Txnip Enhances Fitness of Dnmt3a -Mutant Hematopoietic Stem Cells via p21

No direct experiments have been performed to quantify fitness between mutant and wild-type hematopoietic stem cells in response to IFNγ.

“Although the competitive advantage of Dnmt3a-mutant HSCs during inflammation has been implied (20,23), no direct experiments have been performed to quantify fitness between mutant and WT HSCs in response to IFNγ.”

• cites [20] Hormaechea-Agulla, 2021, Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNgamma signaling, Cell Stem Cell sourceDisputed

  The Results section ('Dnmt3a loss of function blunts HSC response to infection'; page unavailable) reports that daily recombinant IFNγ injections induced expansion of Dnmt3a-mutant HSC clones. The Methods describe the corresponding mosaic-mouse experiment, in which mutant marrow competed against WT marrow. Thus, the paper reports a direct comparison under IFNγ treatment, contrary to the claim that no such experiment had been performed. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [23] Zhang, 2019, Inflammatory cytokines promote clonal hematopoiesis with specific mutations in ulcerative colitis patients, Exp Hematol sourceSupported

Titration of doxycycline concentration in chow identified a dose that approximated serum levels of IFNγ in mice infected with the Mycobacterium avium pathogen.

“Titration of doxycycline concentration in chow (Supplementary Fig. S4A) identified a dose that approximated serum levels of IFNγ in mice infected with the Mycobacterium avium pathogen (29).”

• cites [29] Baldridge, 2010, Quiescent haematopoietic stem cells are activated by IFN-gamma in response to chronic infection, Nature sourcePartly supported

  The passage on IFNγ during M. avium infection (labelled Abstract in our copy; page unavailable) reports increased IFNγ in infected mice’s bone marrow and refers to previous findings in serum. It gives no serum concentration against which a doxycycline dose could be compared. The chow titration is attributed to the citing paper’s own supplementary figure, not to this source. (LLM, conf=0.81)

  Experimental — accuracy not yet measured

Ulcerative colitis patients have a higher incidence of DNMT3A-mutant clonal hematopoiesis associated with increased serum IFNγ.

“We recently showed ulcerative colitis patients have a higher incidence of DNMT3A-mutant CH associated with increased serum IFNγ (23).”

• cites [23] Zhang, 2019, Inflammatory cytokines promote clonal hematopoiesis with specific mutations in ulcerative colitis patients, Exp Hematol sourceSupported

Analysis of DNA methylation and gene expression patterns in mouse and human systems has yet to identify a unifying molecular mechanism to explain the increased fitness of Dnmt3a-mutant hematopoietic stem cells.

“However, analysis of DNA methylation and gene expression patterns in mouse (15,17) and human (17,18) systems have yet to identify a unifying molecular mechanism to explain the increased fitness of these mutant HSCs.”

• cites [15] Challen, 2012, Dnmt3a is essential for hematopoietic stem cell differentiation, Nat Genet sourceSupported
• cites [17] Izzo, 2020, DNA methylation disruption reshapes the hematopoietic differentiation landscape, Nat Genet sourcePartly supported

  The paper reports gene-expression and methylation analyses in Dnmt3a-deficient mice and a human DNMT3A-mutant sample (Abstract section; page unavailable). It proposes a potential mechanism for altered lineage priming, but does not establish whether a unifying molecular mechanism explains the increased fitness of mutant HSCs. That specific fitness claim is not supported by the cited passages. (LLM, conf=0.77)

  Experimental — accuracy not yet measured
• cites [18] Spencer, 2017, CpG island hypermethylation mediated by DNMT3A is a consequence of AML progression, Cell sourceSupported

Control test hematopoietic stem cells possessed an advantage without IFNγ due to the described fitness deficit of Ubc-GFP hematopoietic stem cells.

“Control test HSCs possessed an advantage without IFNγ due to the described fitness deficit of Ubc-GFP HSCs (30).”

• cites [30] Faltusova, 2018, Stem cell defect in ubiquitin-green fluorescent protein mice facilitates engraftment of lymphoid-primed hematopoietic stem cells, Stem Cells sourceSupported

Mouse models show Dnmt3a loss-of-function skews hematopoietic stem cell fate decisions toward self-renewal, facilitating clonal expansion in the bone marrow.

“Mouse models show Dnmt3a loss-of-function skews HSC fate decisions toward self-renewal, facilitating clonal expansion in the bone marrow (BM; ref. 15,16).”

• cites [15] Challen, 2012, Dnmt3a is essential for hematopoietic stem cell differentiation, Nat Genet sourceSupported
• cites [16] Challen, 2014, Dnmt3a and Dnmt3b have overlapping and distinct functions in hematopoietic stem cells, Cell Stem Cell sourceSupported

The competitive advantage of Dnmt3a-mutant hematopoietic stem cells is exacerbated by hematopoietic stress such as serial transplantation and microbial infection.

“The competitive advantage of Dnmt3a-mutant HSCs is exacerbated by hematopoietic stress such as serial transplantation (19) and microbial infection (20).”

• cites [19] Jeong, 2018, Loss of Dnmt3a immortalizes hematopoietic stem cells in vivo, Cell Rep sourceSupported
• cites [20] Hormaechea-Agulla, 2021, Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNgamma signaling, Cell Stem Cell sourceSupported

Dnmt3a heterozygous hematopoietic stem cells produce only modest phenotypes in mice.

“However, we and others have shown that Dnmt3a HET HSCs produce only modest phenotypes in mice (25,26), likely because these variants provide very minor fitness advantages to HSCs (2) that require the extended lifespan of humans to manifest measureable phenotypes.”

• cites [25] Cole, 2017, Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies, J Clin Invest sourceNot checked — no full text of the source
• cites [26] Celik, 2015, Enforced differentiation of Dnmt3a-null bone marrow leads to failure with c-Kit mutations driving leukemic transformation, Blood sourceSupported

In unmanipulated mice, there were few significantly differentially expressed genes between control and Dnmt3a KO hematopoietic stem cells.

“In unmanipulated mice, there were few significantly differentially expressed genes (DEG) between control and Dnmt3a KO HSCs (Fig. 4A), consistent with prior studies (15).”

• cites [15] Challen, 2012, Dnmt3a is essential for hematopoietic stem cell differentiation, Nat Genet sourceCould not verify

  The Results section on multipotency gene upregulation reports 516 differentially expressed genes between transplanted Dnmt3a-null and control HSCs. The Microarrays methods specify that the profiled HSCs came from secondary recipient mice. Our copy does not report the number of differentially expressed genes between these groups in unmanipulated mice, so it cannot verify the cited comparison. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Focal methylation alterations appear physiologically relevant.

“yet focal methylation alterations appear physiologically relevant (17,18,37,41).”

• cites [17] Izzo, 2020, DNA methylation disruption reshapes the hematopoietic differentiation landscape, Nat Genet sourceSupported
• cites [18] Spencer, 2017, CpG island hypermethylation mediated by DNMT3A is a consequence of AML progression, Cell sourcePartly supported

  The Introduction says that focal methylation changes occur in cancer and that, in some cases, altered methylation is associated with expression of genes related to cancer pathogenesis. That supports possible physiological relevance, but the Discussion says the downstream consequences of the changes studied in AML remain unclear and reports few reproducible transcriptional effects. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [37] Ketkar, 2020, Remethylation of Dnmt3a (-/-) hematopoietic cells is associated with partial correction of gene dysregulation and reduced myeloid skewing, Proc Natl Acad Sci U S A sourceSupported
• cites [41] Jeong, 2014, Large conserved domains of low DNA methylation maintained by Dnmt3a, Nat Genet sourceSupported

Genetic deletion of IFNgr1 in Dnmt3a KO hematopoietic stem cells normalized gene expression, including the previously reported upregulation of hematopoietic stem cell stemness genes.

“In support of the functional data, genetic deletion of IFNgr1 in Dnmt3a KO HSCs normalized gene expression, including the previously reported (15) upregulation of HSC “stemness” genes (Supplementary Fig. S5A).”

• cites [15] Challen, 2012, Dnmt3a is essential for hematopoietic stem cell differentiation, Nat Genet sourceSupported

The trend toward impaired peripheral blood engraftment of IFNγ-treated control hematopoietic stem cells was not observed from IFNγ-treated Dnmt3a-mutant hematopoietic stem cells.

“The trend toward impaired peripheral blood engraftment of IFNγ-treated control HSCs was not observed from IFNγ-treated Dnmt3a-mutant HSCs (Supplementary Fig. S1F).”

IFNγ signaling is required for clonal expansion of Dnmt3a-mutant hematopoietic stem cells in vivo.

“We found Dnmt3a-mutant HSCs specifically resist IFNγ-mediated depletion, and IFNγ signaling is required for clonal expansion of Dnmt3a-mutant HSCs in vivo.”

In unperturbed adult mice, genetic loss of IFNgr1 normalized the increased abundance of Dnmt3a-mutant hematopoietic stem cells in the bone marrow.

“In unperturbed adult (∼12-week old) mice, genetic loss of IFNgr1 normalized the increased abundance of Dnmt3a-mutant HSCs in the BM (Supplementary Fig. S3A and S3B).”

The trend toward reduced self-renewal of IFNγ-treated AAVS1-targeted hematopoietic stem cells was not observed in IFNγ-treated DNMT3A-targeted hematopoietic stem cells.

“Again, while peripheral blood engraftment was not different between genotypes (Supplementary Fig. S2D), the trend toward reduced self-renewal of IFNγ-treated AAVS1-targered HSCs was not observed in IFNγ-treated DNMT3A-targeted HSCs (Fig. 1F).”

Treatment with human IFNγ did not influence peripheral blood engraftment of human cells.

“As with mice, treatment with hIFNγ did not influence peripheral blood engraftment of human cells (Supplementary Fig. S2A and S2B).”

Complementary genomic techniques identified a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant hematopoietic stem cells under conditions of inflammatory stress.

“Complementary genomic techniques identified a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant HSCs under conditions of inflammatory stress, which was validated by functional genetic rescue experiments in vivo.”

The apoptotic resistance phenotype of Dnmt3a-mutant hematopoietic stem cells to IFNγ was not observed after exposure to related proinflammatory cytokines.

“Moreover, the apoptotic resistance phenotype of Dnmt3a-mutant HSCs to IFNγ was not observed after exposure to related proinflammatory cytokines (Fig. 2E).”

IFNγ treatment had minimal impact on peripheral blood engraftment.

“IFNγ treatment had minimal impact on peripheral blood engraftment (Supplementary Fig. S1A and S1B).”

The increased abundance of DNMT3A-targeted human hematopoietic stem cells was not influenced by IFNγ treatment.

“Analysis of the BM (Supplementary Fig. S2C) showed the increased abundance of DNMT3A-targeted HSCs was not influenced by IFNγ treatment (Fig. 1E).”

Comparable numbers of control hematopoietic stem cells were found in WT and IFNγ-/- recipients.

“BM analysis posttransplant showed comparable numbers of control HSCs in WT and IFNγ -/-recipients.”

Fitness of Dnmt3a R878 hematopoietic stem cells was enhanced by chronic IFNγ.

“Conversely, fitness of Dnmt3a-mutant HSCs was generally unaffected by chronic IFNγ, and was in fact enhanced for Dnmt3a R878 HSCs (Fig. 3G).”

Dnmt3a HET, Dnmt3a KO, and Dnmt3a R878 hematopoietic stem cells were resistant to depletion in response to IFNγ.

“In contrast, Dnmt3a HET , Dnmt3a KO , and Dnmt3a R878 HSCs (Supplementary Fig. S1D) were resistant to depletion in response to IFNγ (Fig. 1B; Supplementary Fig. S1E).”

In secondary recipients, there was a marked reduction in blood output of control hematopoietic stem cells previously exposed to IFNγ.

“In secondary recipients, there was a marked reduction in blood output of control HSCs previously exposed to IFNγ (Supplementary Fig. S4E).”

IFNγ was detrimental to the self-renewal of control hematopoietic stem cells.

“While IFNγ was detrimental to the self-renewal of control HSCs, clonal expansion of Dnmt3a HET , Dnmt3a KO , and Dnmt3a R878 HSCs was unaffected (Fig. 1C).”

All hematopoietic stem cell genotypes showed equivalent activation of the IFNγ target gene Stat1.

“All HSC genotypes express comparable levels of cell surface IFNgr1 (Fig. 2A) and showed equivalent activation of the IFNγ target gene Stat1 (Fig. 2B).”

The apoptotic response to IFNγ after culture was absent in Dnmt3a-mutant hematopoietic stem cells.

“While IFNγ-treated control HSCs showed significantly elevated apoptosis after culture, this response was absent in Dnmt3a-mutant HSCs (Fig. 2D).”

Transfer of 1 × 10^6 hCD34+ cells from primary recipients via intratibial injection was required for robust engraftment in secondary recipients.

“We found transfer of 1 × 10 6 hCD34 + cells from primary recipients via intratibial injection was required for robust engraftment in secondary recipients.”

Loss of systemic IFNγ reduced blood chimerism of both control and Dnmt3a KO cells compared with transplantation into IFNγ-replete environments.

“Loss of systemic IFNγ reduced blood chimerism of both control and Dnmt3a KO cells compared with transplantation into IFNγ-replete environments (Fig. 3D).”

All hematopoietic stem cell genotypes express comparable levels of cell surface IFNgr1.

“All HSC genotypes express comparable levels of cell surface IFNgr1 (Fig. 2A) and showed equivalent activation of the IFNγ target gene Stat1 (Fig. 2B).”

Dnmt3a-mutant hematopoietic stem cells specifically resist IFNγ-mediated depletion.

“We found Dnmt3a-mutant HSCs specifically resist IFNγ-mediated depletion, and IFNγ signaling is required for clonal expansion of Dnmt3a-mutant HSCs in vivo.”

• Also stated in: § INTRODUCTION · hedged

  “Cumulatively, these data suggest that Dnmt3a-mutant HSCs are specifically resistant to IFNγmediated depletion.”

Loss of IFNgr1 had no effect on blood contribution from control hematopoietic stem cells.

“While loss of IFNgr1 had no effect on blood contribution from control HSCs, Dnmt3a KO and Dnmt3a R878H HSCs lacking IFNgr1 showed significantly reduced myeloid cell output (Fig. 3B).”

Self-renewal of Dnmt3a-mutant hematopoietic stem cells with prior IFNγ treatment was enhanced postsecondary transplant.

“Self-renewal of Dnmt3a-mutant HSCs with prior IFNγ treatment was enhanced postsecondary transplant (Fig. 3H; Supplementary Fig. S4F).”

IFNγ-treated control hematopoietic stem cells showed significantly elevated apoptosis after culture.

“While IFNγ-treated control HSCs showed significantly elevated apoptosis after culture, this response was absent in Dnmt3a-mutant HSCs (Fig. 2D).”

Loss of IFNgr1 also hindered lymphoid production from Dnmt3a R878H hematopoietic stem cells.

“Loss of IFNgr1 also hindered lymphoid production from Dnmt3a R878H HSCs (Fig. 3B).”

Dnmt3a KO hematopoietic stem cells were significantly reduced in IFNγ-/- recipients.

“Conversely, Dnmt3a KO HSCs were significantly reduced in IFNγ -/-recipients (Fig. 3E), suggesting that both intrinsic IFNγ signaling and extrinsic IFNγ stimulation are required for clonal expansion of Dnmt3a-mutant HSCs.”

Fitness of Dnmt3a-mutant hematopoietic stem cells was generally unaffected by chronic IFNγ.

“Conversely, fitness of Dnmt3a-mutant HSCs was generally unaffected by chronic IFNγ, and was in fact enhanced for Dnmt3a R878 HSCs (Fig. 3G).”

Control hematopoietic stem cells were reduced following IFNγ treatment in relation to PBS-treated mice.

“Control HSCs (Supplementary Fig. S1C) were reduced following IFNγ treatment in relation to PBS-treated mice (Fig. 1B).”

Dnmt3a KO and Dnmt3a R878H hematopoietic stem cells lacking IFNgr1 showed significantly reduced myeloid cell output.

“While loss of IFNgr1 had no effect on blood contribution from control HSCs, Dnmt3a KO and Dnmt3a R878H HSCs lacking IFNgr1 showed significantly reduced myeloid cell output (Fig. 3B).”

There were no differences in apoptosis between freshly isolated hematopoietic stem cells of any genotype.

“There were no differences in apoptosis (cleaved Caspase 3/7 activity) between freshly isolated HSCs of any genotype (Fig. 2C).”

Peripheral blood engraftment was not different between genotypes in secondary human transplant.

“Again, while peripheral blood engraftment was not different between genotypes (Supplementary Fig. S2D), the trend toward reduced self-renewal of IFNγ-treated AAVS1-targered HSCs was not observed in IFNγ-treated DNMT3A-targeted HSCs (Fig. 1F).”

IFNγ signaling does not appear to be a universal clonal hematopoiesis selection mechanism, as genetic deletion of IFNgr1 from Tet2 KO hematopoietic stem cells had no effect on their competitive advantage in analogous transplantation experiments.

“IFNγ signaling does not appear a universal CH selection mechanism as genetic deletion of IFNgr1 from Vav-Cre;Tet2 fl/fl (= Tet2 KO ) HSCs had no effect on their competitive advantage in analogous transplantation experiments (Supplementary Fig. S3C and S3D).”

Chronic IFNγ exposure did not significantly alter blood chimerism for each donor cell genotype relative to engraftment in TetON chimeras.

“Chronic IFNγ exposure did not significantly alter blood chimerism (Supplementary Fig. S4B) for each donor cell genotype relative to engraftment in TetON chimeras (Supplementary Fig. S4C).”

IFNγ-exposed Dnmt3a-mutant cells showed significantly increased blood engraftment in secondary transplant.

“In contrast, IFNγ-exposed Dnmt3a-mutant cells showed significantly increased blood engraftment in secondary transplant (Supplementary Fig. S4E), showing a fitness advantage over WT cells previously exposed to chronic IFNγ.”

Clonal expansion of Dnmt3a HET, Dnmt3a KO, and Dnmt3a R878 hematopoietic stem cells was unaffected by IFNγ.

“While IFNγ was detrimental to the self-renewal of control HSCs, clonal expansion of Dnmt3a HET , Dnmt3a KO , and Dnmt3a R878 HSCs was unaffected (Fig. 1C).”

The reactive oxygen species response to IFNγ was absent in Dnmt3a HET and Dnmt3a KO hematopoietic stem cells.

“While IFNγ-treatment increased ROS in control HSCs, this response was absent in Dnmt3a HET and Dnmt3a KO HSCs (Supplementary Fig. S7B).”

Proliferation of Dnmt3a KO hematopoietic stem cells was unaltered in response to IFNγ.

“However, Dnmt3a KO HSCs were more quiescent at baseline, and their proliferation was unaltered in response to IFNγ (Fig. 4E).”

Dnmt3a KO hematopoietic stem cells were unaffected by IFNγ treatment in time to first division.

“While IFNγ treatment accelerated time to first division for control HSCs, Dnmt3a KO HSCs were unaffected (Fig. 4G), again indicating Dnmt3a-mutant HSCs resist proliferation following IFNγ exposure.”

The IFNγ exposure regimen used in this study revealed an enforced quiescence phenotype in Dnmt3a-mutant HSCs by in vivo BrdU labeling, cell-cycle analysis and in vitro single-cell division assay.

“The IFNγ exposure regimen used in this study revealed an enforced quiescence phenotype in Dnmt3a-mutant HSCs by both in vivo BrdU labeling, cell-cycle analysis and in vitro single-cell division assay.”

p21 knockdown has no influence on the response of normal HSCs to IFNγ.

“That is, p21 knockdown had no influence on the response of normal HSCs to IFNγ.”

A similar p21 expression profile is observed in DNMT3A-edited cord blood CD34+ cells after transplant.

“A similar expression profile was observed in DNMT3A-edited cord blood CD34 + cells after transplant (Supplementary Fig. S7D).”

Dnmt3a-mutant HSCs show a delayed exit from G0 in response to IFNγ.

“Cell-cycle analysis showed Dnmt3amutant HSCs show a delayed exit from G 0 in response to IFNγ.”

Time to first division was not different between PBS-treated control and Dnmt3a KO hematopoietic stem cells.

“Time to first division was not different between PBS-treated control and Dnmt3a KO HSCs.”

There were no significant cell-cycle differences in freshly analyzed hematopoietic stem cells.

“While there were no significant cellcycle differences in freshly analyzed HSCs (Supplementary Fig. S5D), Dnmt3a R878 and Dnmt3a KO HSCs showed a delayed exit from G 0 after a 26-hour culture period with concomitant deceases of HSCs in G 1 and S/G 2 -M (Fig. 4H).”

Analogous results are observed from p21-shRNA-expressing IFNγ-treated Dnmt3a KO HSCs.

“Analogous results were observed from p21-shRNA-expressing IFNγ-treated Dnmt3a KO HSCs (Fig. 7F).”

There is an IFNγ-dependent increase in binding of Txnip to p53 specifically in Dnmt3a-null 32D cells.

“This showed an IFNγ-dependent increase in binding of Txnip to p53 specifically in Dnmt3anull 32D cells (Fig. 6G).”

• AssumptionRests on an assumption: The mouse myeloid progenitor 32D cell line is a valid surrogate model for primary Dnmt3a-mutant HSCs for biochemical studies.

The increased chimerism of Dnmt3a KO and Dnmt3a R878 HSCs after IFNγ is completely reversed following p21 knockdown.

“In contrast, the increased chimerism of Dnmt3a KO and Dnmt3a R878 HSCs after IFNγ (Fig. 6C) was completely reversed following p21 knockdown (Fig. 6C-E).”

In hematopoietic stem cells at baseline, p53 pathway targets were activated in Dnmt3a KO hematopoietic stem cells.

“In HSCs at baseline, gene-set enrichment analysis (GSEA) revealed suppression of cell-cycle networks (E2F targets, G 2 -M checkpoint) in Dnmt3a KO HSCs, whereas p53 pathway targets were activated (Fig. 4B).”

p53 binding is significantly increased at the p21 promoter in WT 32D cells following Txnip knockdown, opposite to Renilla-transduced cells.

“We did note that p53 binding was significantly increased at the p21 promoter in WT 32D cells following Txnip knockdown which was the opposite response to Renilla-transduced cells (Fig. 6H).”

• AssumptionRests on an assumption: The mouse myeloid progenitor 32D cell line is a valid surrogate model for primary Dnmt3a-mutant HSCs for biochemical studies.

The p53 target gene Cdkn1a (p21) was significantly upregulated in Dnmt3a KO hematopoietic stem cells at baseline.

“However, the p53 target gene Cdkn1a (p21) was significantly upregulated in Dnmt3a KO HSCs at baseline and further increased after IFNγ treatment (Fig. 6A).”

Dnmt3a R878 and Dnmt3a KO hematopoietic stem cells showed a delayed exit from G0 after a 26-hour culture period.

“While there were no significant cellcycle differences in freshly analyzed HSCs (Supplementary Fig. S5D), Dnmt3a R878 and Dnmt3a KO HSCs showed a delayed exit from G 0 after a 26-hour culture period with concomitant deceases of HSCs in G 1 and S/G 2 -M (Fig. 4H).”

Dnmt3a R878 and Dnmt3a KO hematopoietic stem cells showed concomitant decreases of hematopoietic stem cells in G1 and S/G2-M after a 26-hour culture period.

“While there were no significant cellcycle differences in freshly analyzed HSCs (Supplementary Fig. S5D), Dnmt3a R878 and Dnmt3a KO HSCs showed a delayed exit from G 0 after a 26-hour culture period with concomitant deceases of HSCs in G 1 and S/G 2 -M (Fig. 4H).”

The DNA methylation level in the Txnip regulatory region was not altered by IFNγ treatment.

“Dnmt3amutant cells showed a hypomethylated DMR in the Txnip regulatory region (Supplementary Fig. S6F), although the DNA methylation level was not altered by IFNγ treatment, suggesting this loss of methylation was a canonical consequence to Dnmt3a loss-of-function.”

The same Txnip regulatory region was also hypomethylated specifically in Dnmt3a KO and Dnmt3a R878 hematopoietic stem cells.

“The same region was also hypomethylated specifically in Dnmt3a KO and Dnmt3a R878 HSCs (Fig. 5F).”

There is reduced p53 binding at the p21 promoter in WT 32D cells treated with IFNγ.

“In contrast, there was reduced p53 binding at the p21 promoter in WT 32D cells treated with IFNγ.”

• AssumptionRests on an assumption: The mouse myeloid progenitor 32D cell line is a valid surrogate model for primary Dnmt3a-mutant HSCs for biochemical studies.

Dnmt3a KO IFNgr1 KO hematopoietic stem cells remained insensitive to IFNγ.

“Dnmt3a KO IFNgr1 KO HSCs remained insensitive to IFNγ, suggesting the effect is cell intrinsic.”

Dnmt3a-mutant cells showed a hypomethylated differentially methylated region in the Txnip regulatory region.

“Dnmt3amutant cells showed a hypomethylated DMR in the Txnip regulatory region (Supplementary Fig. S6F), although the DNA methylation level was not altered by IFNγ treatment, suggesting this loss of methylation was a canonical consequence to Dnmt3a loss-of-function.”

Only a fraction of Txnip appears to bind p53.

“Moreover, only a fraction of Txnip appears to bind p53”

Dnmt3a KO hematopoietic stem cells were more quiescent at baseline.

“However, Dnmt3a KO HSCs were more quiescent at baseline, and their proliferation was unaltered in response to IFNγ (Fig. 4E).”

Cell cycle-related genesets remained suppressed in IFNγ-stimulated Dnmt3a KO hematopoietic stem cells.

“Comparison of IFNγ-stimulated HSCs showed a more robust IFN response in Dnmt3a KO HSCs, but cell cycle-related genesets remained suppressed (Fig. 4C), suggesting Dnmt3a KO HSCs may have a different proliferative response to IFNγ exposure.”

There are no significant differences in apoptosis between any shRNA-transduced HSCs from PBS-treated mice after culture.

“There were no significant differences in apoptosis between any shRNA-transduced HSCs from PBS-treated mice after culture (Fig. 7D), similar to previous observations (Fig. 2D).”

Control and Dnmt3a KO hematopoietic stem cells displayed a similar transcriptional response to IFNγ.

“Control and Dnmt3a KO HSCs displayed a similar transcriptional response to IFNγ (Supplementary Fig. S5B).”

Exposure to chronic IFNγ did not dramatically alter the methylome of any genotype.

“Exposure to chronic IFNγ did not dramatically alter the methylome of any genotype (Supplementary Fig. S6B).”

In the absence of DNA methylation, the differentially methylated region functioned as an active regulatory element.

“In the absence of DNA methylation, the DMR functioned as an active regulatory element (Fig. 5G).”

Approximately 30% of Dnmt3a KO hematopoietic stem cells had not divided by 72 hours in culture after IFNγ treatment.

“The single-cell division assay also revealed another interesting observation: while virtually all IFNγ-treated control HSCs had divided by 72 hours in culture, approximately 30% of Dnmt3a KO HSCs had not divided by this timepoint, suggesting some clonal heterogeneity even within this highly purified cell population.”

Analysis of single-cell transcriptomic data identified only approximately 50 differentially expressed genes in the hematopoietic stem cell clusters between genotypes and treatments.

“Similar to the bulk HSC RNA-seq data, analysis of single-cell transcriptomic data identified only approximately 50 DEGs in the HSC clusters between genotypes and treatments.”

Dnmt3a KO cells showed global hypomethylation.

“Dnmt3a KO cells showed global hypomethylation, whereas the overall genomic DNA methylation profile of Dnmt3a HET cells was virtually indistinguishable from control cells (Supplementary Fig. S6A).”

Hematopoietic progenitor-enriched BM cells from Dnmt3a-mutant mice show upregulation of p21 associated with p53 stabilization following IFNγ.

“Hematopoietic progenitor-enriched BM cells (Lineage -c-Kit + ) from Dnmt3a-mutant mice also showed upregulation of p21 associated with p53 stabilization following IFNγ (Fig. 6B; Supplementary Fig. S7E).”

UMAP clustering showed treatment was a stronger influence on transcriptome than genotype.

“UMAP clustering showed treatment was a stronger influence on transcriptome than genotype (Fig. 5A, left).”

IFNγ-treatment increased reactive oxygen species in control hematopoietic stem cells.

“While IFNγ-treatment increased ROS in control HSCs, this response was absent in Dnmt3a HET and Dnmt3a KO HSCs (Supplementary Fig. S7B).”

The overall genomic DNA methylation profile of Dnmt3a HET cells was virtually indistinguishable from control cells.

“Dnmt3a KO cells showed global hypomethylation, whereas the overall genomic DNA methylation profile of Dnmt3a HET cells was virtually indistinguishable from control cells (Supplementary Fig. S6A).”

Txnip shRNA-transduced Dnmt3a KO or Dnmt3a R878 HSCs phenocopied p21 knockdown whereby the competitive advantage and clonal expansion of these HSCs under IFNγ challenge was mitigated by Txnip inhibition.

“However, Txnip shRNA-transduced Dnmt3a KO or Dnmt3a R878 HSCs phenocopied p21 knockdown whereby the competitive advantage and clonal expansion of these HSCs under IFNγ challenge was mitigated by Txnip inhibition (Fig. 7B).”

IFNγ treatment accelerated time to first division for control hematopoietic stem cells.

“While IFNγ treatment accelerated time to first division for control HSCs, Dnmt3a KO HSCs were unaffected (Fig. 4G), again indicating Dnmt3a-mutant HSCs resist proliferation following IFNγ exposure.”

In hematopoietic stem cells at baseline, gene-set enrichment analysis revealed suppression of cell-cycle networks (E2F targets, G2-M checkpoint) in Dnmt3a KO hematopoietic stem cells.

“In HSCs at baseline, gene-set enrichment analysis (GSEA) revealed suppression of cell-cycle networks (E2F targets, G 2 -M checkpoint) in Dnmt3a KO HSCs, whereas p53 pathway targets were activated (Fig. 4B).”

There were no IFNγ-induced differences in transcript levels of p53 itself, or major p53 targets for prosurvival (Bcl2, Mcl1) or proapoptosis (Bax, Puma) across hematopoietic stem cell genotypes.

“There were no IFNγ-induced differences in transcript levels of p53 itself, or major p53 targets for prosurvival (Bcl2, Mcl1) or proapoptosis (Bax, Puma) across HSC genotypes (Supplementary Fig. S7C).”

The upregulation of p21 in Dnmt3a KO HSCs is p53-dependent.

“Gene expression analysis showed the upregulation of p21 in Dnmt3a KO HSCs was p53-dependent (Fig. 6A).”

Txnip expression pattern was hematopoietic stem cell-predominant.

“Thioredoxin interacting protein (Txnip) was one of the few genes upregulated specifically in Dnmt3a KO HSCs (Fig. 5C), and its expression pattern was HSC-predominant (Fig. 5D).”

Targeted qPCR confirmed the upregulation of Txnip in Dnmt3a KO hematopoietic stem cells at baseline.

“Targeted qPCR confirmed the upregulation of Txnip in Dnmt3a KO HSCs at baseline, and while there was a trend for reduced Txnip in IFNγ-treated control HSCs, there was no diminution of the levels in IFNγtreated Dnmt3a KO HSCs (Fig. 5E).”

Proliferation of IFNgr1 KO hematopoietic stem cells was not different than control hematopoietic stem cells in mice treated with PBS.

“Proliferation of IFNgr1 KO HSCs was not different than control HSCs in mice treated with PBS, and remained unchanged following IFNγ treatment.”

Txnip knockdown does not influence the impaired self-renewal of control HSCs exposed to IFNγ.

“Similar to inhibition of p21, Txnip knockdown did not influence the impaired self-renewal of control HSCs exposed to IFNγ (Fig. 7A).”

p21 expression is reduced in Txnip shRNA-transduced Dnmt3a KO or Dnmt3a R878 HSCs.

“p21 expression was reduced in Txnip shRNA-transduced Dnmt3a KO or Dnmt3a R878 HSCs (Supplementary Fig. S7G)”

Dnmt3a HET hematopoietic stem cells were resistant to IFNγ-induced proliferation.

“Dnmt3a HET HSCs were not more quiescent than the control HSCs at baseline, but like Dnmt3a KO HSCs were resistant to IFNγ-induced proliferation (Fig. 4E).”

Deletion of IFNgr1 in Dnmt3a KO hematopoietic stem cells restored their proliferation rate at baseline to that of control hematopoietic stem cells.

“Deletion of IFNgr1 in Dnmt3a KO HSCs restored their proliferation rate at baseline to that of control HSCs (Fig. 4E).”

HSCs transduced with control Renilla shRNA recapitulate the native setting with Dnmt3a KO HSCs resistant to IFNγ-induced apoptosis.

“HSCs transduced with the control Renilla shRNA recapitulated the native setting with Dnmt3a KO HSCs resistant to IFNγ-induced apoptosis (Fig. 7D).”

The increased p53 binding in WT 32D cells after Txnip knockdown did not translate to increased p21 transcription.

“However, this did not translate to increased p21 transcription (Fig. 6I)”

• AssumptionRests on an assumption: The mouse myeloid progenitor 32D cell line is a valid surrogate model for primary Dnmt3a-mutant HSCs for biochemical studies.

Comparison of IFNγ-stimulated hematopoietic stem cells showed a more robust IFN response in Dnmt3a KO hematopoietic stem cells.

“Comparison of IFNγ-stimulated HSCs showed a more robust IFN response in Dnmt3a KO HSCs, but cell cycle-related genesets remained suppressed (Fig. 4C), suggesting Dnmt3a KO HSCs may have a different proliferative response to IFNγ exposure.”

Proliferation of IFNgr1 KO hematopoietic stem cells remained unchanged following IFNγ treatment.

“Proliferation of IFNgr1 KO HSCs was not different than control HSCs in mice treated with PBS, and remained unchanged following IFNγ treatment.”

In PBS-treated mice, Txnip inhibition has minimal effect on time to first division in both HSC genotypes compared with respective Renilla controls.

“In PBS-treated mice, Txnip inhibition had minimal effect on time to first division in both HSC genotypes compared with respective Renilla controls (Supplementary Fig. S7H).”

Cdkn1a (p21) was further increased after IFNγ treatment in Dnmt3a KO hematopoietic stem cells.

“However, the p53 target gene Cdkn1a (p21) was significantly upregulated in Dnmt3a KO HSCs at baseline and further increased after IFNγ treatment (Fig. 6A).”

Control hematopoietic stem cells were stimulated to proliferate in response to IFNγ.

“Control HSCs were stimulated to proliferate in response to IFNγ (Fig. 4E).”

There was no diminution of Txnip levels in IFNγ-treated Dnmt3a KO hematopoietic stem cells.

“Targeted qPCR confirmed the upregulation of Txnip in Dnmt3a KO HSCs at baseline, and while there was a trend for reduced Txnip in IFNγ-treated control HSCs, there was no diminution of the levels in IFNγtreated Dnmt3a KO HSCs (Fig. 5E).”

Txnip was one of the few genes upregulated specifically in Dnmt3a KO hematopoietic stem cells.

“Thioredoxin interacting protein (Txnip) was one of the few genes upregulated specifically in Dnmt3a KO HSCs (Fig. 5C), and its expression pattern was HSC-predominant (Fig. 5D).”

Dnmt3a-null 32D cells replicate the IFNγ-dependent increase in p21 seen in Dnmt3a-mutant BM cells.

“After CRISPR/ Cas9-mediated Dnmt3a deletion, we confirmed these cells replicated the IFNγ-dependent increase in p21 seen in Dnmt3amutant BM cells (Fig. 6F).”

• AssumptionRests on an assumption: The mouse myeloid progenitor 32D cell line is a valid surrogate model for primary Dnmt3a-mutant HSCs for biochemical studies.

There was a trend for reduced Txnip in IFNγ-treated control hematopoietic stem cells.

“Targeted qPCR confirmed the upregulation of Txnip in Dnmt3a KO HSCs at baseline, and while there was a trend for reduced Txnip in IFNγ-treated control HSCs, there was no diminution of the levels in IFNγtreated Dnmt3a KO HSCs (Fig. 5E).”

Virtually all differences in DNA methylation were driven by genotype, rather than treatment.

“Identification of differentially methylated regions (DMR) showed virtually all differences in DNA methylation were driven by genotype, rather than treatment (Supplementary Fig. S6C-S6E).”

IFNγ treatment of control HSCs transduced with Renilla shRNA reduces time to first division, unlike Dnmt3a KO HSCs.

“In terms of cell cycle, IFNγ treatment of control HSCs transduced with Renilla shRNA reduced time to first division, unlike Dnmt3a KO HSCs (Fig. 7E), analogous to the native setting (Fig. 4G).”

Analysis of IFNγ-treated cells confirmed increased IFNγ signaling in Dnmt3a KO hematopoietic stem cells.

“GSEA comparison of PBS-treated HSCs replicated many of the findings of bulk RNA-seq such as downregulation of “E2F targets” and “MYC targets” in Dnmt3a KO HSCs, whereas analysis of IFNγ-treated cells confirmed the increased IFNγ signaling in Dnmt3a KO HSCs (Supplementary Fig. S5E).”

GSEA comparison of PBS-treated hematopoietic stem cells replicated many findings of bulk RNA-seq such as downregulation of E2F targets and MYC targets in Dnmt3a KO hematopoietic stem cells.

“GSEA comparison of PBS-treated HSCs replicated many of the findings of bulk RNA-seq such as downregulation of “E2F targets” and “MYC targets” in Dnmt3a KO HSCs, whereas analysis of IFNγ-treated cells confirmed the increased IFNγ signaling in Dnmt3a KO HSCs (Supplementary Fig. S5E).”

Knockdown of p53 reduces apoptosis in both IFNγ-treated control and Dnmt3a KO HSCs.

“Knockdown of p53 reduced apoptosis in both IFNγ-treated control and Dnmt3a KO HSCs”

Artificial methylation of the reporter plasmid with M.SssI ablated this regulatory element activity.

“Artificial methylation of the reporter plasmid with the bacterial CpG methyltransferase M.SssI ablated this regulatory element activity (Fig. 5G).”

Dnmt3a HET hematopoietic stem cells were not more quiescent than control hematopoietic stem cells at baseline.

“Dnmt3a HET HSCs were not more quiescent than the control HSCs at baseline, but like Dnmt3a KO HSCs were resistant to IFNγ-induced proliferation (Fig. 4E).”

The Txnip locus has a hypomethylated DMR in Dnmt3a-mutant HSCs.

“Black box denotes hypomethylated DMR in Dnmt3a-mutant HSCs.”

Dnmt3a-mutant HSCs retain quiescence after IFNγ challenge.

“Dnmt3a-mutant HSCs retain quiescence after IFNγ challenge.”

DNMT3A-mutant HSCs are insensitive to the deleterious effects of IFNγ in vivo.

“DNMT3A-mutant HSCs are insensitive to the deleterious effects of IFNγ in vivo.”

DNMT3A-mutant HSCs are resistant to IFNg-induced apoptosis.

“DNMT3A-mutant HSCs are resistant to IFNg-induced apoptosis.”

The Txnip-p53-p21 pathway was validated by functional genetic rescue experiments in vivo.

“Complementary genomic techniques identified a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant HSCs under conditions of inflammatory stress, which was validated by functional genetic rescue experiments in vivo.”

Dnmt3a mutations render hematopoietic stem cells specifically insensitive to IFNγ-induced apoptosis.

“These results demonstrate that Dnmt3a mutations render HSCs specifically insensitive to IFNγ-induced apoptosis.”

These findings highlight a novel mechanistic basis to explain the increased fitness of Dnmt3a-mutant hematopoietic stem cells.

“These findings highlight a novel mechanistic basis to explain the increased fitness of Dnmt3a-mutant HSCs, and supports rationale for developing interventions to mitigate expansion of premalignant clones as a method of blood cancer prevention.”

Dnmt3a heterozygous variants provide very minor fitness advantages to hematopoietic stem cells that require the extended lifespan of humans to manifest measurable phenotypes.

“However, we and others have shown that Dnmt3a HET HSCs produce only modest phenotypes in mice (25,26), likely because these variants provide very minor fitness advantages to HSCs (2) that require the extended lifespan of humans to manifest measureable phenotypes.”

• cites [2] Watson, 2020, The evolutionary dynamics and fitness landscape of clonal hematopoiesis, Science sourceNot checked — no full text of the source

IFNγ-exposed Dnmt3a-mutant cells showed a fitness advantage over wild-type cells previously exposed to chronic IFNγ.

“In contrast, IFNγ-exposed Dnmt3a-mutant cells showed significantly increased blood engraftment in secondary transplant (Supplementary Fig. S4E), showing a fitness advantage over WT cells previously exposed to chronic IFNγ.”

Any differences between Dnmt3a-mutant and control hematopoietic stem cells should not result from the inability of the mutant hematopoietic stem cells to sense or respond to IFNγ.

“Thus, any differences should not result from the inability of the mutant HSCs to sense or respond to IFNγ.”

IFNγ signaling is required for the enhanced self-renewal of Dnmt3a-mutant hematopoietic stem cells.

“These data show IFNγ signaling is required for the enhanced self-renewal of Dnmt3a-mutant HSCs.”

In mice, complete loss of Dnmt3a function is required to generate robust phenotypes.

“In mice, complete loss of Dnmt3a function is required to generate robust phenotypes.”

There is a fundamental difference in the molecular response to IFNγ between control and Dnmt3a-mutant cells.

“and implies a fundamental difference in the molecular response to IFNγ between control and Dnmt3a-mutant cells.”

• AssumptionRests on an assumption: The mouse myeloid progenitor 32D cell line is a valid surrogate model for primary Dnmt3a-mutant HSCs for biochemical studies.

This study demonstrates the first in vivo evidence that human DNMT3A-mutant HSCs are sustained in response to episodic IFNγ exposure.

“Importantly, this study demonstrates the first in vivo evidence that human DNMT3A-mutant HSCs are sustained in response to episodic IFNγ exposure.”

The effect of IFNγ insensitivity in Dnmt3a KO IFNgr1 KO hematopoietic stem cells is cell intrinsic.

“Dnmt3a KO IFNgr1 KO HSCs remained insensitive to IFNγ, suggesting the effect is cell intrinsic.”

Quiescence-enforcing mechanisms may delay the proliferative activation of Dnmt3a-mutant hematopoietic stem cells following IFNγ exposure.

“This suggests quiescence-enforcing mechanisms may delay the proliferative activation of Dnmt3a-mutant HSCs following IFNγ exposure.”

Dnmt3a KO hematopoietic stem cells may have a different proliferative response to IFNγ exposure.

“Comparison of IFNγ-stimulated HSCs showed a more robust IFN response in Dnmt3a KO HSCs, but cell cycle-related genesets remained suppressed (Fig. 4C), suggesting Dnmt3a KO HSCs may have a different proliferative response to IFNγ exposure.”

The Txnip-p53-p21 pathway cannot be the only molecular mechanism that increases the fitness of Dnmt3a-mutant HSCs.

“But while the importance of the Txnip-p53-p21 mechanism was functionally validated in vivo, this pathway cannot be the only molecular mechanism that increases the fitness of Dnmt3a-mtuant HSCs.”

p21 has genotype-specific functions in the stress response of HSCs to inflammation.

“These data suggest that p21 has genotype-specific functions in the stress response of HSCs to inflammation.”

Txnip has p53-independent actions.

“suggesting Txnip has p53-independent actions.”

These data implicate a protective function for Txnip and p21 in preventing stress-induced apoptosis in Dnmt3a-mutant HSCs in response to IFNγ.

“These data again highlight genotype-specific HSC responses and implicate a protective function for Txnip and p21 in preventing stress-induced apoptosis in Dnmt3amutant HSCs in response to IFNγ.”

There is clonal heterogeneity even within the highly purified Dnmt3a KO hematopoietic stem cell population.

“The single-cell division assay also revealed another interesting observation: while virtually all IFNγ-treated control HSCs had divided by 72 hours in culture, approximately 30% of Dnmt3a KO HSCs had not divided by this timepoint, suggesting some clonal heterogeneity even within this highly purified cell population.”

The Txnip-p53-p21 mechanism has a genotype-specific effect in preserving the functional integrity of Dnmt3a-mutant HSCs in response to IFNγ.

“we demonstrate a genotype-specific effect in preserving the functional integrity of Dnmt3a-mutant HSCs in response to IFNγ.”

Txnip is an important regulator of p21 in Dnmt3a-mutant HSCs under IFNγ challenge.

“implicating Txnip as an important regulator of p21 in these mutant HSCs under IFNγ challenge.”

There is an uncoupling of the molecular response of Dnmt3a KO hematopoietic stem cells to IFNγ exposure with the normal functional effect of increased proliferation.

“This suggests an uncoupling of the molecular response of Dnmt3a KO HSCs to IFNγ exposure with the normal functional effect of increased proliferation.”

The differences of Dnmt3a-mutant hematopoietic stem cells in response to IFNγ were not associated with DNA methylome remodeling.

“This suggests the differences of Dnmt3a-mutant HSCs in response to IFNγ were not associated with DNA methylome remodeling.”

The loss of methylation in the Txnip regulatory region was a canonical consequence to Dnmt3a loss-of-function.

“Dnmt3amutant cells showed a hypomethylated DMR in the Txnip regulatory region (Supplementary Fig. S6F), although the DNA methylation level was not altered by IFNγ treatment, suggesting this loss of methylation was a canonical consequence to Dnmt3a loss-of-function.”

The relatively small survival benefit observed in this model supports the premise that the fitness advantage conveyed by DNMT3A mutations in HSCs requires cumulative selection pressure over decades to manifest human CH.

“The relatively small survival benefit observed in this model supports the premise that the fitness advantage conveyed by DNMT3A mutations in HSCs requires cumulative selection pressure over decades to manifest human CH.”

The reduction of apoptosis by p53 knockdown is likely because the genetic inhibition nonspecifically altered a myriad of p53-related cellular functions, including induction of apoptosis.

“likely because the genetic inhibition nonspecifically altered a myriad of p53-related cellular functions, including induction of apoptosis (40).”

• cites [40] Moll, 2005, Transcription-independent pro-apoptotic functions of p53, Curr Opin Cell Biol sourceNot checked — no full text of the source

Dnmt3a-mutant hematopoietic stem cells resist proliferation following IFNγ exposure.

“While IFNγ treatment accelerated time to first division for control HSCs, Dnmt3a KO HSCs were unaffected (Fig. 4G), again indicating Dnmt3a-mutant HSCs resist proliferation following IFNγ exposure.”

The subsequent increased p21 expression hinders proliferation of Dnmt3a-mutant cells to preserve functional potential.

“The subsequent increased p21 expression (Fig. 6I) hinders proliferation of Dnmt3a-mutant cells to preserve functional potential.”

• AssumptionRests on an assumption: p53 regulates p21 expression by transcriptional activation.
• AssumptionRests on an assumption: The mouse myeloid progenitor 32D cell line is a valid surrogate model for primary Dnmt3a-mutant HSCs for biochemical studies.

This study identified a Txnip-p53-p21 mechanism that protects Dnmt3a-mutant HSCs from the deleterious effects of IFNγ.

“This study identified a Txnip-p53-p21 mechanism that protects Dnmt3a-mutant HSCs from the deleterious effects of IFNγ.”

Txnip preserves functional integrity of Dnmt3a-mutant HSCs via p21.

“Txnip preserves functional integrity of Dnmt3a-mutant HSCs via p21.”

Txnip upregulates p21 in Dnmt3a-mutant HSCs in response to IFNγ.

“Txnip upregulates p21 in Dnmt3a-mutant HSCs in response to IFNγ.”

This is one of the first studies to link changes in an environmental selection pressure with a fitness advantage for HSCs bearing a specific mutation.

“To our knowledge, this is one of the first studies to link changes in an environmental selection pressure with a fitness advantage for HSCs bearing a specific mutation.”