Cancer sequencing studies have implicated regulators of pre-mRNA splicing as important disease determinants in acute myeloid leukemia (AML), but the underlying mechanisms have remained elusive. We ...hypothesized that "non-mutated" splicing regulators may also play a role in AML biology and therefore conducted an in vivo shRNA screen in a mouse model of CEBPA mutant AML. This has led to the identification of the splicing regulator RBM25 as a novel tumor suppressor. In multiple human leukemic cell lines, knockdown of RBM25 promotes proliferation and decreases apoptosis. Mechanistically, we show that RBM25 controls the splicing of key genes, including those encoding the apoptotic regulator BCL-X and the MYC inhibitor BIN1. This mechanism is also operative in human AML patients where low RBM25 levels are associated with high MYC activity and poor outcome. Thus, we demonstrate that RBM25 acts as a regulator of MYC activity and sensitizes cells to increased MYC levels.
Transcription factors PU.1 and CEBPA are required for the proper coordination of enhancer activity during granulocytic-monocytic (GM) lineage differentiation to form myeloid cells. However, precisely ...how these factors control the chronology of enhancer establishment during differentiation is not known. Through integrated analyses of enhancer dynamics, transcription factor binding, and proximal gene expression during successive stages of murine GM-lineage differentiation, we unravel the distinct kinetics by which PU.1 and CEBPA coordinate GM enhancer activity. We find no evidence of a pioneering function of PU.1 during late GM-lineage differentiation. Instead, we delineate a set of enhancers that gain accessibility in a CEBPA-dependent manner, suggesting a pioneering function of CEBPA. Analyses of Cebpa null bone marrow demonstrate that CEBPA controls PU.1 levels and, unexpectedly, that the loss of CEBPA results in an early differentiation block. Taken together, our data provide insights into how PU.1 and CEBPA functionally interact to drive GM-lineage differentiation.
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•Characterization of the enhancer landscape during GM-lineage differentiation•CEBPA controls PU.1 levels•PU.1 binding to enhancers is diminished in Cebpa null GM progenitors•Loss of CEBPA leads to a differentiation block upstream of CMPs/preGMs
Granulocytic-monocytic differentiation is governed by the PU.1 and CEBPA transcription factors (TFs). Here, Pundhir et al. use histone and TF ChIP-seq to demonstrate an unexpected role for CEBPA in controlling PU.1 function. They show that loss of CEBPA results in an early GM lineage differentiation block upstream of CMPs/preGMs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific ...transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of Cdk1/2/4 inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements.Here the authors show that differentiation of haematopoietic stem cells into mature blood cells is primed by cell type-specific transcription factors at the enhancer level during early differentiation, before they confere promoter-driven growth arrest, and activate post-mitotic terminal differentiation.
Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) ...suppressor of variegation 3-9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP. Indeed, loss of H3K9me2 correlated with the activation of key interferon pathway genes, and interference with the activities of G9A/GLP largely phenocopied loss of SUV39H1. Last, we demonstrate that inhibition of G9A/GLP synergized with DNA demethylating agents and that SUV39H1 constitutes a potential biomarker for the response to hypomethylation treatment. Collectively, we uncovered a clinically relevant role for H3K9me2 in safeguarding cancer cells against activation of the interferon pathway.
Hematopoiesis relies on a well-regulated balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) to maintain the lifelong regeneration of blood cells. This homeostasis is ...highly dependent on regulated gene expression both at transcriptional and post-transcriptional levels. Epigenetic regulation has been shown to significantly affect regulation and maintenance of HSC potential. Through an shRNA screen, we were able to identify KDM5C, a H3K4me2/3-demethylase, as a tumour suppressor in acute myeloid leukemia. However, the role of KDM5C in normal hematopoiesis has yet to be investigated.
To explore this question, we generated a conditional Kdm5c knock-out (KO) mouse. Preliminary data shows that loss of Kdm5c perturbs normal hematopoiesis via reduced HSC-fitness. This project aims to gain insight into the mechanisms underlying this phenotype. However, loss of HSCs resulting from Kdm5c-KO, limits the amount material available for downstream analysis, such as ChIPseq, ATACseq etc. To counteract this limitation, we have successfully expanded Kdm5c-WT and -KO HSCs ex vivo. Although both Kdm5c-KO and WT cells were able to expand, expanded KO HSCs presented significantly higher numbers of apoptotic cells, thus reflecting the above-mentioned reduced in vivo fitness. Additionally, upon transplantation, cultured Kdm5c KO cells display significantly reduced engraftment capacity.
Briefly, in our study, we use a promising tool to elucidate the role of KDM5C in HSC regulation in normal hematopoiesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific ...transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of
Cdk1/2/4
inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements.
The CCATT/enhancer binding protein alpha, C/EBPα, is a key transcription factor involved in late differentiation events of several cell types. Besides acting as a classical transcription factor, ...C/EBPα is also a well-characterized inhibitor of mitotic growth in most cell lines tested. In line with its anti-mitotic properties, C/EBPα has been shown to interact with, and alter the activities of, several cell cycle related proteins and a number of models as to the mechanistics of C/EBPα-mediated growth repression have been proposed. More recently, several reports have indicated that C/EBPα acts as a tumour suppressor in the hematopoietic system and that mutation within C/EBPα is sufficient to induce tumourigenesis. Here, we will review these data and probe the possibility that C/EBPα also act as a tumour suppressor in other C/EBPα-expressing tissues.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic ...control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.
•JMJD1C is required for leukemia maintenance.•JMJD1C is a potential therapeutic target in leukemia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting ...AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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