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Gene regulation in hematopoietic cells by Pcgf1 in a Runx1 null background

ABSTRACT: The transcription factor Runx1 is essential for the establishment of definitive hematopoiesis during embryonic development. In adult blood homeostasis, Runx1 plays a pivotal role in the maturation of lymphocytes and megakaryocytes. Furthermore, Runx1 is required for the regulation of hematopoietic stem and progenitor cell (HSPC) pools. However, how Runx1 orchestrates self-renewal and lineage choices in combination with other factors is not well understood. Here we describe a genome-scale RNAi screen to detect genes that cooperate with Runx1 in regulating HSPCs. We identify the polycomb group protein Pcgf1 as an epigenetic regulator involved in hematopoietic cell differentiation. We show that simultaneous depletion of Runx1 and Pcgf1 allows sustained self-renewal while blocking differentiation of HSPCs in vitro. We find an upregulation of HoxA cluster genes upon Pcgf1 knockdown that possibly accounts for the increase in self-renewal. Further, our data suggest that cells lacking both Runx1 and Pcgf1 are blocked at an early progenitor stage, indicating that a concerted action of the transcription factor Runx1 together with the epigenetic repressor Pcgf1 is necessary for terminal differentiation. Thus, our work discovers a genetic link between transcriptional and epigenetic regulation that is required for hematopoietic differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE33280 | GEO | 2012/06/18

SECONDARY ACCESSION(S): PRJNA149081

REPOSITORIES: GEO

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Gene regulation in hematopoietic cells by Pcgf1 in a Runx1 null background

Project description:The transcription factor Runx1 is essential for the establishment of definitive hematopoiesis during embryonic development. In adult blood homeostasis, Runx1 plays a pivotal role in the maturation of lymphocytes and megakaryocytes. Furthermore, Runx1 is required for the regulation of hematopoietic stem and progenitor cell (HSPC) pools. However, how Runx1 orchestrates self-renewal and lineage choices in combination with other factors is not well understood. Here we describe a genome-scale RNAi screen to detect genes that cooperate with Runx1 in regulating HSPCs. We identify the polycomb group protein Pcgf1 as an epigenetic regulator involved in hematopoietic cell differentiation. We show that simultaneous depletion of Runx1 and Pcgf1 allows sustained self-renewal while blocking differentiation of HSPCs in vitro. We find an upregulation of HoxA cluster genes upon Pcgf1 knockdown that possibly accounts for the increase in self-renewal. Further, our data suggest that cells lacking both Runx1 and Pcgf1 are blocked at an early progenitor stage, indicating that a concerted action of the transcription factor Runx1 together with the epigenetic repressor Pcgf1 is necessary for terminal differentiation. Thus, our work discovers a genetic link between transcriptional and epigenetic regulation that is required for hematopoietic differentiation. Hematopoietic stem and precursor cells freshly isolated from mice were transduced with an shRNA targeting Pcgf1 or a control shRNA. Cells were selected with puromycin for 36 h before total mRNA was isolated.

2012-06-17 | E-GEOD-33280 | biostudies-arrayexpress

Transcriptommic profiling of hematopoietic progenitor cells following PCGF1 loss

Project description:Polycomb proteins are epigenetic regulators important for cell fate decision and maintenance. Here we show that PCGF1, one of components of Polycomb repressive complex (PRC) 1 is important for the differentiation of hematopoietic stem/progenitor cells (HSPCs) towards lymphoid lineage. The deletion of Pcgf1 in HSPCs lead to severe defects in B cell development with an expansion of myeloid progenitors. The blockade of B cell differentiation occurred at the lymphoid-primed multipotent progenitor (LMPP) stage. Chromatin immunoprecipitation followed by DNA sequencing analysis of normal LMPP-like cells demonstrated that PCGF1 colocalized with RING1A/B and PRC2. Upon deletion of Pcgf1, we found dramatic decline of H3K27me3 levels, resulting in de-repression of target genes. Strikingly, the occupancy of RING1A/B, PRC2 and monoubiquitination of histone H2A at lysin 119 on the target loci was not affected. These results demonstrate that the PCGF1 safeguards the immune system development by directly regulating the PRC2-mediated H3K27me3 levels.

2022-10-19 | GSE141523 | GEO

Gene regulation by PCGF1 in hematopoietic progenitor cells [ChIP, MNaseSeq]

2022-10-19 | GSE141559 | GEO

Gene regulation by PCGF1 in hematopoietic progenitor cells [ChIP and MNaseSeq]

2022-10-19 | GSE207719 | GEO

Transcriptommic profiling of hematopoietic progenitor cells following PCGF1 loss [RNA-Seq]

2022-10-19 | GSE207478 | GEO

Expression regulation by Pcgf1 in mouse embryonic stem cells

Project description:The Polycomb repressive complex 1 (PRC1) is essential for fate decisions of embryonic stem (ES) cells. Emerging evidence suggests that six major variants of PRC1 complex, defined by the mutually exclusive presence of Pcgf subunit, regulate distinct biological processes, yet very little is known about the mechanism by which each version of PRC1 instructs and maintains cell fate. Here, we disrupted the Pcgf1, also known as Nspc1 and one of six Pcgf paralogs, in mouse ES cells by the CRISPR/Cas9 technology. We showed that although these mutant cells were viable and retained normal self-renewal, they displayed severe defects in differentiation in vitro. To gain a better understanding of the role of Pcgf1 in transcriptional control of differentiation, we analysed mRNA profiles from Pcgf1 deficient cells using RNA-seq. In contrast to the canonical role of PRC1 in gene repression, we found that Pcgf1 mainly functioned as a transcriptional activator to drive expression of many genes involved in ectoderm and mesoderm differentiation. Chromatin immunoprecipitation experiments demonstrated that Pcgf1 deletion caused a decrease in Ring1B and its associated H2AK119ub1 mark binding to target genes. Altogether, our results revealed an unexpected function of Pcgf1 in gene activation during ES cell maintenance.

2017-06-13 | GSE95383 | GEO

Next-Generation Sequencing Facilitates Quantitative Analysis of WT, Ring1a-/-, Ring1b-/-, Ring1a/b-/-, Pcgf1/3/5/6-/- and Pcgf1/2/3/4/5/6-/- Embryonic Stem Cells Transcriptomes

Project description:Polycomb repressive complexes (PRC) are epigenetic transcriptional repressors by establishing facultative heterochromatin repressive domains and play an essential role in numerous developmental processes. PRC1, which contains the E3 ligases RING1A/B and deposits H2AK119ub, forms six biochemically subcomplexes (PRC1.1-PRC1.6) based on the assembled PCGF protein (PCGF1–PCGF6), and has been involved in the maintenance of embryonic stem (ES) cell state. More recent evidence indicates that PCGF family participates in the self-renewal and pluripotent maintenance. However, it is still enigmatic that how the PCGF family engaged in the controlling process. Here, we generate the Ring1a-/-, Ring1b-/-, Ring1a/b-/-, Pcgf1/3/5/6-/- and Pcgf1/2/3/4/5/6-/- ES cells using CRISPR/Cas9 technique and CRE-LoxP recombination system to discover the central determinants in PCGF family-related biological process.

2022-09-01 | GSE183646 | GEO

Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding [expression array data]

Project description:The t(8;21) translocation fuses the DNA binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape we measured genome-wide RUNX1- and RUNX1/ETO bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide re-distribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML. Total RNA were obtained using 8 samples over four time courses (mismatch control and knock-down)

2012-03-27 | E-GEOD-29223 | biostudies-arrayexpress

Ubiquitin E3 ligase FBXO21 regulates cytokine mediated signaling pathways, but is dispensable for steady-state hematopoiesis

Project description:Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways. One pathway, the ubiquitin proteasome system (UPS), controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. Ubiquitin E3 ligases serve as the substrate-recognition component of the UPS. Through investigating the FBOX family of E3 ligases, we discovered that Fbxo21 was highly expressed in the hematopoietic stem and progenitor cell (HSPC) population, and showed low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a hematopoietic specific Fbxo21 conditional knockout (cKO) mouse model. We found that silencing FBXO21 in HSPCs led to a loss in colony formation and an increase in cell differentiation in vitro. Additionally, stressing the HSPC populations in our Fbxo21 cKO mouse with 5-FU injections resulted in a decrease in survival, despite these populations showing minimal alterations during steady-state hematopoiesis. Although FBXO21 has previously been proposed to regulate cytokine signaling via ASK and p38, our results show that depletion of FBXO21 led to altered ERK signaling in vitro. Together, these findings suggest ubiquitin E3 ligase FBXO21 regulates HSPCs through cytokine mediated pathways.

2022-11-11 | PXD035283 | Pride

Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding (Illumina expression)

Project description:The t(8;21) translocation fuses the DNA binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape we measured genome-wide RUNX1- and RUNX1/ETO bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide re-distribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML. Total RNA were obtained using 8 samples over a time course of 10 days or using two samples two days after siRNA electroporation (mismatch control siRNA and RUNX1/ETO knock-down)

2012-03-27 | E-GEOD-34594 | biostudies-arrayexpress

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