Mechanisms of Action of Hematopoietic Transcription Factor PU.1 in Initiation of T-Cell Development

In the paper

“A very intriguing new prospect for fast antagonism of PU.1 activity is the discovery of small-molecule inhibitors, some of which are highly potent and specific at blocking PU.1 action in leukemia cells; however, these have not yet been tested for effects on normal T-cell development (89).”

What the sources say

• Disputed

  The Introduction supports the discovery of small-molecule PU.1 inhibitors that block PU.1 activity in AML cells. But the section “Inhibitors show on-target PU.1-inhibitory activity in AML cells” explicitly reports testing their effects on T-cell differentiation in OP9-Delta1 culture, contrary to the claim that they had not yet been tested for effects on normal T-cell development. (LLM, conf=0.92)

  Checked by a reasoning model — may be wrong.

  cites [89] Antony-Debré, 2017, Pharmacological inhibition of the transcription factor PU.1 in leukemia sourceExperimental — accuracy not yet measured

In the paper

“The best current candidates for this silencing activity include GATA-3 (104, 105), TCF-1 or LEF-1 (37), and especially Runx1 (80, 150–152), probably working in a dose-dependent combination, although the mechanism through which they finally achieve the ability to repress PU.1 has not yet been reported.”

What the sources say

• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [104] Scripture-Adams, 2014, GATA-3 dose-dependent checkpoints in early T cell commitment source
• ?Not checked — no full text of the sourcecites [105] Taghon, 2007, Mast cell lineage diversion of T lineage precursors by the essential T cell transcription factor GATA-3 source
• ?Not checked — no full text of the sourcecites [37] Rosenbauer, 2006, Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU source
• ?Could not verify

  The Results section on PU.1 association in primary DN cells says Runx1 becomes essential around the time PU.1 is downregulated, but does not say Runx1 silences PU.1. The Results and Discussion passages instead examine how PU.1 redirects Runx1 binding. Searches for Runx1-mediated PU.1 silencing and Spi1 downregulation found no passage supporting the proposed dose-dependent combination or the claim about its unknown mechanism. (LLM, conf=0.73)

  cites [80] Hosokawa, 2018, Transcription factor PU.1 represses and activates gene expression in early T cells by redirecting partner transcription factor binding sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [150] Huang, 2008, PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis source
• Disputed

  Split into 2 atoms — 0 confirmed, 0 partially supported, 1 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  Checked by a reasoning model — may be wrong.

  cites [151] Hoogenkamp, 2009, Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program sourceExperimental — accuracy not yet measured
• Disputed

  Split into 2 atoms — 0 confirmed, 1 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Checked by a reasoning model — may be wrong.

  cites [152] Zarnegar, 2010, Cell type-specific activation and repression of PU.1 by a complex of discrete, functionally specialized cis-regulatory elements sourceExperimental — accuracy not yet measured

In the paper

“or as a collaboration-dependent partner in binding complexes, either with activation-dependent factors like NF-κB or with lineage-defining partners like C/EBPα (or β) or IRF4/8 (13–15)”

What the sources say

• Partly supported

  The section “PU.1 binds in the vicinity of other lineage-determining transcription factors” (page unavailable) reports nearby PU.1 and C/EBPα/β binding sites and describes a PU.1–IRF4/8 ternary complex. “Combinatorial interplay delineates stage-specific cistromes” (page unavailable) further reports reciprocal effects of PU.1 and C/EBPβ on binding. For NF-κB, however, the paper reports nearby motifs and its activation in TLR4 signaling, not a collaboration-dependent PU.1–NF-κB binding complex. (LLM, conf=0.87)

  cites [13] Heinz, 2010, Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities sourceExperimental — accuracy not yet measured
• Partly supported

  In “Variation and collaborative LDTF binding” (page not provided), the paper reports that C/EBPα motif mutations can reduce nearby PU.1 binding, supporting collaboration between those factors. In “LDTF motif mutations affect NF-κB binding” (page not provided), it instead describes NF-κB binding at sites already bound by PU.1 and/or C/EBPα; it does not establish that PU.1 binding depends on NF-κB. The supplied passages and searches do not support the specified C/EBPβ or IRF4/8 partnerships. (LLM, conf=0.86)

  cites [14] Heinz, 2013, Effect of natural genetic variation on enhancer selection and function sourceExperimental — accuracy not yet measured
• Partly supported

  The Mechanistic Aspects section (p. 163) describes PU.1 recruitment as potentially dependent on cooperation with a stimulus-activated partner or constitutively expressed transcription factors. The Introduction also links PU.1-controlled enhancers with recruitment of stimulus-induced NF-κB. Our copy does not identify C/EBPα/β or IRF4/8 as PU.1 binding partners, and the proposed cooperative mechanisms are not established for those named factors. (LLM, conf=0.86)

  cites [15] Ostuni, 2013, Latent enhancers activated by stimulation in differentiated cells sourceExperimental — accuracy not yet measured

In the paper

“PU.1 can work in an all-or-none gene network switch through mutual antagonism with GATA-1 (20–24)”

What the sources say

• Partly supported

  The section “Mechanism of PU.1-mediated block to differentiation in erythroleukemia” (page unavailable) says PU.1 and GATA-1 can antagonize each other’s actions and suggests their relative levels could regulate lineage commitment. The paper does not establish an all-or-none gene-network switch; its lineage-commitment proposal is conditional. (LLM, conf=0.85)

  cites [20] Rekhtman, 1999, Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells sourceExperimental — accuracy not yet measured
• Partly supported

  The Results (page unavailable) show reciprocal inhibition: GATA-1 represses PU.1 transactivation, and PU.1 inhibits GATA-1 activation in reporter assays. The Discussion links this negative regulation to hematopoietic differentiation, but the paper does not establish an all-or-none gene-network switch. (LLM, conf=0.84)

  cites [21] Zhang, 1999, Negative cross-talk between hematopoietic regulators: GATA proteins repress PU sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [22] GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription254351 NerlovC QuerfurthE KulessaH GrafT 10753833Blood952000
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [23] PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding264148 ZhangP ZhangX IwamaA YuC SmithKA MuellerBU 11023493Blood962000
• ?Not checked — no full text of the sourcecites [24] Stopka, 2005, PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure source

In the paper

“This is part of a general downregulation of stem/progenitor associated regulatory genes (“phase 1 genes”) (34, 43) and a major reorganization of active chromatin and chromatin interactions, genome-wide, that occurs during this transition (44).”

What the sources say

• Supported

  Section III.A (page unavailable) describes silencing of stem-progenitor regulatory genes, including a round that occurs during commitment. In the citing sentence, the chromatin-reorganization claim is attached to citation 44, not to the citations for phase 1 gene downregulation, so it is not assessed here. (LLM, conf=0.90)

  cites [34] Rothenberg, 2016, Forging T-Lymphocyte identity: intersecting networks of transcriptional control source
• ?Could not verify

  Citation (44) is attached to the claim of genome-wide reorganization of active chromatin and chromatin interactions. Our copy discusses downregulation of stem/progenitor-associated genes in “Unleashing the expression of T-cell identity by negative regulation?” and local chromatin opening at TCR loci in “Emergence of T-cell identity,” but neither establishes the cited genome-wide reorganization. Searches for chromatin reorganization and chromatin interactions found no passage addressing it; page numbers were unavailable. (LLM, conf=0.77)

  cites [43] Rothenberg, 2010, Multilayered specification of the T-cell lineage fate sourceExperimental — accuracy not yet measured
• Supported

  The Discussion describes genome-wide reorganization of the regulome and 3D nucleome at the DN2-to-DN3 transition. The Results also report abrupt global changes in chromatin accessibility and TAD connectivity. This source is cited for the chromatin-reorganization clause; the phase 1 gene clause has separate citations. (LLM, conf=0.94)

  cites [44] Hu, 2018, Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells source

In the paper

“typically (A/G)AGGAAGTG motifs [e.g., (10, 11)]”

What the sources say

• ?Not checked — no full text of the sourcecites [10] Poon, 2004, A thermodynamic basis of DNA sequence selectivity by the ETS domain of murine PU source
• Supported

  The Discussion (page unavailable) identifies AAAGAGGAAGTG as a PU.1 consensus sequence and reports that 80% of its genomic occurrences overlap PU.1 ChIP-seq peaks. That sequence contains GAGGAAGTG, one of the stated (A/G)AGGAAGTG motifs. The paper also notes that PU.1 recognizes many other sequences, so the motif is not exclusive. (LLM, conf=0.86)

  cites [11] Pham, 2013, Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1 source

In the paper

“re-expression of PU.1 after T-cell commitment turns on myeloid genes and readily transforms later pro-T cells into dendritic cells, macrophages, or promyelocytic-like cells (46–50).”

What the sources say

• ?Not checked — no full text of the sourcecites [46] Anderson, 2002, Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage source
• Partly supported

  The Results report that PU.1 induced myeloid-associated genes in a pro-T cell line, and the section on diverted gene expression reports that committed DN3 thymocytes acquired Mac1 expression and non-lymphoid morphology after PU.1 was restored. The paper describes this as diversion to a myeloid-like phenotype, not demonstrated transformation of later pro-T cells into dendritic cells, macrophages, or promyelocytic-like cells. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  cites [47] Dionne, 2005, Subversion of T lineage commitment by PU.1 in a clonal cell line system sourceExperimental — accuracy not yet measured
• Partly supported

  The organ-culture results (page not available) report that PU.1 expression induced a dendritic-cell marker and diverted thymocytes toward that lineage, but identify the affected DN1–DN2 cells as not yet committed to the T-cell lineage. The Discussion attributes diversion of committed DN3 cells to Spi-B, not PU.1. The paper does not establish that PU.1 re-expression in later committed pro-T cells activates myeloid genes or produces macrophages or promyelocytic-like cells. (LLM, conf=0.87)

  cites [48] Lefebvre, 2005, Enforced expression of Spi-B reverses T lineage commitment and blocks β-selection sourceExperimental — accuracy not yet measured
• Partly supported

  In the Results (p. 4), PU.1-transduced pro-T and pre-T cells activate a broad spectrum of myeloid genes and can acquire myeloid markers. The paper does not establish that later committed cells readily become each of the three named cell types: it describes myeloid-like cells, cites earlier work for a dendritic-cell fate (p. 1), and reports that Notch/Delta signaling blocks diversion in the specified cells (p. 4). (LLM, conf=0.88)

  cites [49] Franco, 2006, Notch/Delta signaling constrains reengineering of pro-T cells by PU.1 sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page not available) says enforced PU.1 expression in committed pre-T cells induced cells resembling myeloid dendritic cells, and that PU.1 induces dendritic genes and a subset of myeloid genes. It distinguishes this from macrophage formation, which it attributes to C/EBPα and C/EBPβ; the cited passages do not report PU.1 producing promyelocytic-like cells. (LLM, conf=0.90)

  cites [50] Laiosa, 2006, Reprogramming of committed T cell progenitors to macrophages and dendritic cells by C/EBPα and PU.1 transcription factors sourceExperimental — accuracy not yet measured

In the paper

“if Spi1 is conditionally deleted in T-lineage cells only after the cells have passed the DN2 stage, there are very modest effects on T cells as a whole, apparently limited to selective reduction of IL-9-producing T-cells (74), and some loss of restraint on γδ T cells and T follicular helper cell activity (75, 76).”

What the sources say

• Partly supported

  The Results (‘PU.1 promotes the development of IL-9–secreting T cells’; page unavailable) report reduced IL-9 in PU.1-deficient T-cell cultures, supporting the finding attached to citation 74. But the Results (‘TH9 cells and allergic inflammation’) also report reduced production of the chemokines Ccl17 and Ccl22 from those T cells, so the effects are not shown to be limited to IL-9-producing cells. The γδ T-cell and Tfh findings are attached to other citations, not this one. (LLM, conf=0.84)

  cites [74] Chang, 2010, The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation sourceExperimental — accuracy not yet measured
• Partly supported

  The Results report that Lck-Cre deletion of Sfpi1 leaves αβ T-cell profiles largely unchanged but increases γδ T-cell numbers, supporting the portion attributed to this paper (Results, ‘γδ T cell numbers are expanded in the absence of PU.1’; page unavailable). The Discussion places deletion after Sfpi1 expression declines between DN1 and DN2, but does not establish that deletion occurs only after cells have passed DN2. The IL-9 and Tfh portions carry other citations in the citing sentence. (LLM, conf=0.82)

  cites [75] Jabeen, 2011, The transcription factor PU.1 regulates γδ T cell homeostasis sourceExperimental — accuracy not yet measured
• Supported

  The Introduction states that Sfpi1 deletion with Lck-Cre occurs at DN3–DN4, after DN2, and that thymic and peripheral T-cell populations are normal. The Results report increased Tfh-cell numbers and activity after deletion, supporting the loss of restraint on Tfh activity attributed to this paper. The IL-9 and γδ T-cell points have separate citations in the citing sentence. (LLM, conf=0.91)

  cites [76] Awe, 2015, PU.1 Expression in T follicular helper cells limits CD40L-dependent germinal center B Cell development source

In the paper

“PU.1 is indeed important within the T-cell program as well as before thymic entry, as shown by using in vitro differentiation to provide conditions where PU.1 could be removed acutely in a synchronized cohort of precursors and the fates of the cells could be monitored immediately afterwards.”

What the sources say

• Supported

  The Discussion states that endogenous PU.1 supports proliferation and regulates developmental timing within the early T-cell pathway, while the Abstract notes its importance in prethymic precursors. The Results describe deleting PU.1 at specific precursor stages in an in vitro T-cell differentiation culture and tracking subsequent development. (LLM, conf=0.93)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU source
• Partly supported

  The Results section on endogenous PU.1 in pro-T cells reports that acute disruption during in vitro differentiation altered proliferation, developmental progression, and gene expression, supporting its importance within the T-cell program. The paper does not establish PU.1’s importance before thymic entry; its fetal-liver precursors were used to initiate cultures, but the reported disruption occurred when cells were at the DN1 stage. The cited Results passage also assesses cells four days after disruption, rather than immediately afterward. (LLM, conf=0.84)

  cites [80] Hosokawa, 2018, Transcription factor PU.1 represses and activates gene expression in early T cells by redirecting partner transcription factor binding sourceExperimental — accuracy not yet measured

In the paper

“Whereas ETP and DN2 cells can generate myeloid cells if removed from the thymus, pro-T cells that have differentiated past the stage of PU.1 expression in the thymus (i.e., from the DN2 stage to the DN3 stage or later) do not make myeloid cells under these conditions”

What the sources say

• Partly supported

  The Introduction says DN2 cells retain possible myeloid potential while DN3 cells are committed to the T lineage. The Results section on DC and NK potential reports that ETP and DN2a cells produce alternative lineages without Notch signaling, whereas DN2b and DN3a cells produce few, if any, DCs or NK cells. It does not directly test myeloid production at those stages, and the gene-expression Results place the first decline in PU.1 at DN2b, before DN3. (LLM, conf=0.84)

  cites [33] Yui, 2010, Fine-scale staging of T cell lineage commitment in adult mouse thymus sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [40] Bell, 2008, The earliest thymic progenitors for T cells possess myeloid lineage potential source
• ?Not checked — no full text of the sourcecites [41] Wada, 2008, Adult T-cell progenitors retain myeloid potential source
• Partly supported

  In ‘Development of Lymphoid and Myeloid Cells from Intrathymic Precursors’ (pp. 905–906), the authors report that after intravenous transfer, the early low-CD4 precursors produced no discrete myeloid population and the CD44+CD25+ pro-T precursors produced myeloid cells only at background levels. They also report that the later CD44−CD25+ pre-T population could not form myeloid cells. Thus the later-stage finding is supported, but the claimed myeloid capacity of the earlier cells under these conditions is contrary to the reported results; the copy does not establish a PU.1-defined boundary. (LLM, conf=0.88)

  cites [51] Wu, 1996, Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section ('Both Pro-T1 and T2 cells can generate macrophages'; page not supplied) reports that pro-T1 and pro-T2 cells generated macrophages in culture, whereas pro-T3 and pro-T4 cells did not under those conditions. The paper does not establish the claim's PU.1-expression boundary or test stages later than pro-T4. (LLM, conf=0.86)

  cites [52] Lee, 2001, Generation of macrophages from early T progenitors in vitro sourceExperimental — accuracy not yet measured
• Partly supported

  The Results (page not specified) show that isolated proT1 and proT2 cells generated granulocyte/macrophage cells, whereas TN3 and TN4 cells did not, in the tested cultures. But proT2 myeloid development required ectopic IL-2 receptor expression and added human IL-2; removal from the thymus alone was not sufficient. The cited passages also do not establish PU.1 expression as the reason for the DN2-to-DN3 boundary. (LLM, conf=0.88)

  cites [53] King, 2002, Lineage infidelity in myeloid cells with TCR gene rearrangement: a latent developmental potential of proT cells revealed by ectopic cytokine receptor signaling sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [54] Balciunaite, 2005, The earliest subpopulation of mouse thymocytes contains potent T, significant macrophage, and natural killer cell but no B-lymphocyte potential source
• ?Not checked — no full text of the sourcecites [55] Ikawa, 2010, An essential developmental checkpoint for production of the T cell lineage source
• Partly supported

  The Results section on ETPs and bone-marrow progenitors reports that DN2 cells retain T-cell and granulocyte–monocyte potential, while DN3 cells are the first T-cell-restricted progenitors (page unavailable). The Results on neonatal ETPs demonstrate myeloid potential in cultures outside the thymus. Our copy does not establish that the DN2-to-DN3 change coincides with the end of PU.1 expression or directly test DN2 and DN3 cells under the same removal conditions. (LLM, conf=0.87)

  cites [56] Luc, 2012, The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential sourceExperimental — accuracy not yet measured

In the paper

“the effects on target gene regulation appear very weak at timepoints up to 2 days after PU.1 deletion, despite the fact that the reduced cell yields from the knockout cells show that PU.1 is biologically important (79).”

What the sources say

• Supported

  In Results (‘PU.1 temporally restricts activation of the T-lineage developmental program’), cells were analyzed two days after Cre transduction, and the paper describes the gene-expression effects of acute deletion as weak. Results (‘PU.1 is required for DN progression and survival of lymphoid precursors in T-cell differentiation culture’) reports that PU.1-deleted cells subsequently generated considerably fewer progeny; it does not say cell yield fell immediately after deletion. (LLM, conf=0.90)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU source

In the paper

“whereas PU.1 itself can upregulate progenitor-associated genes Bcl11a, Lmo2, Mef2c, and Hhex above their normal levels in DN2a and DN2b primary cells, PU.1-ENG can downregulate them (79).”

What the sources say

• Partly supported

  In Results, under “Selective roles for PU.1 in the progenitor-associated regulatory gene network” (page unavailable), the paper reports that added wild-type PU.1 up-regulated Bcl11a, Lmo2, and Mef2c, consistent with effects of PU.1-Eng. It does not name Hhex among those genes, so the claim about all four is not fully supported by our copy. (LLM, conf=0.86)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU sourceExperimental — accuracy not yet measured

In the paper

“However, deletion and clearance of PU.1 protein in this system still require analysis >2 days after introduction of the guide RNAs (80, 85), and the continuing developmental progression of both knockout samples and controls needs to be taken into account in interpreting the results.”

What the sources say

• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [80] Hosokawa, 2018, Transcription factor PU.1 represses and activates gene expression in early T cells by redirecting partner transcription factor binding sourceExperimental — accuracy not yet measured
• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) sourceExperimental — accuracy not yet measured

In the paper

“The gene expression profiles of cells losing CD25 and upregulating CD11b are radically transformed from the state of newly-committed pro-T cells within 2 days after transduction, with widespread repression of T-lineage-affiliated transcription factor genes and Notch target genes as well as upregulation of multiple Cebp and Irf family transcription factor genes (Figure 4) (85).”

What the sources say

• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) sourceExperimental — accuracy not yet measured

In the paper

“This construct has been useful to reveal quick impacts on expression of positively regulated PU.1 target genes, many of which have been confirmed later by other approaches (85).”

What the sources say

• Partly supported

  The Results section on PU.1’s non-DNA-binding domains (page unavailable) reports that PU1ENGHA represses several genes positively regulated by full-length PU.1. The Discussion (page unavailable) says that gain- and loss-of-function approaches defined a core group of positive PU.1 targets. Our copy does not establish that the PU.1-ENG construct revealed quick expression impacts or that many targets identified specifically through it were later confirmed by other approaches. (LLM, conf=0.78)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) sourceExperimental — accuracy not yet measured

In the paper

“The sites occupied by exogenous PU.1 in Scid.adh.2C2 cells overlap highly with the sites occupied by endogenous PU.1 in normal pro-T cells, but the match is by no means complete (85).”

What the sources say

• Supported

  In Results, under “PU.1 requires its non-DNA-binding domains to occupy sites in closed chromatin” (page unavailable), the paper says full-length exogenous PU.1 in Scid.adh.2C2 cells bound normal pro-T-cell PU.1 sites and additional sites. This supports substantial, but incomplete, overlap with endogenous binding. (LLM, conf=0.89)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) source

In the paper

“The target genes stimulated by PU.1 include both pro-proliferative and G1-prolonging cell cycle effectors, whereas some important proliferative genes are repressed when PU.1 levels are high (82, 83).”

What the sources say

• Partly supported

  In the passage on regulatory gene expression (indexed as Abstract; page unavailable), PU.1 transduction reduced Cyclin D2, Cdc25a, Myb, and Myc, supporting the repression of proliferative genes. The paper says PU.1 promotes growth in some progenitors and notes that p21 and Gfi1 are up-regulated by PU.1 in stem cells, but it does not establish here that PU.1 stimulates both pro-proliferative and G1-prolonging target genes. (LLM, conf=0.87)

  cites [82] Kueh, 2013, Positive feedback between PU.1 and the cell cycle controls myeloid differentiation sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section, “PU.1 Transcriptionally Induces Cell-Cycle Inhibitors and Represses Cell-Cycle Activators” (page unavailable), supports PU.1 stimulation of cell-cycle inhibitors and repression of proliferative genes. It does not support the additional claim that PU.1 stimulates pro-proliferative cell-cycle effectors; the activators examined here were repressed. (LLM, conf=0.88)

  cites [83] Staber, 2013, Sustained PU.1 levels balance cell-cycle regulators to prevent exhaustion of adult hematopoietic stem cells sourceExperimental — accuracy not yet measured

In the paper

“Even with PU.1 transduction, Scid.adh.2C2 cells do not restore the full chromatin accessibility landscape of ETP and DN2a stage pro-T cells, and despite detectable upregulation of a few other early pro-T cell genes (e.g., Bcl11a and Lyl1), the transduced cells as a whole reactivate little of the program that forms the normal context for endogenous PU.1 activity in pro-T cells (58, 79, 85).”

What the sources say

• Partly supported

  The section on the Scid.adh.2C2 cell line describes it as DN3-like and reports that PU.1 transduction upregulated Mac1 in only a fraction of cells. The paper also reports PU.1-dependent repression of T-cell genes, which is consistent with an incomplete early pro-T-cell response. But our copy does not compare these cells’ chromatin accessibility with ETP or DN2a cells, or establish how much of the normal early pro-T-cell program they reactivate. (LLM, conf=0.74)

  cites [58] Del Real, 2013, Architecture of a lymphomyeloid developmental switch controlled by PU.1, Notch and Gata3 sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Results section on PU.1-Eng specificity reports that wild-type PU.1 transduction of Adh.2C2 cells causes myeloid diversion and represses many T-cell genes. It does not assess whether those cells restore ETP/DN2a chromatin accessibility or how much of the early pro-T-cell program they reactivate. Searches for chromatin accessibility and early-program reactivation in Adh.2C2 cells did not find a passage establishing either point. (LLM, conf=0.73)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section on pioneering in DN3-like cells (page unavailable) shows that PU.1 transduction opens previously closed sites and activates some linked genes. The paper identifies Scid.adh.2C2 cells as DN3-like, but the passages do not establish that transduction fails to restore the full ETP/DN2a accessibility landscape or that the cells reactivate little of the normal early pro-T-cell program. (LLM, conf=0.76)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) sourceExperimental — accuracy not yet measured

In the paper

“although the RNA transcript levels are modest in absolute terms, the impact of PU.1 on the cells can be magnified by the high stability of PU.1 protein (82).”

What the sources say

• Partly supported

  The passage discussing PU.1 removal (Fig. 1E; page unavailable) says its protein half-life is substantially longer than the progenitor cell cycle. The passages discussing cell-cycle lengthening (Figs. 1F–H and 3C–D; page unavailable) show that PU.1 can accumulate to higher levels without increased synthesis, supporting the proposed magnifying effect. Our copy does not establish that PU.1 RNA transcript levels are modest in absolute terms. (LLM, conf=0.84)

  cites [82] Kueh, 2013, Positive feedback between PU.1 and the cell cycle controls myeloid differentiation sourceExperimental — accuracy not yet measured

In the paper

“They respond in an all-or-none way to forced expression of PU.1, coordinately upregulating myeloid- or dendritic-cell associated genes and downregulating T-cell genes in a discrete fraction of the cells that increases with increasing levels of PU.1 (47, 58)”

What the sources say

• Supported

  The Discussion (page unavailable) describes the response of cloned pre-T cells to PU.1 as an all-or-none choice. The Results and Fig. 3 report dose-dependent Mac1 upregulation and CD25 downregulation in a subset of adh.2C2 cells; the gene-expression analysis shows that diverted cells repress pro-T-cell genes while inducing Mac1 and other genes associated with diversion. The paper also notes that PU.1 level is not the only determinant of which cells divert. (LLM, conf=0.92)

  cites [47] Dionne, 2005, Subversion of T lineage commitment by PU.1 in a clonal cell line system source
• Partly supported

  The section on Scid.adh.2C2 cells reports an all-or-none response to forced PU.1, with Mac1 upregulated in a fraction of cells; the gene-expression sections link diversion to repression of T-cell genes and induction of myeloid markers (page unavailable in our copy). However, the passage relating PU.1 protein levels to the fraction of Mac1-positive cells concerns fetal thymocytes, not Scid.adh.2C2 cells. This source therefore does not establish that the responding fraction of Scid.adh.2C2 cells increases with PU.1 level. (LLM, conf=0.85)

  cites [58] Del Real, 2013, Architecture of a lymphomyeloid developmental switch controlled by PU.1, Notch and Gata3 sourceExperimental — accuracy not yet measured

In the paper

“The obligate repressor should affect PU.1 positive regulation targets in the opposite direction from wildtype PU.1, and in theory should affect PU.1 negative regulation targets in the same direction, an “algebraic sign” distinction that could be used in principle to dissect indirect effects as well (79).”

What the sources say

• Partly supported

  The Results section describes PU.1-Eng as an obligate repressor that antagonizes PU.1-dependent activation, supporting the predicted opposite effect on positively regulated targets. In “Negative regulation of T-cell development by PU.1 is indirect” (page unavailable), it predicts the same effect as wild-type PU.1 only for directly repressed genes; genes repressed indirectly through an intermediate repressor would instead be up-regulated by PU.1-Eng. (LLM, conf=0.86)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU sourceExperimental — accuracy not yet measured

In the paper

“However, PU.1-ENG also has some spurious effects and cannot access closed chromatin sites as well as full-length PU.1 (79, 85).”

What the sources say

• Partly supported

  The Discussion (page unavailable) acknowledges caveats of forced PU.1-Eng expression and reports that some genes bound by endogenous PU.1 were unaffected by PU.1-Eng. The Results section on specificity (page unavailable) tests for side effects but finds the construct selective. Neither passage establishes that PU.1-Eng has spurious effects or that its weaker response at some sites is due to an inability to access closed chromatin relative to full-length PU.1. (LLM, conf=0.82)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section on PU.1’s non-DNA-binding domains (page unavailable) reports that PU1ENGHA bound comparably to full-length PU.1 at open sites but much less at closed sites. The paper describes PU1ENGHA as primarily repressing targets rather than activating them, but the passages do not establish that it has spurious effects. (LLM, conf=0.86)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) sourceExperimental — accuracy not yet measured

In the paper

“These cells were a subclone derived from the Scid.adh cell line (87) and are similar to developmentally arrested versions of committed DN3 pro-T cells, lacking any expression of endogenous PU.1 (47).”

What the sources say

• Partly supported

  The Introduction identifies adh.2C2 as a SCID.adh subclone with a DN3-like phenotype, and the Results describe the SCID.adh cells as arrested at a developmental stage. The Introduction says the subclone has ‘little if any’ endogenous PU.1 expression; the DN3-comparison section says it does not express PU.1 significantly. Those passages support very low expression, but not the categorical claim that it lacks any expression. (LLM, conf=0.82)

  cites [47] Dionne, 2005, Subversion of T lineage commitment by PU.1 in a clonal cell line system sourceExperimental — accuracy not yet measured

In the paper

“One problem is that the long half-life of PU.1 protein (82) can mask some loss effects at time points <2 days after deletion, while development of the pro-T cells can proceed to new stages if time windows are extended further.”

What the sources say

• Supported

  The citation is attached to the statement that PU.1 protein has a long half-life. The passage on PU.1 synthesis and removal states that its protein half-life is substantially longer than the progenitor cell-cycle length. The proposed masking of deletion effects and pro-T-cell development are the citing authors’ subsequent reasoning, not claims directly attributed by this citation. (LLM, conf=0.92)

  cites [82] Kueh, 2013, Positive feedback between PU.1 and the cell cycle controls myeloid differentiation source

In the paper

“Second, in pro-T cells forced to express PU.1, those that make the lineage jump (i.e. lose CD25, gain CD11b) not only silence Notch1 but also activate myeloid regulatory genes (85).”

What the sources say

• Partly supported

  The Results section on PU.1 pioneering in DN3-like cells (page unavailable) reports that induced PU.1 lowers CD25 and later raises CD11b. The Discussion reports positive regulation of genes by PU.1 in pro-T cells, but our copy does not establish that the CD25-losing, CD11b-gaining cells silence Notch1 or activate myeloid regulatory genes specifically. (LLM, conf=0.84)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) sourceExperimental — accuracy not yet measured

In the paper

“Although PU.1 occupancy of genomic sites declines as development proceeds, PU.1 occupancy is still detectable through T-lineage commitment at ~5,000 sites before disappearing (84).”

What the sources say

• Partly supported

  The Results section, “Early T-cell specific sites for PU.1: a positive role” (page unavailable), reports reduced PU.1 binding intensity in FLDN2b cells at T-lineage commitment and absence of PU.1 in later DP cells. It does not report approximately 5,000 occupied sites at commitment; the site count it gives is approximately 34,000 in DN T cells. (LLM, conf=0.88)

  cites [84] Zhang, 2012, Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity sourceExperimental — accuracy not yet measured

In the paper

“However, approximately half of the PU.1 occupancy sites in early pro-T cells appear to be relatively “inaccessible” in chromatin by the criterion of ATAC-seq at the stages when PU.1 is seen to be binding there, showing that PU.1 binding can occur without opening the chromatin.”

What the sources say

• Supported

  The Results section, “Dynamic PU.1 occupancy at open and closed chromatin sites in precommitment pro-T cells,” states that PU.1 occupied similar numbers of ATAC-closed and ATAC-open sites in DN1 and DN2a cells. It concludes that PU.1 binding does not always cause chromatin opening as scored by ATAC-seq. (LLM, conf=0.94)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) source

In the paper

“Full-length PU.1 binds closed sites nearly as well as open sites, whereas the isolated PU.1 DNA binding domain binds open sites as well as full-length but selectively fails to bind at closed sites (85) (Figure 6).”

What the sources say

• Partly supported

  In Results (‘PU.1 requires its non-DNA-binding domains to occupy sites in closed chromatin,’ Fig. 5E–F), the truncated constructs match full-length PU.1 at open sites but bind substantially less at closed sites. The claim that full-length PU.1 binds closed sites ‘nearly as well’ as open sites overstates the finding: the Results describe an affinity cost for entering closed chromatin. (LLM, conf=0.86)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) sourceExperimental — accuracy not yet measured

In the paper

“while the obligate repressor downregulated genes that are positive regulatory targets of wildtype PU.1, it actually upregulated many genes that wildtype PU.1 represses, completely inconsistent with a direct repression mechanism (79).”

What the sources say

• Supported

  The Results sections on PU.1-Eng target genes report that genes downregulated by the obligate repressor are enriched for direct, functional targets of endogenous PU.1. The Results section on T-lineage development says PU.1-Eng instead upregulates T-cell genes that endogenous PU.1 normally represses, supporting an indirect rather than direct repression mechanism. Page numbers are unavailable in our copy. (LLM, conf=0.91)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU source

In the paper

“This is consistent with PU.1's activity as a site-specific chromatin opening factor in B cells (96, 97), with the ability of PU.1 to eject nucleosomes from sites where it binds in macrophage lines (12), and with its ability to cause rapid increases in ATAC accessibility at the sites it occupies when introduced into Scid.adh.2C2 cells (85).”

What the sources say

• Supported

  This source is cited for the B-cell chromatin-opening clause, not the separate macrophage or ATAC-accessibility claims. In the Discussion (page not provided), the authors report that PU.1 increases accessibility at the IgH μ locus and propose that it opens the locus during B-cell development. (LLM, conf=0.87)

  cites [96] Nikolajczyk, 1999, ETS protein-dependent accessibility changes at the immunoglobulin æ heavy chain enhancer source
• ?Not checked — no full text of the sourcecites [97] Marecki, 2004, PU.1 as a chromatin accessibility factor for immunoglobulin genes source
• Partly supported

  The Results section ('Selective masking of engaged Pu.1 sites by nucleosomes'; page unavailable) reports removal of a centrally positioned nucleosome at some Pu.1-bound sites, but only partial displacement and repositioning at others. The Introduction identifies the study system as primary mouse macrophages, not macrophage cell lines. The B-cell and Scid.adh.2C2 statements have separate citations and are not assessed here. (LLM, conf=0.87)

  cites [12] Barozzi, 2014, Coregulation of transcription factor binding and nucleosome occupancy through DNA features of mammalian enhancers sourceExperimental — accuracy not yet measured
• Supported

  Citation 85 supports the Scid.adh.2C2 clause, not the separately cited B-cell and macrophage clauses. In the Results section on pioneering in DN3-like cells (page unavailable), the paper reports that introduced PU1-ERT2 rapidly made occupied, previously closed sites ATAC-accessible, with the response maximal within 2 hours. (LLM, conf=0.94)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) source

In the paper

“It is also reported to act as a repressor by direct recruitment of Dnmt3b (102).”

What the sources say

• Supported

  The Discussion reports that PU.1 associates with genes repressed through hypermethylation and has a direct role in recruiting DNMT3b to target promoters during monocyte-to-osteoclast differentiation. The paper also describes a separate activating role for PU.1, but that does not conflict with the claim that it can act as a repressor. (LLM, conf=0.93)

  cites [102] de la Rica, 2013, PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation source

In the paper

“At high levels, PU.1 has been found to block DNA binding by GATA-1 (23), while at lower levels it is reported to antagonize GATA-1-mediated transactivation by forming complexes with it that recruit Rb through the PU.1 acidic transactivation domain (101).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [23] PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding264148 ZhangP ZhangX IwamaA YuC SmithKA MuellerBU 11023493Blood962000
• Supported

  The citation to this paper is attached to the lower-level mechanism, not the preceding high-level DNA-binding claim. The Results (pp. 3–5) describe PU.1 binding to GATA-1 on DNA and identify its acidic transactivation subdomain as necessary for pRB binding and GATA-1 repression. The Discussion model (p. 13) states that PU.1 tethers pRB to repress transcription. (LLM, conf=0.92)

  cites [101] Rekhtman, 2003, PU.1 and pRB interact and cooperate to repress GATA-1 and block erythroid differentiation source

In the paper

“For PU.1 in pro-T cells, IRF and C/EBP family partners are mostly not available, but a key positive regulatory partner is Runx1 (previously known as AML1 or CBFα2), which has long been known to interact with PU.1 (and C/EBPα) to form a functional complex at its myeloid positive regulatory target sites (9, 112, 113).”

What the sources say

• Partly supported

  The ‘Developmental-stage-specific RUNX1 and PU.1 function’ section says RUNX1 and PU.1 form a synergistic complex in myeloid cells that regulates lineage-specific genes. The ‘RUNX1 and PU.1 in T and B-cells’ section discusses each factor’s role in early T-cell development but does not establish that RUNX1 is a key positive partner of PU.1 in pro-T cells. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  cites [9] Imperato, 2015, The RUNX1-PU.1 axis in the control of hematopoiesis sourceExperimental — accuracy not yet measured
• Not checked yetcites [112] Petrovick, 1998, Multiple functional domains of AML1: PU
• Partly supported

  The section on regulation of PU.1 in mice says RUNX1 activates the PU.1 upstream regulatory element early in development, supporting a positive regulatory role. It does not establish that Runx1 is a key PU.1 partner in pro-T cells or that the two form a functional complex at myeloid target sites; the paper examines regulation at the PU.1 locus in B and myeloid cells. Page numbers are unavailable in our copy. (LLM, conf=0.84)

  cites [113] Leddin, 2011, Two distinct auto-regulatory loops operate at the PU.1 locus in B cells and myeloid cells sourceExperimental — accuracy not yet measured

In the paper

“PU.1 itself is functionally important for the open status of these chromatin sites in the early stages, for many of these sites in fact do not remain as open if the PU.1 is removed acutely from primary pro-T cells by Cas9-mediated deletion (85).”

What the sources say

• Supported

  The Results section, Figure 2 legend (page not supplied), describes Cas9-mediated disruption of PU.1 in DN2 pro-T cells and identifies sites that lose accessibility after deletion. The Discussion (page not supplied) states that PU.1 is continuously required to maintain accessibility at many target sites. (LLM, conf=0.94)

  cites [85] Ungerbäck, 2018, Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1) source

In the paper

“Egr2, for example, can collaborate with PU.1 in positive regulation when co-bound with it (106), but has also been implicated as a PU.1-stimulated repressor of the mir17~92 complex (107).”

What the sources say

• ?Not checked — no full text of the sourcecites [106] Krysinska, 2007, A two-step, PU.1 dependent, mechanism for developmentally regulated chromatin remodelling and transcription of the c-fms gene source
• Supported

  The Results section, “Downregulation of miR-17-92 is mediated by Egr2” (page not provided), reports that PU.1 activates Egr2 during macrophage differentiation and that Egr2 represses the miR-17-92 cluster. Citation 107 is attached to this repressor claim, not to the preceding claim about co-bound positive regulation. (LLM, conf=0.93)

  cites [107] Pospisil, 2011, Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation source

In the paper

“However, the aspect of this redistribution that is most notable is that Runx1 is depleted in the process from alternative sites, and the sites that it abandons are themselves highly functional sites.”

What the sources say

• Supported

  In Results, “PU.1 redirects transcription factor ensembles across the genome” reports that introducing PU.1 reduced Runx1 binding at about half of the non-promoter sites occupied in control cells. “Functional importance of Satb1 and Runx1 in gene regulation by PU.1” links the abandoned ‘old’ sites to genes whose expression falls when Runx1 is disrupted, supporting their functional importance. Page numbers are not available in our copy. (LLM, conf=0.88)

  cites [80] Hosokawa, 2018, Transcription factor PU.1 represses and activates gene expression in early T cells by redirecting partner transcription factor binding source

In the paper

“PU.1 is not uniquely responsible for the Phase 1 gene expression pattern, however, for other Phase 1 genes are either unaffected or moderately inhibited by PU.1, as discussed elsewhere (79, 123).”

What the sources say

• Partly supported

  The Discussion (page unavailable) rules out PU.1 as a global positive regulator of Phase 1 genes and says it blunts expression of one group. The Results section on the progenitor-associated regulatory gene network (page unavailable) likewise reports repression of several Phase 1 genes, but the cited passages do not establish that other Phase 1 genes are unaffected or that the inhibition is moderate. (LLM, conf=0.82)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU sourceExperimental — accuracy not yet measured
• Partly supported

  In “Explaining Bcl11b Impact on Development” (page unavailable), the paper finds that PU.1-dependent genes form a subset of early-expressed genes and that PU.1 and Bcl11b provide mostly orthogonal inputs. It also reports weak PU.1 repression of Kit, but does not establish that the other Phase 1 genes are generally unaffected or moderately inhibited by PU.1. (LLM, conf=0.78)

  cites [123] Longabaugh, 2017, Bcl11b and combinatorial resolution of cell fate in the T-cell gene regulatory network sourceExperimental — accuracy not yet measured

In the paper

“First, PU.1 does not appear to bind, even transiently, at the sites from which Runx1 is lost: Runx1 is competitively redistributed, but is not displaced.”

What the sources say

• Supported

  In Results, the paper reports that most sites losing Runx1 showed no direct PU.1 binding and that early transient PU.1 binding did not account for Runx1 removal. It concludes that PU.1 recruits Runx1 elsewhere rather than displacing it from the old sites. (LLM, conf=0.90)

  cites [80] Hosokawa, 2018, Transcription factor PU.1 represses and activates gene expression in early T cells by redirecting partner transcription factor binding source

In the paper

“Additional factors also probably collaborate with PU.1 to fine-tune the responses of Mef2c and Lmo2, for they are already declining by the end of ETP stage, multiple cell cycles before DN2b stage when PU.1 itself declines (65, 67, 84, 121, 124) (Figure 8A).”

What the sources say

• Partly supported

  The ‘Transition to phase 2’ section says Spi1 (PU.1) is turned off as cells progress through DN2b, and ‘Checkpoint failure and T-cell leukemias’ says some progenitor genes terminate expression within the ETP stage. Neither passage identifies Mef2c and Lmo2 as declining by the end of ETP or says that other factors collaborate with PU.1 to fine-tune their responses. The paper describes several cell cycles before DN2b in relation to TCF-1 and GATA-3 activation, not the proposed decline of those two genes. (LLM, conf=0.83)

  cites [65] Yui, 2014, Developmental gene networks: a triathlon on the course to T cell identity sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Discussion describes division-linked changes in DN1 cells’ competence to progress to DN2, but does not address Mef2c or Lmo2 expression or PU.1’s decline. Searches for those factors and the claimed ETP-to-DN2b timing found no passage in our copy that verifies the comparison. (LLM, conf=0.75)

  cites [67] Manesso, 2013, Computational modelling of T-cell formation kinetics: output regulated by initial proliferation-linked deferral of developmental competence sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section on transcription-factor expression dynamics reports that Mef2c, Lmo2, and Sfpi1 (PU.1) decrease between FLDN1 and FLDN2b. The Results section on early T-cell-specific PU.1 sites says PU.1 is markedly downregulated at DN2b, and the binding-dynamics section suggests PU.1 can act through combinations of binding complexes. Our copy does not establish that Mef2c and Lmo2 begin declining by the end of ETP, multiple cell cycles before PU.1, or that other factors specifically collaborate with PU.1 to regulate those two genes. (LLM, conf=0.83)

  cites [84] Zhang, 2012, Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity sourceExperimental — accuracy not yet measured

In the paper

“However, cells in these stages are hard to visualize in the intact thymus, as only a few cells per day are granted regulated entry into the thymic antechamber (127), then migrate slowly through the cortex, dispersing among a vast excess of more advanced T-cell precursors, as they begin to differentiate toward commitment (128).”

What the sources say

• Partly supported

  The Abstract (page unavailable) says that low numbers of progenitors enter the thymus, making them difficult to characterize, and describes thymus colonization as tightly controlled. Its estimate of 10–200 cells per day, however, is explicitly for irradiated adult thymi; it does not establish that daily rate for the intact, unirradiated thymus. The later migration through the cortex is attached to citation (128), not this source. (LLM, conf=0.84)

  cites [127] Zietara, 2015, Multicongenic fate mapping quantification of dynamics of thymus colonization sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page unavailable) reports that precursors migrate outward across the cortex while progressing through defined developmental stages, supporting the migration-and-differentiation portion attributed to this paper. It describes the stages as concentrated in distinct cortical regions, however, and does not establish that the cells disperse among a vast excess of more advanced precursors. The separate claim about a few cells entering per day is attached to citation 127, not this source. (LLM, conf=0.84)

  cites [128] Lind, 2001, Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development sourceExperimental — accuracy not yet measured

In the paper

“It probably extends for over 10 days for the thymocytes in young adult mice (153), where it is crucial for successful T-cell generation (2, 147).”

What the sources say

• Partly supported

  The Results and Discussion (page unavailable) report that thymic progenitors take about 15 days to reach the double-positive stage, supporting a developmental interval longer than 10 days. The paper does not establish that PU.1 remains active throughout that interval. The statement about PU.1 being crucial for T-cell generation is attached to other citations, not this one. (LLM, conf=0.85)

  cites [153] Porritt, 2003, Kinetics of steady-state differentiation and mapping of intrathymic-signaling environments by stem cell transplantation in nonirradiated mice sourceExperimental — accuracy not yet measured
• Partly supported

  The Results (‘PU.1-deficient stem cells do not generate lymphoid progeny’; page unavailable) report that PU.1-deficient progenitors were essentially unable to contribute to T-cell production, supporting its importance for T-cell generation. The paper does not establish that PU.1 remains active in thymocytes for over 10 days; the Results (‘Generation and validation of a PU.1 conditional allele’) instead state that most thymocytes do not express it. Its 10–14-day interval refers to when mice were analyzed after induced deletion, not a duration of thymocyte activity. (LLM, conf=0.87)

  cites [2] Dakic, 2005, PU.1 regulates the commitment of adult hematopoietic progenitors and restricts granulopoiesis sourceExperimental — accuracy not yet measured
• Supported

  The Discussion (page unavailable) reports that adult T-cell development requires higher PU.1 expression and that thymopoiesis was absent in adult mice with reduced PU.1 expression. This supports the claim that PU.1 is crucial for adult T-cell generation. The separate claim about activity lasting over 10 days is attributed to citation 153, not this source. (LLM, conf=0.91)

  cites [147] Montecino-Rodriguez, 2018, Differential expression of PU.1 and Key T lineage transcription factors distinguishes fetal and adult T Cell development source

In the paper

“In postnatal mice, the entry point is thought to be formed by specialized endothelial cells at the cortical/medullary border of the thymus (129).”

What the sources say

• Supported

  The paper states that thymus-seeding progenitors enter at the cortico-medullary junction (Introduction; page unavailable). The Results identify a distinct vascular endothelial-cell subset on its cortical side, in the area of progenitor immigration, and the Discussion suggests that progenitors’ association with these cells may reflect their point of entry. This supports the citing sentence’s hedged description. (LLM, conf=0.86)

  cites [129] Buono, 2016, A dynamic niche provides Kit ligand in a stage-specific manner to the earliest thymocyte progenitors source

In the paper

“Following an unknown triggering signal, after a variable delay (125), the cells in each cohort then begin to migrate centrifugally toward the outer thymic cortex, and cell surface marker expression patterns imply that it is somewhere midway in the course of this migration that the individual cells undergo lineage commitment [reviewed by (60, 130)].”

What the sources say

• ?Not checked — no full text of the sourcecites [60] Petrie, 2007, Zoned out: functional mapping of stromal signaling microenvironments in the thymus source
• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [130] Love, 2011, Signal integration and crosstalk during thymocyte migration and emigration sourceExperimental — accuracy not yet measured

In the paper

“This is now understood to be intrinsically programmed (136) and due to an altered pathway of differentiation in the first-wave cells, which results in T-cell lineage commitment even before entry into the thymus (137–139).”

What the sources say

• Supported

  The citation to this paper is attached to the claim that the difference is intrinsically programmed, not to the later claim about T-cell commitment before thymus entry. In the Abstract, the paper states that fetal-liver HSCs are intrinsically programmed to generate B-1a B cells and Vγ3+ γδ T cells. (LLM, conf=0.90)

  cites [136] Yuan, 2012, Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis source
• ?Not checked — no full text of the sourcecites [137] Harman, 2005, T/B lineage choice occurs prior to intrathymic Notch signalling source
• Partly supported

  The Results report that commitment to the T/NK/DC lineage occurs in extrathymic organs, and the Discussion describes prethymic progenitors as genetically distinct from intrathymic ones. But the Discussion says these progenitors *may* represent the first phase of thymic immigration; it does not establish an altered differentiation pathway specific to first-wave cells or exclusive commitment to the T-cell lineage. The separate claim of intrinsic programming is attached to citation 136, not this source. (LLM, conf=0.85)

  cites [138] Masuda, 2005, Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [139] Berthault, 2017, Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver source

In the paper

“ETPs derived from these precursors naturally express lower levels of PU.1 than adult ETPs, but they are almost unchanged in their ability to generate early fetal T cells when the level of PU.1 is reduced still further (~5 fold) by deletion of the major upstream regulatory element of PU.1 (147).”

What the sources say

• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [147] Montecino-Rodriguez, 2018, Differential expression of PU.1 and Key T lineage transcription factors distinguishes fetal and adult T Cell development sourceExperimental — accuracy not yet measured

In the paper

“It has long been recognized that the earliest fetal thymocytes are different from later waves of developing thymocytes in terms of their abilities to generate particular classes of TCRγδ cells (134) and in terms of their extremely fast differentiation kinetics, both in vivo and in fetal thymic organ culture or stromal coculture systems (104, 133, 135).”

What the sources say

• ?Not checked — no full text of the sourcecites [134] Ikuta, 1990, A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells source
• Partly supported

  Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  cites [104] Scripture-Adams, 2014, GATA-3 dose-dependent checkpoints in early T cell commitment sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [133] Ramond, 2014, Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus source
• ?Not checked — no full text of the sourcecites [135] Kawamoto, 1997, Hemopoietic progenitors in the murine fetal liver capable of rapidly generating T cells source

In the paper

“These stages span multiple cell cycles in vivo and in vitro (33, 39, 67, 124).”

What the sources say

• Partly supported

  The Results section, “DN2b cells are distinct from DN3 cells” (page unavailable), reports that cultured DN2b cells retained CD25 after 5–8 cell divisions, supporting the in vitro portion. Our copy does not establish that these stages span multiple cell cycles in vivo. (LLM, conf=0.84)

  cites [33] Yui, 2010, Fine-scale staging of T cell lineage commitment in adult mouse thymus sourceExperimental — accuracy not yet measured
• Supported

  The Discussion reports more than ten cell divisions during the DN1 and DN2 stages in fetal thymus, supporting the in vivo part. The in vitro transition experiments report approximately fourfold and fivefold increases in cell number during culture, supporting multiple divisions in vitro. Page numbers are not provided in our copy. (LLM, conf=0.90)

  cites [39] Masuda, 2007, T cell lineage determination precedes the initiation of TCRβ rearrangement source
• Supported

  The Introduction states that cells undergo varying numbers of proliferation cycles within thymocyte developmental stages. Results §2.4 reports multiple divisions by DN1 cells over four days in vitro, while the paper’s in vivo model describes successive generations within the DN1 stage. Page numbers are unavailable in our copy. (LLM, conf=0.87)

  cites [67] Manesso, 2013, Computational modelling of T-cell formation kinetics: output regulated by initial proliferation-linked deferral of developmental competence source

In the paper

“For example, the crucial T-lineage transcription factor TCF-1, which plays roles in numerous phases of thymocyte development (95, 140–145), is essential to maintain adult T-cell production but dispensable in fetal and early postnatal T cell development (146).”

What the sources say

• Partly supported

  The Discussion reports a lack of adult T-cell production without Tcf-1, supporting the adult portion of the claim. But the ‘Rapid involution of the adult thymus with age’ section reports fewer fetal thymocytes and changes in thymocyte populations by day 10 after birth; the fetal-progenitor experiments also show a developmental defect. The paper therefore does not support calling Tcf-1 dispensable in fetal and early postnatal development. Page numbers were not available in our copy. (LLM, conf=0.90)

  cites [146] Schilham, 1998, Critical involvement of Tcf-1 in expansion of thymocytes sourceExperimental — accuracy not yet measured

In the paper

“In contrast, the same five-fold diminished level of PU.1 sharply degrades the ability of later fetal hematopoietic stem and progenitor cells to generate T cells at all, in vivo or in vitro, with functional and phenotypic defects evident in the mutants in both multipotent progenitors and newly-entered intrathymic ETPs, as early as in the late fetus.”

What the sources say

• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [147] Montecino-Rodriguez, 2018, Differential expression of PU.1 and Key T lineage transcription factors distinguishes fetal and adult T Cell development sourceExperimental — accuracy not yet measured

In the paper

“In contrast, for many first-wave fetal thymocytes it may last only a day or two, and is mostly or entirely dispensable (71, 147).”

What the sources say

• ?Not checked — no full text of the sourcecites [71] Spain, 1999, T cell development in PU.1-deficient mice source
• Partly supported

  The Discussion (page unavailable) says the early fetal T-cell developmental wave is largely PU.1 independent, supporting the claim’s qualified dispensability point. The study used mice with reduced, not absent, PU.1 expression, and our copy does not establish that PU.1 activity lasts only a day or two in first-wave fetal thymocytes. (LLM, conf=0.86)

  cites [147] Montecino-Rodriguez, 2018, Differential expression of PU.1 and Key T lineage transcription factors distinguishes fetal and adult T Cell development sourceExperimental — accuracy not yet measured

In the paper

“Indeed, pro-T cells that have PU.1 acutely deleted at an early stage do not only differentiate faster along the T lineage; they also tend to shift to a natural killer-like program more readily than controls (79).”

What the sources say

• Partly supported

  The Results section reports that deleting PU.1 at the ETP or DN2a stage made early T cells differentiate faster than controls. It also reports greater NK-cell production under permissive conditions, but that result concerns progeny of PU.1-deficient fetal liver precursors, not the same early-stage pro-T cells. The claim conflates those populations and omits the NK-development condition. (LLM, conf=0.88)

  cites [79] Champhekar, 2015, Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU sourceExperimental — accuracy not yet measured

In the paper

“The highest confidence list of potential PU.1 target genes in pro-T cells could be defined as genes that responded reciprocally to gain and loss of PU.1 function within the same DN2a-DN2b developmental interval, and these genes are listed in Table 1.”

In the paper

“Genes responding to the addition of exogenous PU.1 usually had the exogenous PU.1 binding to distal (intronic or neighboring intergenic) sites, whereas genes that had PU.1 binding only to their promoter regions usually did not change expression at all.”

In the paper

“They also begin to express multiple transcription factors of the Egr and IRF families, and in the case of primary cells, they also upregulate C/EBP family factors.”

In the paper

“In the “blank slate” background of the Scid.adh.2C2 cell line, exogenous PU.1 binding opened chromatin at its non-promoter sites within 2 h, increasing the “activating” H3K27Ac marks at these sites a few hours later, and the linked genes were predominantly upregulated within 8–24 h.”

In the paper

“In the aggregate, most of the responses of genes linked directly to PU.1 binding sites were positive; direct repression targets were much rarer.”

In the paper

“Potentially important clues to PU.1 roles are the prominence among positively regulated PU.1 targets of genes encoding specific cytokine receptors not yet studied in T-cell biology (e.g., Pdgfrb); multiple protein tyrosine kinases (Btk, Syk, Hck, Lyn); and G-protein coupled receptors (Ffar2, P2ry13, P2ry14) and G protein signaling mediators (Gng2 and Rgs18); while PU.1 represses other G protein s”

In the paper

“Hes1, Tcf12 (HEB), Ets1, and Lef1 are strongly affected in cells making a lineage switch, but none of these are measurably repressed in cells within the T-cell path.”

In the paper

“In addition, PU.1 directly promotes expression of cell surface molecules (CD33, CD34, CD44) used as markers for stages in early T-cell development, but which in vivo work to mediate environmental interactions, and it drives expression of adhesion molecules (integrins and Siglecs) as well as cytoskeletal components such as Coro2a and Myo1f.”

In the paper

“It is a lineage-specifying transcription factor that positively regulates many genes in the macrophage, granulocyte, dendritic-cell and B-cell lineages.”

In the paper

“Thus, B, NK, and T cell development are all affected by defects in PU.1 activity, despite partial complementation by the related factor SpiB”

In the paper

“the downregulation of PU.1 is important to avoid malignancy in human T cells: a specifically aggressive class of human T-acute lymphoblastic leukemias results from translocations that promote abnormally sustained and elevated PU.1 expression (38).”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Seki, 2017, Recurrent SPI1 (PU.1) fusions in high-risk pediatric T cell acute lymphoblastic leukemia source

In the paper

“PU.1, encoded by the Spi1 gene, is an ETS-family transcription factor with multiple roles in hematopoiesis.”

In the paper

“It slows developmental progression of pro-T cells even as it supports their early proliferation.”

In the paper

“The effect of PU.1 on proliferation is conditional and dose-dependent, however.”

In the paper

“If timepoints are taken too long after deletion, the controls progress to the point when endogenous PU.1 is downregulated, so that any truly PU.1-dependent targets are expressed weakly in the controls, and comparisons with the knockout samples again lose statistical power.”

In the paper

“Thus, the effect of PU.1 expression per se may be part of the normal T-cell program, but under high-level expression conditions it combines with additional, conditionally induced mechanisms to produce a much broader spectrum of developmental effects that may not only be direct responses to PU.1 itself.”

In the paper

“Cre-dependent deletion of a loxP-flanked Spi1 allele (Spi1fl.fl) is asynchronous, and in an early T-cell population with mixed degrees of Spi1 deletion, cells with inadequate PU.1 levels appear to be at a selective disadvantage, even in vitro.”

In the paper

“In newly-committed primary pro-T cells forced to express PU.1, the cells crossing this developmental boundary are seen to downregulate the Notch-dependent DN2/DN3 stage marker, CD25 (Il2ra), and often upregulate the myeloid-associated marker CD11b (Mac1; Itgam).”

In the paper

“While this list under-represents some PU.1 targets that are only expressed in ETP stage”

In the paper

“The strongest evidence for specific physiological PU.1 effects therefore comes from the consensus results from two or more of these perturbation systems.”

In the paper

“rigorous definition of the genes that are directly regulated by PU.1 in pro-T cells has made it possible to investigate the range of mechanisms used by the PU.1 protein to exert these transcriptional effects.”

In the paper

“It is possible that the fast downregulation of Notch response genes, including Hes1, Nrarp, Dtx1, Lef1, and Il2ra, by overexpressed PU.1 is due to the loss of positive Notch signaling input rather than to a gene-specific mechanism.”

In the paper

“This Notch-inhibitory mechanism is not operating in cells that remain within the T-cell pathway, but it becomes prominent in cells that PU.1 causes to transdifferentiate, and would be expected to affect all T-cell genes that use Notch signaling as an obligate positive input, whether or not PU.1 binds them directly.”

In the paper

“Data from multiple studies show that PU.1 regulates a subset of developmental control genes but is not alone in its actions.”

In the paper

“The PU.1-high stages of thymocyte development are relatively obscure in the context of the whole thymus, yet their accurate regulation is crucial for establishment of immune system homeostasis and avoidance of leukemia (125, 126).”

What the sources say

• ?Not checked — no full text of the sourcecites [125] Goldschneider, 2006, Cyclical mobilization and gated importation of thymocyte progenitors in the adult mouse: evidence for a thymus-bone marrow feedback loop source
• ?Not checked — no full text of the sourcecites [126] Martins, 2014, Cell competition is a tumour suppressor mechanism in the thymus source

In the paper

“Thus, within the T-cell pathway, PU.1 has a specific role in promoting maintenance of certain Phase 1 regulatory genes before commitment, but little role in repressing T-cell differentiation regulators directly.”

In the paper

“With respect to PU.1-mediated repression, some T-cell factors are already upregulated while PU.1 is still highly expressed (GATA-3, TCF-1, and the Notch target Hes1), but others are upregulated only during the period when PU.1 declines (Bcl11b, Ets1, Lef1)”

In the paper

“While dysregulated PU.1 can cause trans-differentiation to myeloid or dendritic-cell fates, endogenous PU.1 normally plays a protracted role within early T-cell development.”

In the paper

“PU.1 is a broad regulator of the properties of the cells that first enter the thymus, and it helps to determine their proliferation and rate of progression to commitment after they arrive.”

In the paper

“The first-wave precursor cells, also uniquely, enter the thymus by a different route than all subsequent waves. Instead of entering through the blood vessels near the cortical-medullary junction, these early cells migrate through cervical-region mesenchyme to the thymic anlage before it is vascularized.”

In the paper

“Its target genes are occasionally repressed but mostly activated by PU.1 binding, and they confer on the cells distinctive stage-specific transcription factor expression patterns as well as a rich array of stage-specific cell biological features that await proper functional analysis.”

In the paper

“Because of the extreme rarity of these very immature cells relative to the later-stage thymocytes at any given time, they were almost impossible to study in depth before the development of in vitro culture systems (88), which have continued to be informative to the present.”

What the sources say

• ?Not checked — no full text of the sourcecites [88] Schmitt, 2002, Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro source

In the paper

“Thus, not only is the first-wave fetal program less dependent on PU.1, but also it may rely on relatively low PU.1 activity for its very distinctiveness.”

In the paper

“This positive regulatory role is one result of PU.1's strong pattern of binding across the genome, its prominent occupancy of open chromatin sites, and the evidence that it helps to maintain the open chromatin states at bound regulatory sites as long as it is expressed.”

In the paper

“These results therefore support a role for PU.1 in delaying differentiation in order to allow more extended proliferation before commitment, showing how the importance of this role is ontogenically scaled to the needs of the developing organism.”

In the paper

“Thus, postnatal thymocytes and any in vitro differentiation cultures seeded with cells from bone marrow or fetal liver from E15 onward are likely to come from true stem cell origins.”

In the paper

“The tests of PU.1 function in these early pro-T cells that have been done so far present the cells with Notch ligands and cytokines, but could be fundamentally lacking in other molecules presented by the normal thymic environment.”

In the paper

“The cells used for in vitro differentiation as well as in vivo analysis in the work reviewed above have all been derived from waves of hematopoiesis that begin with definitive hematopoietic stem cells, which first appear in the mouse fetal liver by about day 11.5 of gestation and may be followed by additional stem-cell waves through the end of gestation (day 20) (131).”

What the sources say

• ?Not checked — no full text of the sourcecites [131] Dzierzak, 2008, Of lineage and legacy: the development of mammalian hematopoietic stem cells source

In the paper

“The first wave of T cell development in the fetus is now thought to arise from these non-stem-cell precursors in the yolk sac (132, 133).”

What the sources say

• ?Not checked — no full text of the sourcecites [132] Yoshimoto, 2012, Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence source
• ?Not checked — no full text of the sourcecites [133] Ramond, 2014, Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus source

In the paper

“The number of genes that respond quickly to changes in PU.1 activity may only account for a minority of all the genomic sites where PU.1 is found engaged; at other sites, its role could be structural or redundant with other factors.”

In the paper

“Not only are these cells intrinsically programmed to cut short the stages supported by PU.1-dependent transcriptional regulators, but also they can dispense with many of the cell biological tools that PU.1 may provide to later-wave successors to navigate the adult or late-fetal thymus.”

In the paper

“The system-wide impact of PU.1 on other factors suggests that in scenarios where PU.1 is absent, the same T-cell transcription factors might initially choose different binding sites.”

In the paper

“Hematopoietic progenitors express PU.1 before they enter the thymus, but the pattern of its occupancy is not well defined at that stage, so the onset of PU.1's pioneering activity in precursors that will eventually generate T cells is not easy to study.”