Mechanisms of PU.1 binding site selection in-vivo
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE43098 | GEO | 2013/05/15
SECONDARY ACCESSION(S): PRJNA185267
REPOSITORIES: GEO
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Project description:The majority of sequence-specific transcription factors bind genomic DNA only at a fraction of their potential binding sites and the ‘rules’ for binding or not-binding are only partially understood. Here, we studied the binding properties of the myeloid and B-cell specific transcription factor PU.1 in-vivo and in-vitro to unveil basic features of occupied vs. non-occupied consensus sites. In addition to published PU.1 ChIP-seq data we mapped CTCF binding sites in monocytes and macrophages to determine chromatin domain boundaries and performed MCIp-seq in monocytes to reveal DNA methylation patterns across the genome. ChIP-seq of CTCF in human monocytes and human monocyte-derived macrophages as well as MCIp-seq in human monocytes
2013-05-15 | E-GEOD-43098 | biostudies-arrayexpress
Project description:Spi-B and PU.1 are highly related members of the E26-transformation-specific (ETS) family of transcription factors that have similar, but not identical, functions in B cell development. PU.1 and Spi-B are both expressed at high levels in lymphoma cell lines. We hypothesized that Spi-B and PU.1 occupy similar sites in the genome. To determine binding sites of Spi-B and PU.1, WEHI-279 mouse lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Both transcription factors occupied approximately 2000 sites in the genome, and approximately half of these sites were bound by both factors while the other sites were unique to each factor.
2014-11-10 | GSE58128 | GEO
Project description:We used the myelogenous leukemia line K562 as a model of HDACi-induced differentiation to investigate chromatin accessibility (DNase-seq) and expression (RNA-seq) changes associated with this process. We identified several thousand specific regulatory elements (~10% of total DHS sites) that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid (SAHA) 72-hour treatments. Most of the differential DNase-hypersensitive (DHS) sites display hallmarks of enhancers; including being enriched for non-promoter regions, associated with nearby gene expression changes, and capable of increasing luciferase reporter expression in K562 cells. Differential DHS sites were enriched for key hematopoietic lineage transcription factor motifs, including SPI1 (PU.1), a known pioneer factor. We found PU.1 becomes up-regulated and increases binding at opened DHS sites by ChIP-seq with HDACi treatment, but show that increased PU.1 protein levels alone are sufficient for only modest increases in chromatin accessibility. PU.1 knockdown by shRNA failed to block the HDACi-induced chromatin accessibility and expression changes in K562, suggesting factors other than PU.1 are responsible for establishment of active enhancers in the HDACi induced differentiation process.
2016-04-10 | GSE74999 | GEO
Project description:This study aims to understand the binding preferences of WT vs Q226E mutant versions of PU.1 throughout the genome.
2023-06-28 | GSE211518 | GEO
Project description:Downregulation of the hematopoietic transcription factor PU.1 in PU.1 low acute myeloid leukemia cells (AML) by novel heterocyclic diamidines or PU.1 inhibitors leads to decrease cell proliferation and apoptosis, representing a new therapeutic strategy for AML treatment. These inhibitors induces decreased PU.1 binding on its target sites, as well as deregulation in PU.1 canonical target genes We used microarray to identify the pathways deregulated after drug treatment.
2017-12-07 | GSE77651 | GEO
Project description:Runx1 and Runx3 function redundantly in early T-development, and together drive T-lineage developmental progression by regulating distinct sets of genes in different stages. Context-specific Runx target genes are particularly enriched near Runx binding sites that dynamically shift from pre-T-commitment stages (Phase 1) to post-T-commitment stages (Phase 2). As total Runx activities (Runx1+Runx3) are maintained stably throughout early T-development stages, yet Runx factors physically interact with multiple collaborators, we hypothesized that different Runx-collaborating transcription factors compete to recruit a limited pool of Runx transcription factors. To test whether increasing Runx availability can alter Runx DNA-binding site choices, Runx1 overexpression vectors were introduced to two different systems. First, we increased Runx1 expression in a DN3-like cell line (representing a post-commitment, Phase 2 stage) in the presence or absence of exogenous Phase 1 co-factor, PU.1. Second, we increased Runx1 expression in Phase 1 primary cells which naturally express PU.1 and other Phase 1 collaborators. Then, Runx1 binding behaviors were measured using ChIP-seq or CUT&RUN (C&R). A modest increase of Runx1 levels (about 2-3 fold increase) substantially increased the number of Runx binding sites seen and the intensity of occupancy in both systems. When Runx expression was at physiological levels, PU.1 dominated Runx1 site choice before T commitment by recruiting Runx1 to PU.1 sites. The Phase 2 cell line system showed that it did this while depleting Runx1 from alternative high quality Runx motif sites. However, when Runx1 was overexpressed, Runx1 was still recruited to PU.1 sites, but this recruitment did not evacuate Runx1 occupancy from default preferred sites. Notably, Runx1 overexpression in primary Phase 1 cells caused precocious occupancy of post-commitment, Phase 2-specific sites. We found that these are often co-occupied with TCF1, E2A, and HEB, but have minimal co-binding with PU.1. In addition, Runx1 overexpression resulted in new binding sites that were not normally observed in pro-T cells, which are mostly sequestered by closed chromatin normally although they harbor more numerous Runx motifs. Thus, these data suggest that Runx DNA binding site choices are sensitive to Runx concentration and co-factors during early T-development.
2023-07-09 | GSE218147 | GEO
Project description:Master transcription factors are the gatekeepers of lineage identity. As such, they have been a major focus of efforts to manipulate cell fate for therapeutic purposes. The ETS transcription factor PU.1 has a potent ability to confer macrophage phenotypes on cells already committed to a different lineage, but how it overcomes the presence of other master regulators is not known. The nuclear receptor PPARγ is the master regulator of the adipose lineage, and its genomic binding pattern is well characterized in adipocytes. Here, we show that when expressed at macrophage levels in mature adipocytes, PU.1 bound a large fraction of its macrophage sites, where it induced chromatin opening and the expression of macrophage target genes. Strikingly, PU.1 markedly reduced the genomic binding of PPARγ without changing its abundance. PU.1 expression repressed genes with nearby adipocyte-specific PPARγ binding sites, while a common macrophage-adipocyte gene expression program was retained. Together, these data reveal unexpected lability within the adipocyte PPARγ cistrome and show that even in terminally differentiated cells, PU.1 can remodel the cistrome of another master regulator. Microarray expression profiling was performed on 3T3-L1 adipocytes from two treatment groups: (1) adipocytes transduced with a control adenovirus expressing beta-galactosidase (LACZ-Ads) and (2) adipocytes transduced with an adenovirus expressing full-length murine PU.1 cDNA (PU.1-Ads). Each sample group consists of four biological replicates which are here defined as separate differentiations of mature 3T3-L1 adipocytes and adenoviral infections. Each replicate was hybridized to an individual array for a total of eight arrays.
2013-06-27 | E-GEOD-48344 | biostudies-arrayexpress
Project description:"Master" transcription factors are the gatekeepers of lineage identity. As such, they have been a major focus of efforts to manipulate cell fate for therapeutic purposes. The ETS transcription factor PU.1 has a potent ability to confer macrophage phenotypes on cells already committed to a different lineage, but how it overcomes the presence of other master regulators is not known. The nuclear receptor PPARM-NM-3 is the master regulator of the adipose lineage, and its genomic binding pattern is well characterized in adipocytes. Here, we show that when expressed at macrophage levels in mature adipocytes, PU.1 bound a large fraction of its macrophage sites, where it induced chromatin opening and the expression of macrophage target genes. Strikingly, PU.1 markedly reduced the genomic binding of PPARM-NM-3 without changing its abundance. PU.1 expression repressed genes with nearby adipocyte-specific PPARM-NM-3 binding sites, while a common macrophage-adipocyte gene expression program was retained. Together, these data reveal unexpected lability within the adipocyte PPARM-NM-3 cistrome and show that even in terminally differentiated cells, PU.1 can remodel the cistrome of another master regulator. ChIP-seq was performed on 3T3-L1 adipocytes from two treatment groups: (1) adipocytes transduced with a control adenovirus expressing beta-galactosidase (LACZ-Ads) and (2) adipocytes transduced with an adenovirus expressing full-length murine PU.1 cDNA (PU.1-Ads). Nuclear lysates from each group were used for PPARg ChIP. For PU.1-Ads, PU.1 ChIP was also performed. To generate chromatin for ChIP-seq, DNA from three immunoprecipitations per condition was pooled. This process was repreated from a second set of L1 adipocytes to generate two biological replicates for sequencing. Genomic input DNA was sequenced from the first biological replicate only.
2013-06-27 | E-GEOD-48343 | biostudies-arrayexpress
Project description:PU.1 is a prototype master transcription factor (TF) of hematopoietic cell differentiation with diverse roles in different lineages. Analysis of its genome-wide binding pattern across PU.1 expressing cell types revealed manifold cell type-specific binding patterns. They are not consistent with the epigenetic and chromatin constraints to PU.1 binding observed in vitro, suggesting that PU.1 requires auxiliary factors to access DNA in vivo. Using a model of transient mRNA expression we show that PU.1 induction leads to the extensive remodeling of chromatin, redistribution of partner transcription factors and rapid initiation of a myeloid gene expression program in heterologous cell types. By probing PU.1 mutants for defects in chromatin access and screening for PU.1 proximal proteins in vivo, we found that its N-terminal acidic domain was required for the recruitment of SWI/SNF remodeling complexes, de novo chromatin access and stable binding as well as the redistribution of partner TFs.
2019-11-20 | PXD013167 | Pride
Project description:A major problem with linking transcription factor binding to function is that many factors bind to a large number of, at least in any given cell type, seemingly irrelevant regions. This makes it hard to filter out which binding sites are responsible for the regulation of a given gene. PU.1 (Spi1, Sfpi1) is an excellent example of a transcription factor that works both to mediate developmental choices and to serve the alternative developmental fates that emerge from these choices. Its role in T-cell development is confined to the early stages where high PU.1 expression persists through multiple cell divisions and is sharply downregulated over the DN2a-to-DN2b T-cell commitment stage. Even though it is known that PU.1 is necessary for the survival of the earliest T-cell progenitors, where it is needed for optimal proliferation, control of alternative lineage genes, and correct timing of the access to T-lineage genes, it has not been well-studied how PU.1 finds it targets and exerts its functions within this context. Here, we show in detail how PU.1 selects its binding sites and subsequently regulate gene expression. We show that PU.1 has two effective affinity thresholds for occupancy depending on current chromatin openness as measured by ATAC-sequencing, but that it is easily capable of binding sites in closed chromatin. Unexpectedly, although its binding to promoters is least constrained, promoters are the only major class of sites where it exerts a predominantly negative effect; otherwise it works locally as an activator, mainly mediated through binding to sites in closed or dynamically closing distal enhancer elements, where it can rapidly open chromatin and induce histone acetylation. However, its ability to open the chromatin depends not only on its own affinity but also on the presence of collaborating factors, because we show that PU.1 introduction into PU.1-negative cells triggers a massive reorganization of occupancy patterns of at least three other factors: Runx1, Satb1, and Gata3. Most strikingly, PU.1’s “theft” of Runx1 and Satb1 from many sites where they were binding in the absence of PU.1 enables PU.1 to exert a novel form of repression even on genes where it has no binding sites itself. We show here that PU.1 requires domains outside of its DNA binding domain to properly open chromatin, and this structural requirement is directly connected with its ability to bind to Runx1 and to Satb1, and moreover, Runx1, in particular, is an important collaborator to activate many of its target genes in the context of early T-cell development. Thus, PU.1 regulates gene expression via two distinct mechanisms. First, PU.1 steals Satb1 and Runx1 from many genomic sites, thereby repressing T cell gene expression indirectly. Second, PU.1, opens chromatin, recruits Satb1 and Runx1 to new sites, and directly activates its target gene expression. In summary, we here present a model where a transcription factor can work through redeployment of other factors and not only through sites that it binds itself.
2018-06-21 | GSE93755 | GEO