829. source
Not checked in the free run.
May combine two claims.
Findingown dataHypermethylated genes showed highly significant enrichment of signal transduction (FDR = 4.09E-17) and enrichment of categories related to other hematopoietic cell types.
“In the case of hypermethylated genes, we also found highly significant enrichment of signal transduction (FDR = 4.09E-17), and enrichment of categories related to other hematopoietic cell types”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGene ontology analysis reveals that all relevant functional categories and the majority of key genes for differentiation or activity of functional OCs undergo DNA methylation changes.
“reveals that all relevant functional categories and the majority of key genes for differentiation or the activity of functional OCs undergo DNA methylation changes and that genes within all relevant functional categories undergo DNA methylation changes.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAnalysis of transcription factor binding sites in sequences undergoing DNA methylation changes shows significant enrichment in binding motifs of transcription factors key in OC differentiation, some of which were validated for selected putative target genes.
“Interestingly, the analysis of the presence of transcription factor binding sites in sequences that undergo DNA methylation changes shows a significant enrichment in binding motifs of transcription factors that are key in OC differentiation, some of which we have validated for a selection of putative target genes.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataPU.1 directly interacts with both DNMT3b and TET2 in osteoclastogenesis samples.
“Our results showed that PU.1 directly interacted with both DNMT3b and TET2 (Figure 4C).”
- Also stated in: § PU.1 recruits DNMT3b and TET2 to hypermethylated and hypomethylated genes · boosted
“These confirmed the direct interaction with PU.1 (Figure 4C).”
Quote in the paperSubstantial claim- Also stated in: § PU.1 recruits DNMT3b and TET2 to hypermethylated and hypomethylated genes · boosted
May combine two claims.
Findingown data452 genes were both hypomethylated and overexpressed and 280 genes were both hypermethylated and repressed at selected thresholds.
“Comparing DNA methylation and expression data revealed that 452 genes were both hypomethylated and overexpressed and 280 genes were both hypermethylated and repressed at the selected thresholds”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data39% of all hypomethylated genes had binding motifs for AP-1, 15% had NF-kB binding motifs, and 15% had binding motifs for PU.1 or other ETS-related factors (PU.1 alone, 9%).
“In fact, 39% of all hypomethylated genes had binding motifs for AP-1, 15% genes had NF-kB binding motifs, and another 15% genes had binding motifs for PU.1 or other ETS-related factors (PU.1 alone, 9%)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFor hypermethylated genes CX3CR1 and NR4A2, PU.1 is associated at both 0 and 2 days, with a slight decrease at 2 days.
“In genes that become hypermethylated (CX3CR1 and NR4A2), we also observed association of PU.1 at both 0 and 2 days. However we again observed a slight decrease at 2 days together.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataDNMT3b and TET2 are present during osteoclastogenesis.
“we also confirmed the presence of DNMT3b, a de novo DNA methyltransferase, and the ten eleven translocation (TET) protein TET2, as enzymatic activities potentially related with DNA demethylation (Figure 4A and 4B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe two DNA methylation activities are very specific to particular gene sets and there are no indications of changes in repetitive elements.
“The two activities are very specific to particular gene sets and there are no indications of changes in repetitive elements.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataDNA methylation changes are closely correlated with gene expression changes, and a majority of genes that undergo hypomethylation or hypermethylation at promoters or gene bodies also experience a change in expression, although the relationship varies between gene sets.
“These data are closely correlated with gene expression changes, and a majority of genes that undergo hypomethylation or hypermethylation at their promoters or gene bodies also experience a change in expression, although the relationship varies between different gene sets.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe effects of PU.1 downregulation on DNA methylation occur only in PU.1 targets and not in non-target genes PLA2G4E and FSCN3.
“At any rate, the observed effects only occurred in PU.1 targets. It did not affect genes that are not targeted by PU.1 (PLA2G4E, FSCN3).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataTET1 and TET3 are undetectable by western blot and have low mRNA levels in this cell type.
“In fact TET1 and TET3 were undetectable in western blot (not shown) and qRT-PCR evidenced their low levels in this cell type (Figure 4A, only shown for TET1).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataHypomethylation takes place in relevant functional categories related with OC differentiation, and most genes necessary for OC function undergo hypomethylation, including ACP5, CTSK, and TM7SF4.
“Hypomethylation takes place in relevant functional categories related with OC differentiation and most of the genes that are necessary for OC function undergo hypomethylation including ACP5, CTSK, and TM7SF4 among others.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationMonocytes stimulated with RANKL and M-CSF generate functional osteoclasts that degrade bone and express osteoclast markers.
“MOs stimulated with RANKL and M-CSF generate functional OCs [27], which degrade bone and express OC markers [28].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [27] Nicholson GC, Malakellis M, Collier FM, Cameron PU, Holloway WR, Gough TJ, Gregorio-King C, Kirkland MA, Myers DE: Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL). Clin Sci (Lond). 2000, 99: 133-140. 10.1042/CS19990355. source
Not checked in the free run.
- cites [28] Sorensen MG, Henriksen K, Schaller S, Henriksen DB, Nielsen FC, Dziegiel MH, Karsdal MA: Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood. J Bone Miner Metab. 2007, 25: 36-45. source
Not checked in the free run.
May combine two claims.
PremisecitationVery few reports have analyzed the role of epigenetic changes during osteoclastogenesis, and these focus mainly on histone modifications.
“however very few reports have analyzed the role of epigenetic changes during osteoclastogenesis, and these focus mainly on histone modifications [29,30].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [29] Youn MY, Takada I, Imai Y, Yasuda H, Kato S: Transcriptionally active nuclei are selective in mature multinucleated osteoclasts. Genes Cells. 2010, 15: 1025-1035. 10.1111/j.1365-2443.2010.01441.x. source
Not checked in the free run.
- cites [30] Saltman LH, Javed A, Ribadeneyra J, Hussain S, Young DW, Osdoby P, Amcheslavsky A, van Wijnen AJ, Stein JL, Stein GS, Lian JB, Bar-Shavit Z: Organization of transcriptional regulatory machinery in osteoclast nuclei: compartmentalization of Runx1. J Cell Physiol. 2005, 204: 871-880. 10.1002/jcp.20329. source
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May combine two claims.
Study design and conductown dataThe quality of mature, bone-resorbing osteoclasts obtained under these conditions was confirmed by several methods, including presence of more than three nuclei in TRAP-positive cells, upregulation of OC markers CA2, CTSK, ACP5/TRACP, MMP9, and downregulation of CX3CR1.
“The quality of mature, bone-resorbing OCs obtained under these conditions was confirmed by several methods, including the presence of more than three nuclei in TRAP-positive cells (in some cases, up to 40 nuclei per cell were counted), the upregulation of OC markers, such as CA2, CTSK, ACP5/TRACP, and MMP9, and downregulation of the monocytic gene CX3CR1”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationOsteoclasts differentiate from monocyte/macrophage progenitors following M-CSF and RANKL stimulation.
“OCs differentiate from monocyte/macrophage progenitors following M-CSF [5] and RANKL [6] stimulation.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [5] Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW, Ahmed-Ansari A, Sell KW, Pollard JW, Stanley ER: Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci USA. 1990, 87: 4828-4832. 10.1073/pnas.87.12.4828. source
Not checked in the free run.
- cites [6] Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, et al: Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998, 93: 165-176. 10.1016/S0092-8674(00)81569-X. source
Not checked in the free run.
May combine two claims.
Interpretationown dataDNA hypomethylation is targeted to genomic regions that are activated during osteoclastogenesis, and hypermethylation silences alternative lineage genes that are not expressed in osteoclasts.
“Together, these data indicate that DNA hypomethylation is targeted to genomic regions that are activated during osteoclastogenesis, and hypermethylation silences alternative lineage genes that are not expressed in OCs.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataPU.1 is associated with genes that become repressed through hypermethylation and directly interacts with DNMT3b in osteoclastogenesis.
“First, we have identified the association of PU.1 with genes that become repressed through hypermethylation and describe its direct interaction with DNMT3b in the context of osteoclastogenesis.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationTET-dependent active DNA demethylation is supported by recent evidence.
“Recent evidences support a role for TET-dependent active DNA demethylation process [42,43].”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [42] Shen L, Wu H, Diep D, Yamaguchi S, D’Alessio AC, Fung HL, Zhang K, Zhang Y: Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell. 2013, 153: 692-706. 10.1016/j.cell.2013.04.002. source
Not checked in the free run.
- cites [43] Cortellino S, Xu J, Sannai M, Moore R, Caretti E, Cigliano A, Le Coz M, Devarajan K, Wessels A, Soprano D, Abramowitz LK, Bartolomei MS, Rambow F, Bassi MR, Bruno T, Fanciulli M, Renner C, Klein-Szanto AJ, Matsumoto Y, Kobi D, Davidson I, Alberti C, Larue L, Bellacosa A: Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair. Cell. 2011, 146: 67-79. 10.1016/j.cell.2011.06.020. source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationTET2 is required for active DNA demethylation in primary human monocytes.
“recent data shows that TET2 is required for active DNA demethylation in primary human MOs [45].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [45] Klug M, Schmidhofer S, Gebhard C, Andreesen R, Rehli M: 5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes. Genome Biol. 2013, 14: R46-10.1186/gb-2013-14-5-r46. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAn over-representation of ETS transcription factor binding sites was found in genome-wide DNA methylation profiling comparing cell types across the hematopoietic differentiation system.
“This is compatible with previous data on genome wide DNA methylation profiling comparing cell types across the hematopietic differentiation system where an over-representation of ETS transcription factor binding sites was found [2].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [2] Hogart A, Lichtenberg J, Ajay SS, Anderson S, Margulies EH, Bodine DM: Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites. Genome Res. 2012, 22: 1407-1418. 10.1101/gr.132878.111. source
Not checked in the free run.
May combine two claims.
Interpretationown dataTRANSFAC motif searches have weak predictive power and need experimental validation.
“however, it also reveals the weakness in the predictive power of TRANSFAC motif searches and the need of experimental validation of its results.”
Quote in the paperSubstantial claimMay combine two claims.
Study design and conductcitationThe initial analysis was validated in several candidate genes and using ChIPseq data for human primary monocytes.
“This initial analysis was validated in several candidate genes and using ChIPseq data for human primary monocytes [1].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [1] Pham TH, Benner C, Lichtinger M, Schwarzfischer L, Hu Y, Andreesen R, Chen W, Rehli M: Dynamic epigenetic enhancer signatures reveal key transcription factors associated with monocytic differentiation states. Blood. 2012, 119: e161-e171. 10.1182/blood-2012-01-402453. source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationTET proteins convert 5mC to 5hmC, 5fC, and 5cac.
“TET proteins are responsible for conversion of 5mC in 5hmC [34], 5fC, and 5cac [36].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [34] Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A: Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009, 324: 930-935. 10.1126/science.1170116. source
Not checked in the free run.
- cites [36] Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, He C, Zhang Y: Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011, 333: 1300-1303. 10.1126/science.1210597. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationTET2 plays a role in derepressing genes during pre-B cell to macrophage differentiation.
“we have recently reported that TET2 plays a role in derepressing genes in pre-B cell to macrophage differentiation [44]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [44] Kallin EM, Rodriguez-Ubreva J, Christensen J, Cimmino L, Aifantis I, Helin K, Ballestar E, Graf T: Tet2 facilitates the derepression of myeloid target genes during CEBPalpha-induced transdifferentiation of pre-B cells. Mol Cell. 2012, 48: 266-276. 10.1016/j.molcel.2012.08.007. source
Not checked in the free run.
May combine two claims.
Interpretationown dataPU.1 interacts with TET2 and is associated with genes that become demethylated.
“Second, we identify a novel interaction between PU.1 and TET2 and their association with genes that become demethylated.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe PU.1 knockdown experiments in primary human monocytes were performed with three biological replicates.
“These experiments were performed with three biological replicates.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenFor repetitive elements (Sat2, D4Z4, NBL2, 18S rRNA, and 28 rRNA), standard bisulfite sequencing of a minimum of 10 clones was performed.
“In the case of repetitive elements (Sat2, D4Z4, NBL2, 18S rRNA, and 28 rRNA) we performed standard bisulfite sequencing of a minimum of 10 clones.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPU.1 acts in concert with MITF to recruit co-repressors to osteoclast-specific genes in committed myeloid precursors capable of forming either macrophages or OC.
“PU.1 has been shown to act in concert with MITF to recruit co-repressors to osteoclast-specific in committed myeloid precursors capable of forming either macrophages or OC [50].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [50] Hu R, Sharma SM, Bronisz A, Srinivasan R, Sankar U, Ostrowski MC: Eos, MITF, and PU.1 recruit corepressors to osteoclast-specific genes in committed myeloid progenitors. Mol Cell Biol. 2007, 27: 4018-4027. 10.1128/MCB.01839-06. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenIgG was used as a negative control in the ChIP assays.
“IgG was used as a negative control.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataExpression data were validated by quantitative RT-PCR.
“Validation of expression data was performed by quantitative RT-PCR.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPU.1 can form a complex with DNMT3a and DNMT3b.
“previous studies have shown that PU.1 can form a complex with DNMT3a and DNMT3b [46].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [46] Suzuki M, Yamada T, Kihara-Negishi F, Sakurai T, Hara E, Tenen DG, Hozumi N, Oikawa T: Site-specific DNA methylation by a complex of PU.1 and Dnmt3a/b. Oncogene. 2006, 25: 2477-2488. 10.1038/sj.onc.1209272. source
Not checked in the free run.
May combine two claims.
Interpretationown dataOur study identifies key DNA methylation changes during OC differentiation and reveals an implication of PU.1 in the acquisition of DNA methylation and expression changes as well as novel interactions with DNMT3b and TET2.
“Our study has allowed us to identify key DNA methylation changes during OC differentiation and has revealed an implication of PU.1 in the acquisition of DNA methylation and expression changes as well as identifying novel interactions with DNMT3b and TET2.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationTET2-mediated hydroxylation of 5-methylcytosine is an intermediate step towards demethylation.
“Several recent reports have pointed at TET2-mediated hydroxylation of 5-methylcytosine as an intermediate step towards demethylation [51]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [51] Vincent JJ, Huang Y, Chen PY, Feng S, Calvopina JH, Nee K, Lee SA, Le T, Yoon AJ, Faull K, Fan G, Rao A, Jacobsen SE, Pellegrini M, Clark AT: Stage-specific roles for tet1 and tet2 in DNA demethylation in primordial germ cells. Cell Stem Cell. 2013, 12: 470-478. 10.1016/j.stem.2013.01.016. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductreasoningMultiple test correction was applied in the Gene Ontology analysis to account for multiple hypotheses and reduce false-positive results.
“Multiple test correction to account for the multiple hypotheses tested (one for each GO term) was applied to reduce false-positive results.”
Quote in the paperNot a substantial claimMay combine two claims.
Study design and conductown dataOCs were confirmed at the mRNA level by upregulation of key OC markers TRAP/ACP5, CA2, MMP9, and CTSK and downregulation of the MO marker CX3CR1.
“OCs (TRAP-positive cells with more than three nuclei) were also analyzed at the mRNA level: upregulation of key OC markers (TRAP/ACP5, CA2, MMP9, and CTSK) and the downregulation of the MO marker CX3CR1 were confirmed.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataOur data show changes in 5hmC at genes that become demethylated in osteoclastogenesis, reinforcing the possibility that PU.1-mediated recruitment of TET2 leads to 5hmC-mediated demethylation.
“our data show changes in 5hmC at genes that become demethylated in osteoclastogenesis, reinforcing the possibility that PU.1-mediated recruitment of TET2 is leading to 5hmC-mediated demethylation.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFor statistical purposes, M values are more adequate than Beta values for Illumina methylation data.
“however for statistics purposes it is more adequate the use of M values [54].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [54] Du P, Zhang X, Huang CC, Jafari N, Kibbe WA, Hou L, Lin SM: Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinforma. 2010, 11: 587-10.1186/1471-2105-11-587. source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPU.1 can generate a repressive chromatin structure characterized by H3K9me3 in myeloid and erythroid differentiation.
“PU.1 is known to generate a repressive chromatin structure characterized by H3K9me3 in myeloid and erythroid differentiation [49].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [49] Stopka T, Amanatullah DF, Papetti M, Skoultchi AI: PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure. EMBO J. 2005, 24: 3712-3723. 10.1038/sj.emboj.7600834. source
Not checked in the free run.
May combine two claims.
InterpretationreasoningThese DNA methylation changes occur in the virtual absence of DNA replication, suggesting participation of active mechanisms, particularly for hypomethylation events.
“These changes occur in the virtual absence of DNA replication suggesting the participation of active mechanisms, particularly relevant for hypomethylation events, for which the mechanisms are still subject of debate.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenBisulfite pyrosequencing was used to validate CpG methylation changes resulting from the Infinium HumanMethylation450 BeadChip analysis.
“We used bisulfite pyrosequencing to validate CpG methylation changes resulting from the analysis with the Infinium HumanMethylation450 BeadChips.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).
Study design and conductown dataThe ChIP experiments were performed with three biological replicates.
“These experiments were performed with three biological replicates.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenIn transcription factor binding enrichment analysis, a final adjusted FDR P value (with 0.25 cutoff) was used to determine statistical significance.
“We calculated two-tailed probabilities, and a final adjusted FDR P value (with 0.25 cutoff) was considered statistically significant.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataImpaired association of PU.1 results in defective acquisition of DNA methylation changes in both directions and reduced effect on gene expression changes.
“as well as showing how impaired association of PU.1 results in defective acquisition of DNA methylation changes in both directions as well as reduced effect on gene expression changes.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSer phosphorylation of PU.1 has already been shown to play a role in its recruitment to promoters.
“Ser phosphorylation of PU.1 has already been shown to play a role in its recruitment to promoters [52]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [52] Azim AC, Wang X, Park GY, Sadikot RT, Cao H, Mathew B, Atchison M, van Breemen RB, Joo M, Christman JW: NF-kappaB-inducing kinase regulates cyclooxygenase 2 gene expression in macrophages by phosphorylation of PU.1. J Immunol. 2007, 179: 7868-7875. source
Not checked in the free run.
May combine two claims.
Study design and conductown dataThe populations were highly enriched in multinuclear cells, some containing more than 40 nuclei.
“A phalloidin/DAPI stain allowed us to confirm that the populations were highly enriched in multinuclear cells, some of them containing more than 40 nuclei.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
PU.1 and NF-kB (p65) are representative examples of transcription factors involved in the acquisition of DNA methylation changes during osteoclastogenesis.
“To investigate the potential role of the aforementioned transcription factors in the acquisition of DNA methylation changes we chose PU.1 and NF-kB (p65 subunit) as two representative examples.”
The paper relies on this without support.
The Infinium HumanMethylation450 BeadChip interrogates >485,000 methylation sites per sample at single-nucleotide resolution, covering 99% of RefSeq genes, with an average of 17 CpG sites per gene region, and covers 96% of CpG islands plus additional coverage in CpG island shores and flanking regions.
“This array allows interrogating >485,000 methylation sites per sample at single-nucleotide resolution, covering 99% of RefSeq genes, with an average of 17 CpG sites per gene region distributed across the promoter, 5′UTR, first exon, gene body and 3′UTR. It covers 96% of CpG islands, with additional coverage in CpG island shores and the regions flanking them.”
The authors state this as an assumption.
Differentially methylated regions (DMRs) are more robust signals than differentially methylated probes (DMPs).
“We considered that DMR are more robust signals than DMPs.”
The paper relies on this without support.
In many cases, methylation changes are observed over a range of CpGs, which may be identified at shores close to transcription starting sites.
“It has been shown that in many cases methylation changes are observed over a range of CpGs, which may be identified for instance at shores close to transcription starting sites.”
The paper relies on this without support.
Under the conditions used to prepare monocytes for transfection, over 80% of the cells are monocytes.
“Under these conditions over 80% are MOs.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
PU.1 participates in the entire process of osteoclast differentiation and promotes osteoclast commitment and maturity.
“Considering PU.1 participates in the entire process of osteoclast differentiation and promotes osteoclast commitment and maturity of osteoclast33,57,58”
From Targeting Msx2 as a brake in the fusion fate of osteoclasts and an anabolic therapy in pre-clinical models of osteoporosis (2025) · cites this paper as [58]
The Discussion identifies PU.1 as an early influence on osteoclast-lineage commitment, and the Background and Results link it to activation of osteoclast-specific genes during differentiation. These passages support a role in commitment and differentiation, but do not establish that PU.1 participates throughout the entire process or promotes osteoclast maturity specifically. Page numbers are unavailable in our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measuredPU.1 and TET2 form a complex to co-regulate myeloid-specific genes.
“Interestingly, PU.1 and TET2 have been reported to form a complex to co-regulate myeloid-specific genes25,26,32.”
From HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages (2024) · cites this paper as [32]
The Discussion (page not specified) reports a PU.1–TET2 interaction and their association with genes that become demethylated. The Results describe PU.1-dependent recruitment of TET2 and changes in gene expression during monocyte-to-osteoclast differentiation. The evidence supports joint regulation in that setting, but not the broader description of the targets as myeloid-specific genes generally. (LLM, conf=0.81)
Experimental — accuracy not yet measuredIn monocytes, TET2 interacts with PU.1, leading to interaction with genetic targets.
“In monocytes, TET2 interacts with PU.1, leading to interaction with genetic targets32.”
From HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages (2024) · cites this paper as [32]
PU.1 acts as a repressor by direct recruitment of Dnmt3b.
“It is also reported to act as a repressor by direct recruitment of Dnmt3b (102).”
From Mechanisms of Action of Hematopoietic Transcription Factor PU.1 in Initiation of T-Cell Development (2019) · cites this paper as [102]
Distinct mechanisms according to haematopoietic lineages with direct or indirect binding to regulatory regions of repressed genes have been reported.
“Distinct mechanisms according to haematopoietic lineages with direct or indirect binding to regulatory regions of repressed genes have been reported (12,14–16).”
From HDAC1 and PRC2 mediate combinatorial control in SPI1/PU.1-dependent gene repression in murine erythroleukaemia (2022) · cites this paper as [12]
There is significant overlap between vitC-induced DMRs and PU.1-binding sites.
“We observe significant overlap between vitC-induced DMRs and PU.1-binding sites, consistent with previously described physical association between PU.1 and TET2.46”
From Vitamin C-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia (2018) · cites this paper as [46]
PU.1 guides TET2 to a specific subset of genomic regions to facilitate the oxidation of 5mC to 5hmC, a reaction inhibited by 2-hydroxyglutarate and enhanced by vitamin C.
“These observations support a model in which PU.1 guides TET2 to a specific subset of genomic regions to facilitate the oxidation of 5mC to 5hmC, a reaction that is inhibited by 2-hydroxyglutarate47 and enhanced by vitamin C13 (Figure 5d).”
From Vitamin C-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia (2018) · cites this paper as [46]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
5-aza-2-deoxycytidine dose differs by a factor of one million
MinorThe figure caption reports a 500 nM treatment, whereas the methods report 500 mM for the same treatment.
“Effects of 5azadC treatment (50 nM, 500 nM) on osteoclastogenesis monitoring ACP5, CTSK, and CX3CR1 levels and TRAP staining over time.”
“In some cases we performed monocyte-to-osteoclast differentiation experiments in the presence of different subtoxic concentrations of the DNA replication-coupled demethylating drug 5-aza-2-deoxycytidine (at 50 nM, 500 mM) for 72 h.”
Figure 2 attributes 4,753 genes to both methylation and expression changes
MinorFigure 2A says 4,753 genes display significant methylation and expression changes, but the results report only 3,515 differentially methylated genes in total. The 4,753 figure instead equals the reported counts of upregulated and downregulated genes.
“Heatmap showing expression levels on 0, 5, and 20 days for genes displaying significant methylation and expression changes (4,753 in total with FC ≥2 or FC ≤0.5; P ≤0.01 and FDR ≤0.05)”
“Statistical analysis of the combined data from the three pairs of samples revealed that 3,515 genes (8,028 CpGs) displayed differential methylation”
Figure 1 describes a nine-CpG cluster as having more than nine CpGs
MinorThe caption says both illustrated regions have more than nine differentially methylated CpGs, while the text identifies one of the illustrated clusters as having nine.
“Two examples of regions with more than nine consecutive CpGs differentially methylated are shown.”
“including 10 CpGs at the promoter of the TM4SF19 gene, also known as OC maturation-associated gene 4 protein, and nine CpGs in the gene body of ARID5B, the AT-rich interactive domain 5B (MRF1-like) (Figure 1F).”
Categories exceeding the stated FDR threshold are described as significantly enriched
MinorThe text says the hypomethylated-CpG ontology categories are significantly enriched at FDR ≤0.05, but lists ruffle organization at 0.0991 and OC differentiation at 0.194.
“Gene ontology analysis of hypomethylated CpGs revealed significant enrichment (FDR ≤0.05) for a variety of functional categories of relevance in OC differentiation and function (Figure 1D).”
“but also more specific categories such as ruffle organization (FDR = 9.91E-2), calcium ion transport (FDR = 4.6E-2), and OC differentiation (FDR = 1.94E-1).”
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MITF and PU.1 Recruit p38 MAPK and NFATc1 to Target Genes during Osteoclast Differentiation
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“MOs stimulated with RANKL and M-CSF generate functional OCs [27], which degrade bone and express OC markers [28].”
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“Moreover, OCs cause bone complications in several diseases, such as multiple myeloma [24], prostate cancer, and breast cancer [25], and there is also a specific tumor with OC origin, the giant cell tumor of bone [26].”
Site-specific DNA methylation by a complex of PU.1 and Dnmt3a/b
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InfluentialThe Signaling Adapter Protein DAP12 Regulates Multinucleation During Osteoclast Development
Humphrey, M. B., Ogasawara, K., Yao, W., Spusta, S. C., Daws, M. R., Lane, N. E., Lanier, L. L., & Nakamura, M. C. (2004) · Journal of Bone and Mineral Research · doi:10.1359/jbmr.0301234 · Cited by 123 · cited 2× · see citing works
“The signaling pathways activated after M-CSF and RANKL induction have been extensively described, and act through TRAF-6 [8,9], immunoreceptor tyrosine-based activation motif (ITAM) [10] adaptors DAP12 [11] and FcRγ [12] associated with their respective receptors, TREM-2 [13] and OSCAR, as well as calcium oscillations…”
Nicholson, G. C., Malakellis, M., Collier, F., Cameron, P. U., Holloway, W. R., Gough, T. J., Gregorio-King, C. C., Kirkland, M. A., & Myers, D. E. (2000) · PubMed · Cited by 119 · see citing works
Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells
Rodríguez, J., Vives, L., Jordà, M., Morales, C., Muñoz, M., Vendrell, E., & Peinado, M. A. (2007) · Nucleic Acids Research · doi:10.1093/nar/gkm1105 · Cited by 113 · cited 2× · see citing works
“We also performed genome-wide amplification of unmethylated DNA Alu repeats (AUMA), the most common family of repetitive elements that are present in tandem or interspersed in the genome [31].”
NICHOLSON, G. C., MALAKELLIS, M., COLLIER, F. M., CAMERON, P. U., HOLLOWAY, W. R., GOUGH, T. J., GREGORIO-KING, C., KIRKLAND, M. A., & MYERS, D. E. (2000) · Clinical Science · doi:10.1042/cs0990133 · Cited by 107 · cited 1× · see citing works
“MOs stimulated with RANKL and M-CSF generate functional OCs [27], which degrade bone and express OC markers [28].”
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“This is compatible with previous data on genome wide DNA methylation profiling comparing cell types across the hematopietic differentiation system where an over-representation of ETS transcription factor binding sites was found [2].”
InfluentialKlug, M., Schmidhofer, S., Gebhard, C., Andreesen, R., & Rehli, M. (2013) · Genome Biology · doi:10.1186/gb-2013-14-5-r46 · Cited by 101 · cited 2× · see citing works
“Also, we have recently reported that TET2 plays a role in derepressing genes in pre-B cell to macrophage differentiation [44], and recent data shows that TET2 is required for active DNA demethylation in primary human MOs [45].”
Kallin, E. M., Rodríguez-Ubreva, J., Christensen, J., Cimmino, L., Aifantis, I., Helin, K., Ballestar, E., & Graf, T. (2012) · Molecular Cell · doi:10.1016/j.molcel.2012.08.007 · Cited by 92 · see citing works
Self-citationStatistical significance of cis-regulatory modules
Schones, D. E., Smith, A. D., & Zhang, M. Q. (2007) · BMC Bioinformatics · doi:10.1186/1471-2105-8-19 · Cited by 89 · cited 1× · see citing works
“Analysis of transcription factor binding We used the STORM algorithm [65] to identify potential over-representation of transcription factor motifs in the 500-bp region around the center of the hypomethylated/ hypermethylated CpG sites (as well as for all other CpGs-containing probes contained in the array) assuming cu…”
Hu, R., Sharma, S. M., Bronisz, A., Srinivasan, R., Sankar, U., & Ostrowski, M. C. (2007) · Molecular and Cellular Biology · doi:10.1128/mcb.01839-06 · Cited by 86 · cited 1× · see citing works
“1 has been shown to act in concert with MITF to recruit co-repressors to osteoclast-specific in committed myeloid precursors capable of forming either macrophages or OC [50].”
C/EBPα regulates osteoclast lineage commitment
Chen, W., Zhu, G., Hao, L., Wu, M., Ci, H., & Li, Y. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1211383110 · Cited by 80 · see citing works
Gallois, A., Lachuer, J., Yvert, G., Wierinckx, A., Brunet, F., Rabourdin-Combe, C., Delprat, C., Jurdic, P., & Mazzorana, M. (2010) · Journal of Bone and Mineral Research · doi:10.1359/jbmr.090829 · Cited by 75 · cited 2× · see citing works
“E-MEXP-2019) on 0, 5, and 20 days [32].”
Accurate genome-scale percentage DNA methylation estimates from microarray data
Aryee, M. J., Wu, Z. J., Ladd‐Acosta, C., Herb, B. R., Feinberg, A. P., Yegnasubramanian, S., & Irizarry, R. A. (2010) · Biostatistics · doi:10.1093/biostatistics/kxq055 · Cited by 71 · cited 1× · see citing works
“Identification of genomic clusters of differentially methylated CpGs A clustering method was applied to the differenced methylated CpGs from charm package [59].”
Hernando, H., Shannon-Lowe, C. D., Islam, A. B. M. M. K., Al‐Shahrour, F., Rodríguez‐Ubreva, J., Rodríguez-Cortez, V., Javierre, B. M., Mangas, C., Fernández, A. F., Parra, M., et al. (2013) · Genome biology · doi:10.1186/gb-2013-14-1-r3 · Cited by 56 · cited 2× · see citing works
“1, key transcription factors in osteoclastogenesis Different studies have recently shown that transcription factor binding events are associated with changes in the DNA methylation profiles and the response to different situations [2,39,40].”
Self-citationHama, M., Kirino, Y., Takeno, M., Takase, K., Miyazaki, T., Yoshimi, R., Ueda, A., Itoh-Nakadai, A., Muto, A., Igarashi, K., et al. (2012) · Arthritis & Rheumatism · doi:10.1002/art.33497 · Cited by 31 · cited 2× · see citing works
“Other key factors involved in OC differentiation include C/EBPα [47] and Bach1 [48].”
Saltman, L. H., Javed, A., Ribadeneyra, J., Hussain, S., Young, D. W., Osdoby, P., Amcheslavsky, A., van Wijnen, A. J., Stein, J. L., Stein, G. S., et al. (2005) · Journal of Cellular Physiology · doi:10.1002/jcp.20329 · Cited by 28 · see citing works
Complex Regulation of Tartrate-resistant Acid Phosphatase (TRAP) Expression by Interleukin 4 (IL-4)
Yu, M., Moreno, J. L., Stains, J. P., & Keegan, A. D. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m109.001016 · Cited by 28 · cited 1× · see citing works
“These tr anscription factors bind to the promoter and help up-regulating OC markers such as dendritic cel l-specific transmembrane protein (DC-STAMP/TM7SF4) [17], tartrate-resistant acid phosphatase ( TRACP/ACP5 ) [18], cathepsin K ( CTSK) [19], matrix metalloproteinase 9 ( MMP9) [20] and carbonic anhydrase 2 (CA2).”
Transcriptionally active nuclei are selective in mature multinucleated osteoclasts
Youn, M., Takada, I., Imai, Y., Yasuda, H., & Kato, S. (2010) · Genes to Cells · doi:10.1111/j.1365-2443.2010.01441.x · Cited by 22 · cited 1× · see citing works
“As indicated, the involvement of transcription factors in this model has been well studied, however very few reports have analyzed the role of epigenetic changes during osteoclastogenesis, and these focus mainly on histone modifications [29,30].”
Mii, Y., Miyauchi, Y., Morishita, T., Miura, S., Honoki, K., Aoki, M., & Tamai, S. (1991) · Ultrastructural Pathology · doi:10.3109/01913129109023192 · Cited by 16 · cited 1× · see citing works
“Moreover, OCs cause bone complications in several diseases, such as multiple myeloma [24], prostate cancer, and breast cancer [25], and there is also a specific tumor with OC origin, the giant cell tumor of bone [26].”
Azim, A. C., Wang, X., Park, G. Y., Sadikot, R. T., Cao, H., Mathew, B., Atchison, M., van Breemen, R. B., Joo, M., & Christman, J. W. (2007) · The Journal of Immunology · doi:10.4049/jimmunol.179.11.7868 · Cited by 13 · cited 1× · see citing works
“1 has already been shown to play a role in its recruitment to promoters [52] and could also participate in discriminating interaction with epigenetic modifiers.”
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TET-mediated active DNA demethylation: mechanism, function and beyond
Wu, X., & Zhang, Y. (2017) · Nature Reviews Genetics · doi:10.1038/nrg.2017.33 · Cited by 1,661 · see citing works
β-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance
Novakovic, B., Habibi, E., Wang, S.-Y., Arts, R. J. W., Davar, R., Megchelenbrink, W., Kim, B., Kuznetsova, T., Kox, M., Zwaag, J., et al. (2016) · Cell · doi:10.1016/j.cell.2016.09.034 · Cited by 689 · see citing works
Oxidative stress alters global histone modification and DNA methylation
Niu, Y., DesMarais, T. L., Tong, Z., Yao, Y., & Costa, M. (2015) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2015.01.028 · Cited by 351 · see citing works
Sensitivity of transcription factors to DNA methylation
Héberlé, É., & Bardet, A. F. (2019) · Essays in Biochemistry · doi:10.1042/ebc20190033 · Cited by 347 · see citing works
Epigenetic control of myeloid cell differentiation, identity and function
Álvarez‐Errico, D., Vento‐Tormo, R., Sieweke, M. H., & Ballestar, E. (2014) · Nature reviews. Immunology · doi:10.1038/nri3777 · Cited by 328 · see citing works
Self-citationTet2 restrains inflammatory gene expression in macrophages
Cull, A. H., Snetsinger, B., Buckstein, R., Wells, R. A., & Rauh, M. J. (2017) · Experimental Hematology · doi:10.1016/j.exphem.2017.08.001 · Cited by 295 · see citing works
TET (Ten-eleven translocation) family proteins: structure, biological functions and applications
Zhang, X., Zhang, Y., Wang, C., & Wang, X. (2023) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-023-01537-x · Cited by 286 · see citing works
Breton, C. V., Marsit, C. J., Faustman, E. M., Nadeau, K. C., Goodrich, J. M., Dolinoy, D. C., Herbstman, J. B., Holland, N., LaSalle, J. M., Schmidt, R. J., et al. (2017) · Environmental Health Perspectives · doi:10.1289/ehp595 · Cited by 261 · see citing works
Tolerogenic Dendritic Cells in Autoimmunity and Inflammatory Diseases
Morante-Palacios, O., Fondelli, F., Ballestar, E., & Martínez-Cáceres, E. M. (2020) · Trends in Immunology · doi:10.1016/j.it.2020.11.001 · Cited by 263 · see citing works
Self-citationTranscription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate
Sardina, J. L., Collombet, S., Tian, T. V., Gómez, A., Stefano, B. D., Berenguer, C., Brumbaugh, J., Stadhouders, R., Segura-Morales, C., Gut, M., et al. (2018) · Cell stem cell · doi:10.1016/j.stem.2018.08.016 · Cited by 230 · see citing works
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Cross-talk between Site-specific Transcription Factors and DNA Methylation States
Blattler, A., & Farnham, P. (2013) · Journal of Biological Chemistry · doi:10.1074/jbc.r113.512517 · Cited by 200 · see citing works
A Highly Sensitive and Robust Method for Genome-wide 5hmC Profiling of Rare Cell Populations
Han, D., Lu, X., Shih, A. H., Nie, J., You, Q., Xu, M. M., Melnick, A. M., Levine, R. L., & He, C. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.06.028 · Cited by 167 · see citing works
“These observations also open an interesting question about how the binding of PU.1, which was reported to physically interact with TET2 (de la Rica et al., 2013), contributes to 5hmC redistribution induced by inactivation of TET2.”
Mechanisms that regulate the activities of TET proteins
Joshi, K., Liu, S., Breslin, P., & Zhang, J. (2022) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-022-04396-x · Cited by 172 · see citing works
Lio, C.-W., Zhang, J., González-Avalos, E., Hogan, P. G., Chang, X., & Rao, A. (2016) · eLife · doi:10.7554/elife.18290 · Cited by 170 · see citing works
Gene activation precedes DNA demethylation in response to infection in human dendritic cells
Pacis, A., Mailhot-Léonard, F., Tailleux, L., Randolph, H. E., Yotova, V., Dumaine, A., Grenier, J., & Barreiro, L. B. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1814700116 · Cited by 146 · see citing works
EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity
Sun, Z., Xu, X., He, J., Murray, A., Sun, M.-A., Wei, X., Wang, X., McCoig, E., Xie, E., Jiang, X., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-11905-3 · Cited by 146 · see citing works
TET Enzymes and 5hmC in Adaptive and Innate Immune Systems
Lio, C. J., & Rao, A. (2019) · Frontiers in Immunology · doi:10.3389/fimmu.2019.00210 · Cited by 139 · see citing works
Mechanisms and Disease Associations of Haplotype-Dependent Allele-Specific DNA Methylation
Do, C., Lang, C. F., Lin, J. P., Darbary, H. K., Krupska, I., Gaba, A., Petukhova, L., Vonsattel, J., GALLAGHER, M. P., Goland, R., et al. (2016) · The American Journal of Human Genetics · doi:10.1016/j.ajhg.2016.03.027 · Cited by 125 · see citing works
IL-4 orchestrates STAT6-mediated DNA demethylation leading to dendritic cell differentiation
Vento‐Tormo, R., Rodríguez‐Ubreva, J., de la Rica, L., Urquiza, J., Javierre, B. M., Sabarinathan, R., Luque, A., Esteller, M., Aran, J. M., Álvarez‐Errico, D., et al. (2016) · Genome biology · doi:10.1186/s13059-015-0863-2 · Cited by 120 · see citing works
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Playing TETris with DNA modifications
Delatte, B., Deplus, R., & Fuks, F. (2014) · The EMBO Journal · doi:10.15252/embj.201488290 · Cited by 116 · see citing works
“…beyond the neuronal context, reports show that the hydroxy-methylcytosine pattern is highly dynamic during terminal cell differentiation, as described for preadipocytes, germ cells, and differentiating monocytes (Fujiki et al , 2013; Gan et al , 2013; Klug et al , 2013; de la Rica et al , 2013).”
The Roles of Epigenetics Regulation in Bone Metabolism and Osteoporosis
Xu, F., Li, W., Yang, X. Y., Na, L., Chen, L., & Liu, G. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2020.619301 · Cited by 117 · see citing works
Function determinants of TET proteins: the arrangements of sequence motifs with specific codes
Liu, D., Li, G., & Zuo, Y. (2018) · Briefings in Bioinformatics · doi:10.1093/bib/bby053 · Cited by 115 · see citing works
Active DNA demethylation promotes cell fate specification and the DNA damage response
Wang, D., Wu, W., Callen, E., Pavani, R., Zolnerowich, N., Kodali, S., Zong, D., Wong, N., Noriega, S., Nathan, W. J., et al. (2022) · Science · doi:10.1126/science.add9838 · Cited by 113 · see citing works
DNMT3B Functions: Novel Insights From Human Disease
Gagliardi, M., Strazzullo, M., & Matarazzo, M. R. (2018) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2018.00140 · Cited by 107 · see citing works
“…reported that several transcription factors, such as E2F6 (Velasco et al., 2010), MIZ1, and PU.1 (de la Rica et al., 2013) also act as positive regulators of the DNMT3B recruitment, leading to silencing of their target genes (Brenner et al., 2005; Suzuki et al., 2006; de la Rica et al., 2013).”
Toward a Mechanistic Understanding of DNA Methylation Readout by Transcription Factors
Kribelbauer-Swietek, J. F., Lu, X., Rohs, R., Mann, R. S., & Bussemaker, H. J. (2019) · Journal of Molecular Biology · doi:10.1016/j.jmb.2019.10.021 · Cited by 101 · see citing works
Epigenetic and transcriptional regulation of osteoclast differentiation
Kurotaki, D., Yoshida, H., & Tamura, T. (2020) · Bone · doi:10.1016/j.bone.2020.115471 · Cited by 97 · see citing works
TET2 regulates osteoclastogenesis by modulating autophagy in OVX-induced bone loss
Chen, Y., Tao, H., Zhang, H., Xia, Y., Bai, J., Ge, G., Li, W., Zhang, W., Xiao, L., Xu, Y., et al. (2022) · Autophagy · doi:10.1080/15548627.2022.2048432 · Cited by 103 · see citing works
Mechanisms of Action of Hematopoietic Transcription Factor PU.1 in Initiation of T-Cell Development
Rothenberg, E. V., Hosokawa, H., & Ungerbäck, J. (2019) · Frontiers in Immunology · doi:10.3389/fimmu.2019.00228 · Cited by 99 · see citing works
“It is also reported to act as a repressor by direct recruitment of Dnmt3b (102).”
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Vitamin C-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia
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Impact of constitutional TET2 haploinsufficiency on molecular and clinical phenotype in humans
Kaasinen, E., Kuismin, O., Rajamäki, K., Ristolainen, H., Aavikko, M., Kondelin, J., Saarinen, S., Berta, D. G., Katainen, R., Hirvonen, E. A. M., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-09198-7 · Cited by 92 · see citing works
Clinical value of DNA methylation markers in autoimmune rheumatic diseases
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Self-citationUnderstanding the Relevance of DNA Methylation Changes in Immune Differentiation and Disease
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Self-citationThe Role of Host Cell DNA Methylation in the Immune Response to Bacterial Infection
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Epigenetics and Bone Remodeling
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Role of Epigenomics in Bone and Cartilage Disease
van Meurs, J. B. J., Boer, C. G., López-Delgado, L., & Riancho, J. A. (2019) · Journal of Bone and Mineral Research · doi:10.1002/jbmr.3662 · Cited by 74 · see citing works
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Self-citationEffect of diabetes status and hyperglycemia on global DNA methylation and hydroxymethylation
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TET Methylcytosine Oxidases in T Cell and B Cell Development and Function
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Transcriptional determination and functional specificity of myeloid cells: making sense of diversity
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Vitamin D receptor, STAT3, and TET2 cooperate to establish tolerogenesis
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Self-citationTET-Mediated Epigenetic Regulation in Immune Cell Development and Disease
Tsiouplis, N. J., Bailey, D. W., Chiou, L. F., Wissink, F. J., & Tsagaratou, A. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2020.623948 · Cited by 59 · see citing works
Role and Regulation of Transcription Factors in Osteoclastogenesis
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“However, impaired osteoclastogenesis caused by NF-κ Bp50/p52 or c-Fos deletion can be rescued by overexpression of NFATc1 [153].”
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Osteoclasts and osteoarthritis: Novel intervention targets and therapeutic potentials during aging
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“It has been well-documented that excessive RANKL and M-CSF expression, along with a decrease in OPG levels, can be identified in the entire bone and bone marrow cells (Cao et al., 2005; Chung et al., 2014; Makhluf et al., 2000).”
BackgroundInfluentialDekkers, K. F., Neele, A. E., Jukema, J. W., Heijmans, B. T., & de Winther, M. P. J. (2019) · Epigenetics & Chromatin · doi:10.1186/s13072-019-0279-4 · Cited by 56 · see citing works
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Deciphering the genetic code of DNA methylation
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“has been pointed out as the main enzyme involved in multistep active demethylation processes in terminal MO-related differentiation processes (6,10).”
BackgroundSelf-citationZhao, Z., Zhang, Z., Li, J., Dong, Q., Xiong, J., Li, Y., Lan, M., Li, G., & Zhu, B. (2020) · eLife · doi:10.7554/elife.61965 · Cited by 56 · see citing works
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Self-citationNew insights into the epigenetics of inflammatory rheumatic diseases
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“Among these, only the PU.1 binding motif showed signi fi cant enrichment in both hypermethylated and hypomethylated gene sets 193 .”
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Epigenetic control of hematopoiesis: the PU.1 chromatin connection
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5-Hydroxymethylcytosine (5hmC), or How to Identify Your Favorite Cell
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- Authors
- de la Rica, L., Rodríguez-Ubreva, J., García, M., Islam, A. B., Urquiza, J. M., Hernando, H., Christensen, J., Helin, K., Gómez-Vaquero, C., & Ballestar, E.
- Affiliations
- Bellvitge University Hospital (ES)
- Institut d'Investigació Biomédica de Bellvitge (ES)
- University of Copenhagen (DK)
- Corresponding author
- Ballestar, Esteban
- Venue
- Genome Biology
- Published
- 2013-09-12
- Volume
- 14
- Issue
- 9
- Pages
- R99-R99
- ISSN
- 1465-6906 (ISSN-L)
- 1474-760X
- PMC
- PMC4054781
- OpenAlex
- W2123115454
- Semantic Scholar
- 1bf13dc677b7a6bf2895c59587e6626186b9efa9
- Cited by
- 211
- References
- 70
- Citations by year
- 2026: 5 · 2025: 18 · 2024: 12 · 2023: 9 · 2022: 18 · 2021: 21 · 2020: 24 · 2019: 26 · 2018: 13 · 2017: 23 · 2016: 15 · 2015: 14 · 2014: 12 · 2013: 1
- FWCI
- 6.23
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Genome biology (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RECERCAT (Consorci de Serveis Universitaris de Catalunya) (publishedVersion, other-oa)
- Dipòsit Digital de la Universitat de Barcelona (Universitat de Barcelona) (submittedVersion, cc-by)
- Research at the University of Copenhagen (University of Copenhagen) (submittedVersion)
- Repositori digital de la UPF (Universitat Pompeu Fabra) (submittedVersion, cc-by)
- Funders
- Fundación Ramón Areces
- Ministerio de Ciencia e Innovación (2009SGR184, SAF2011-29635)
- Agència de Gestió d'Ajuts Universitaris i de Recerca
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
- Keywords
- PU.1, TET2, DNMT3B, DNA methylation, 5-hydroxymethylcytosine, demethylation, osteoclastogenesis, monocyte differentiation, transcription factor binding motifs, epigenetic regulation, NF-κB, AP-1, transcription factor binding
- MeSH terms
- DNA Methyltransferase 3B, Proto-Oncogene Protein Spi-1, Binding Sites, Cell Differentiation, Cytosine, DNA (Cytosine-5-)-Methyltransferases, DNA-Binding Proteins, Humans, Monocytes, Osteoclasts, Protein Binding, Proto-Oncogene Proteins, Trans-Activators, NF-kappa B, Transcription Factor AP-1, CpG Islands, DNA Methylation, RNA, Small Interfering, Epigenesis, Genetic, 5-Methylcytosine, Dioxygenases, Protein Interaction Domains and Motifs
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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