The Hbo1-Brd1/Brpf2 HAT Complex Is Required for Erythropoiesis In Fetal Liver

Abstract Abstract 3872 Bromodomain-containing protein 1 (Brd1, initially designated as BR140-LIKE; BRL) contains a bromodomain, two plant homology domain (PHD) zinc fingers, and a proline-tryptophan-tryptophan-proline (PWWP) domain, three types of modules characteristic of chromatin regulators. Recently, BRD1 appeared to belong to the BRPF family which includes BRPF1, BRD1/BRPF2, and BRPF3. Among them, BRPF1 is known to be a subunit of the MOZ H3 histone acetyltransferase (HAT) complex. BRD1 has been proposed to be additional subunit of the MOZ H3 HAT complex on the analogy of BRPF1. However, its molecular function remains elusive. To elucidate the biological functions of BRD1, we generated Brd1-null mice and found that they die in utero. Brd1-/- embryos were alive and recovered at nearly the expected Mendelian ratio at 12.5 days postcoitum (dpc) but died by 15.5 dpc. Brd1-/- embryos at 12.5 dpc were pale and the cell number of fetal livers, in which fetal hematopoiesis occurs, was decreased to about 20% of the control. Cytological analysis revealed that Brd1-/- fetal livers had profoundly fewer erythroblasts at maturation stages beyond proerythroblasts compared to wild-type fetal livers. Flow cytometric analysis of Brd1-/- fetal livers revealed a significant accumulation of CD71+Ter119- proerythroblasts and a reduction in CD71+Ter119+ and CD71-Ter119+ maturating erythroblasts. A drastic increase in AnnexinV+ apoptotic cells was detected in the CD71+Ter119+ and CD71-Ter119- cell fractions in Brd1-/- fetal livers. These findings suggested that severe anemia caused by compromised differentiation and/or survival of erythroblasts accounts for embryonic lethality of Brd1-/- embryos. To understand the mechanism underlying defective erythropoiesis in Brd1-null embryos, we performed biochemical analyses and found that Brd1 bridges the HAT, HBO1 but not MOZ, and its activator protein, ING4, to form an enzymatically active HAT complex. Forced expression of Brd1 promoted erythroid differentiation of K562 cells, while Brpf1, which preferentially binds to MOZ, had no significant effect. Correspondingly, depletion of Hbo1 by Hbo1 knockdown perturbed erythroid differentiation of mouse fetal liver progenitors. Of note, the level of global acetylation of histone H3 at lysine 14 (H3K14) was specifically decreased in Brd1-deficient erythroblasts. These results collectively implied that acetylation of H3K14 catalyzed by the Hbo1-Brd1 complex has a crucial role in fetal liver erythropoiesis. To identify the downstream targets for the HBO1-BRD1 complex, we performed the ChIP-on-chip analysis in K562 cells and found that BRD1 and HBO1 largely co-localize on the genome, especially on the promoters of erythroid transcription factor genes. ChIP analysis revealed that acetylation of H3K14 at the promoters of erythroid transcription factor genes, including Gata1, Gata2, Tal1, Stat5a, and ETO2, were profoundly diminished in the Brd1-deficient erythroblasts. Among these target genes, we focused on Gata1, which plays a central role in erythropoiesis, and carried out complementation experiments with Gata1 using a Gata1 retrovirus. Exogenous Gata1, but not Bcl-xL, efficiently improved proliferative capacity and survival of Brd1-deficient erythroid progenitors and also restored, at least partially, their impaired differentiation. These results clearly showed that the Hbo1-Brd1 complex is required for the acetylation of H3K14 at the promoters of erythroid transcription factor genes, thereby is crucial for erythropoiesis in fetal liver. Disclosures: No relevant conflicts of interest to declare.

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Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)

Blood ◽

10.1182/blood.v120.21.982.982 ◽

2012 ◽

Vol 120 (21) ◽

pp. 982-982

Author(s):

Tohru Fujiwara ◽

Haruka Saitoh ◽

Yoko Okitsu ◽

Noriko Fukuhara ◽

Yasushi Onishi ◽

...

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

Conflicts Of Interest ◽

K562 Cells ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Mrna Levels ◽

Wide Range ◽

The Impact ◽

Chip Analysis

Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.

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A Positive Regulatory Feedback Loop Between 1 EKLF/ KLF1 and TAL1/SCL2 Sustaining the Erythropoiesis

10.1101/2021.04.08.439008 ◽

2021 ◽

Author(s):

Chun-Hao Hung ◽

Yu-Szu Huang ◽

Tung-Liang Lee ◽

Kang-Chung Yang ◽

Yu-Chiau Shyu ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Feedback Loop ◽

Target Genes ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Cellular Analysis ◽

Direct Target ◽

Regulatory Feedback Loop

The erythroid Krppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we have identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with molecular/ cellular analysis of mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tall gene promoter in combination with in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates Tall gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors that sustains the mammalian erythropoiesis.

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Stage Selective Requirement For The N-Terminus Of GATA1 During Erythropoiesis

Blood ◽

10.1182/blood.v122.21.3670.3670 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3670-3670

Author(s):

Monika J Stankiewicz ◽

Timothy M Chlon ◽

John D Crispino

Keyword(s):

Down Syndrome ◽

Target Genes ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

Erythroid Differentiation ◽

Full Length ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Megakaryoblastic Leukemia ◽

Exclusive Production

Abstract The X-linked blood transcription factor GATA1 is required for the survival and maturation of erythroid cells. Loss of Gata1 causes profound anemia and related mid-gestation lethality in mouse models and human GATA1 mutations are associated with congenital dyserythropoietic anemia, congenital thrombocytopenia, porphyria, Diamond-Blackfan anemia (DBA) and acute megakaryoblastic leukemia in children with Down syndrome (DS-AMKL). In DBA, DS-AMKL, and a subset of congenital anemia, GATA1 mutations cause the exclusive production of a shorter GATA1 isoform, termed GATA1s. The GATA1s isoform retains both zinc fingers and binds DNA and cofactors accordingly, but lacks the N-terminal 83 residues of full-length GATA1. Intriguingly, exclusive production of GATA1s promotes AMKL in the context of Down syndrome (trisomy 21), while disomic individuals harboring the same type of mutations suffer from anemia and severe red cell defects. A mutation causing exclusive production of Gata1s in mice does not appear to affect adult hematopoiesis, but has been shown to cause a marked expansion of megakaryocytes in the fetal liver. The effects of the replacement of Gata1 with Gata1s during fetal liver erythropoiesis, however, remain uncharacterized. To investigate the effects of exclusive Gata1s production during fetal hematopoiesis, we performed comprehensive phenotypic and mechanistic studies using Gata1s knock-in embryos. We found significant changes in myelo-erythroid differentiation beyond the known expansion of megakaryocytes. Flow cytometric analysis revealed altered erythroid differentiation at embryonic days 12.5 and 14.5, evidenced by decreased Ter119 and increased CD71 expression, characteristic of delayed erythroid maturation. Changes in expression of myelo-erythroid progenitor commitment markers were also discovered. Specifically, we observed marked decreases in pre-CFU-E and CFU-E committed progenitors and an increase in pre-GM and pre-MegE populations. Our findings are consistent with a bias towards megakaryopoiesis at the expense of erythroid commitment caused by the expression of Gata1s in place of full-length Gata1. A shift in differentiation was also observed in the embryonic granulocyte/ macrophage lineage, with an increased generation of macrophages with fewer developing granulocytes. Mechanistically, we found that expression of erythroid -specific Gata1 target genes such as Alas2, Slc4a1 and Klf1 are markedly reduced in the Gata1 knock-in erythroid cells, indicating that Gata1s is a less effective transcriptional activator than full-length Gata1. In particular, given that Klf1 functions to promote erythroid specification downstream of the megakaryocyte-erythrocyte progenitor, our results suggest that Gata1s may promote megakaryocyte differentiation at the expense of erythroid differentiation, in part, by failing to activate Klf1. Taken together, our studies demonstrate a stage-specific requirement for full-length Gata1 during embryonic erythropoiesis. Furthermore, erythroid defects associated with exclusive production of Gata1s in humans may result from an incomplete activation of the erythroid transcriptional program. Disclosures: Crispino: Sanofi: Research Funding.

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A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis

International Journal of Molecular Sciences ◽

10.3390/ijms22158024 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8024

Author(s):

Chun-Hao Hung ◽

Tung-Liang Lee ◽

Anna Yu-Szu Huang ◽

Kang-Chung Yang ◽

Yu-Chiau Shyu ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Feedback Loop ◽

Target Genes ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Cellular Analysis ◽

Direct Target ◽

Regulatory Feedback Loop

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf−/−) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.

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Mef2C Is a Lineage-Restricted Target Gene of Scl/Tal1 and Regulates Megakaryopoiesis and B-Cell Homeostasis

Blood ◽

10.1182/blood.v112.11.278.278 ◽

2008 ◽

Vol 112 (11) ◽

pp. 278-278

Author(s):

Katrin E Rhodes ◽

Christos Gekas ◽

Laurraine Gereige ◽

Hildur Helgadottir ◽

Roberto Ferrari ◽

...

Keyword(s):

Transcription Factor ◽

B Cell ◽

Muscle Development ◽

Target Genes ◽

Fetal Liver ◽

Premature Aging ◽

Bhlh Transcription Factor ◽

Functional Requirements ◽

B Lymphoid ◽

Deficient Mice

Abstract The bHLH transcription factor stem cell leukemia/T-cell acute leukemia gene (Scl/Tal1) is a master regulator for hematopoiesis, essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. To identify Scl target genes in hematopoietic cells, we performed gene expression analysis on HOX11-immortalized Sclfl/fl fetal liver cell lines. Analysis of the top 50 downregulated genes revealed several genes related to hematopoiesis including erythroid and megakaryocyte development, vasculogenesis, as well as genes/unknown ESTs that have not been previously linked to blood development. One of the top downregulated genes was transcription factor myocyte enhancer factor 2C (Mef2C). Mef2C−/− embryos die at E9.5, the same time as Scl−/− embryos, and exhibit severe defects in cardiac and muscle development. Analysis of Mef2C−/− embryos showed that, Mef2C, in contrast to Scl, is not required for specification into primitive or definitive hematopoietic lineages. To bypass the embryonic lethality, we utilized a conditionally targeted Mef2Cfl/fl strain and crossed it with a hematopoietic cell-specific VavCre strain that deactivates Mef2C shortly after the emergence of HSCs. Interestingly, adult VavCre+Mef2Cfl/fl mice exhibited severe platelet defects highly reminiscent to those observed in Scl deficient mice. The platelet counts were reduced, while platelet size was increased and the platelet shape and granularity was altered. Furthermore, megakaryopoiesis was severely impaired in vitro. ChIP-on-chip analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reductions of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.

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Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.1462.1462 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1462-1462

Author(s):

Michael Tallack ◽

Thomas Whitington ◽

Brooke Gardiner ◽

Eleanor Wainwright ◽

Janelle Keys ◽

...

Keyword(s):

Regulatory Networks ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Es Cells ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Gene Promoters ◽

Erythroid Cells ◽

Red Cell Membrane ◽

Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

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Hypoxia-Inducible Factor 1-Mediated Human GATA1 Induction Promotes Erythroid Differentiation Under Hypoxic Conditions

Blood ◽

10.1182/blood.v118.21.4794.4794 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4794-4794

Author(s):

Jun-Wu Zhang ◽

Feng-Lin Zhang ◽

Guo-Min Shen ◽

Xiao-Ling Liu ◽

Fang Wang

Keyword(s):

Rna Interference ◽

Conflicts Of Interest ◽

Hypoxia Inducible Factor ◽

K562 Cells ◽

Erythroid Differentiation ◽

Flanking Sequence ◽

Differentiation Markers ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Hypoxic Conditions

Abstract Abstract 4794 The stimulation of red blood cell (RBC) production is one of the systemic adaptions to hypoxia. Hypoxia-inducible factor (HIF) promotes erythropoiesis through coordinated cell type-specific hypoxia responses. Hematopoietic transcription factor GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells (HPCs) derived from human cord blood. Enforced HIF1Á expression increased GATA1 expression, while HIF1Á knock-down by RNA interference decreased GATA1 expression in K562 cells. We searched the human GATA1 gene sequence on NCBI and identified a putative HRE in the 3'-flanking sequence of the gene. The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1Á or GATA1. Flow cytometry analysis also indicated that hypoxia or desferrioxamine or CoCl2 induced expression of erythroid surface marker CD71 and CD235a, while expression repression of HIF1Á or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 upregulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. Disclosures: No relevant conflicts of interest to declare.

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C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽

10.1182/blood.v120.21.650.650 ◽

2012 ◽

Vol 120 (21) ◽

pp. 650-650

Author(s):

Cailin Collins ◽

Jingya Wang ◽

Joel Bronstein ◽

Jay L. Hess

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Target Genes ◽

De Novo ◽

Conflicts Of Interest ◽

Epigenetic Modifications ◽

Conditional Knockout ◽

Motif Analysis ◽

Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

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ETO-2 Associates with SCL in Erythroid Cells and Megakaryocytes and Provides Repressor Functions in Erythropoiesis

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10235-10250.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10235-10250 ◽

Cited By ~ 101

Author(s):

Anna H. Schuh ◽

Alex J. Tipping ◽

Allison J. Clark ◽

Isla Hamlett ◽

Boris Guyot ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Protein Complexes ◽

Fetal Liver ◽

Regulation Of Gene Expression ◽

Erythroid Differentiation ◽

Cell Types ◽

Erythroid Cells ◽

Proteomic Approach ◽

Endogenous Proteins

ABSTRACT Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

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Gfi1 Upregulates c-Myc Expression and Promotes c-Myc-Driven Cell Proliferation

Blood ◽

10.1182/blood-2019-124833 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3769-3769

Author(s):

Yangyang Zhang ◽

Fan Dong

Keyword(s):

Cell Proliferation ◽

Transcription Factor ◽

Transcriptional Repression ◽

Cell Cycle Progression ◽

Target Genes ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Protein Levels ◽

Enhanced Expression ◽

Ubiquitin Proteasome

Gfi1 is a zinc-finger transcriptional repressor that plays an important role in hematopoiesis. When aberrantly activated, Gfi1 may function as a weak oncoprotein in the lymphoid system, but collaborate strongly with c-Myc in lymphomagenesis. c-Myc is a transcription factor that is frequently activated in human cancers including leukemia and lymphoma mainly due to its overexpression as a result of gene amplifications and chromosomal translocations. c-Myc overexpression may also result from stabilization of c-Myc protein, which is highly unstable and rapidly degraded through the ubiquitin-proteasome pathway. The mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis is incompletely understood. c-Myc activates gene expression by forming a heterodimeric complex with the partner protein Max, but may also repress target genes through interaction with transcription factor Miz-1. We previously showed that Gfi1 indirectly interacts with c-Myc through Miz-1 and collaborates with c-Myc to repress CDK inhibitors p21Cip1 and p15Ink4B. In this study, we show that Gfi1 augmented the level of c-Myc protein transiently expressed in Hela cells and the levels of MycER fusion protein stably expressed in the mouse pro-B Ba/F3 and myeloid 32D cells. The C-terminal ZF domains of Gfi1, but not its transcriptional repression and DNA binding activities, were required for c-Myc upregulation. Notably, although Miz-1 has been shown to stabilize c-Myc protein, the expression of c-Myc V394D mutant, which is defective in Miz-1 interaction, was still upregulated by Gfi1, suggesting that Gfi1-mediated c-Myc upregulation was independent of Miz-1 interaction. We further show that Gfi1 overexpression led to reduced polyubiquitination and increased stability of c-Myc protein. Interestingly, the levels of endogenous c-Myc mRNA and protein were augmented upon induction of Gfi1 expression in Ba/F3 and Burkitt lymphoma Ramos cells transduced with the doxycycline-inducible Gfi1 lentiviral construct, but reduced in Gfi1-knocked down human leukemic HL60 and U937 cells. Additionally, targeted deletion of Gfi1 resulted in reduced c-Myc expression in mouse lineage negative bone marrow cells, which was associated with a decline in the expression of c-Myc-activated target genes. The oncogenic potential of Myc derives from its ability to stimulate cell proliferation. Our results demonstrate that inducible expression of Gfi1 in Ba/F3 cells expressing MycER promoted Myc-driven cell cycle progression and proliferation. Thus, in addition to its role in c-Myc-mediated transcriptional repression, Gfi1 upregulates c-Myc expression at both mRNA and protein levels, leading to enhanced expression of c-Myc-activated genes and augmented cell proliferation driven by c-Myc. Together, these data may reveal a novel mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

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