Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis.
ABSTRACT: During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66?, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.
Project description:An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate ?-type globin gene disorders such as sickle cell anemia and ?-thalassemia through activation of the fetal ?-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD, play a role in ?-globin gene silencing, and Mi2? (CHD4) is a critical component of NuRD complexes. We observed that knockdown of Mi2? relieves ?-globin gene silencing in ?-YAC transgenic murine chemical inducer of dimerization hematopoietic cells and in CD34(+) progenitor-derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2? binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for ?-globin gene silencing during ?-type globin gene switching. Remarkably, <50% knockdown of Mi2? is sufficient to significantly induce ?-globin gene expression without disrupting erythroid differentiation of primary human CD34(+) progenitors. These results indicate that Mi2? is a potential target for therapeutic induction of fetal hemoglobin.
Project description:Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal β-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiled-coil interaction between MBD2 and p66α, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.
Project description:BACKGROUND: ADNP is vital for embryonic development. Is this function conserved for the homologous protein ADNP2? RESULTS: Down-regulation/silencing of ADNP or ADNP2 in zebrafish embryos or mouse erythroleukemia cells inhibited erythroid maturation, with ADNP directly associating with the ?-globin locus control region. CONCLUSION: ADNPs are novel molecular regulators of erythropoiesis. SIGNIFICANCE: New regulators of globin synthesis are suggested. Activity-dependent neuroprotective protein (ADNP) and its homologue ADNP2 belong to a homeodomain, the zinc finger-containing protein family. ADNP is essential for mouse embryonic brain formation. ADNP2 is associated with cell survival, but its role in embryogenesis has not been evaluated. Here, we describe the use of the zebrafish model to elucidate the developmental roles of ADNP and ADNP2. Although we expected brain defects, we were astonished to discover that the knockdown zebrafish embryos were actually lacking blood and suffered from defective hemoglobin production. Evolutionary conservation was established using mouse erythroleukemia (MEL) cells, a well studied erythropoiesis model, in which silencing of ADNP or ADNP2 produced similar results as in zebrafish. Exogenous RNA encoding ADNP/ADNP2 rescued the MEL cell undifferentiated state, demonstrating phenotype specificity. Brg1, an ADNP-interacting chromatin-remodeling protein involved in erythropoiesis through regulation of the globin locus, was shown here to interact also with ADNP2. Furthermore, chromatin immunoprecipitation revealed recruitment of ADNP, similar to Brg1, to the mouse ?-globin locus control region in MEL cells. This recruitment was apparently diminished upon dimethyl sulfoxide (DMSO)-induced erythrocyte differentiation compared with the nondifferentiated state. Importantly, exogenous RNA encoding ADNP/ADNP2 significantly increased ?-globin expression in MEL cells in the absence of any other differentiation factors. Taken together, our results reveal an ancestral role for the ADNP protein family in maturation and differentiation of the erythroid lineage, associated with direct regulation of ?-globin expression.
Project description:The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human ?-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the ?-globin locus control region and ?-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal ?-globin/LCR looping and ?-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing ?-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.
Project description:Erythropoietic and megakaryocytic programs are specified from multipotential progenitors by the transcription factor GATA1. FOG1, a GATA1-interaction partner, is critical for GATA1 function in several contexts by bringing multiple complexes into association with GATA1 to facilitate activation or repression of target genes. To further elucidate regulation of these associations by cellular and extracellular cues, we examined FOG1 for post-translational modifications. We found that FOG1 is SUMOylated and phosphorylated in erythroid cells in a differentiation-dependent manner. Removal of the SUMOylation sites in FOG1 does not impair nuclear localization, protein stability, or chromatin occupancy. However, SUMOylation of FOG1 modulates interactions with C-terminal binding protein family members, specifically promoting CTBP1 binding. Phosphorylation of FOG1 modulates SUMOylation and, therefore, indirectly regulates the CTBP interaction. Post-translational modification of FOG1 may contribute to control of co-occupancy by CTBP family members, the NuRD complex, and GATA1 at differentially regulated genes.
Project description:The chicken embryonic beta-type globin gene, rho, is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated rho-globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zinc-finger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pull-down assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive rho-globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active beta(A)-globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent rho-globin gene in vivo. Knock-down of MBD2 resulted in up-regulation of a methylated rho-gene construct in mouse erythroleukemic (MEL)-rho cells. These results represent the first purification of a MeCP1-like complex from a primary cell source and provide support for a role for MBD2 in developmental gene regulation.
Project description:The epigenetic code of DNA methylation is interpreted chiefly by methyl cytosine binding domain (MBD) proteins which in turn recruit multiprotein co-repressor complexes. We previously isolated one such complex, MBD2-NuRD, from primary erythroid cells and have shown it contributes to embryonic/fetal ?-type globin gene silencing during development. This complex has been implicated in silencing tumor suppressor genes in a variety of human tumor cell types. Here we present structural details of chicken MBD2 bound to a methylated DNA sequence from the ?-globin promoter to which it binds in vivo and mediates developmental transcriptional silencing in normal erythroid cells. While previous studies have failed to show sequence specificity for MBD2 outside of the symmetric mCpG, we find that this domain binds in a single orientation on the ?-globin target DNA sequence. Further, we show that the orientation and affinity depends on guanine immediately following the mCpG dinucleotide. Dynamic analyses show that DNA binding stabilizes the central ?-sheet, while the N- and C-terminal regions of the protein maintain mobility. Taken together, these data lead to a model in which DNA binding stabilizes the MBD2 structure and that binding orientation and affinity is influenced by the DNA sequence surrounding the central mCpG.
Project description:During erythroid development, the embryonic ε-globin gene becomes silenced as erythropoiesis shifts from the yolk sac to the fetal liver where γ-globin gene expression predominates. Previous studies have shown that the ε-globin gene is autonomously silenced through promoter proximal cis-acting sequences in adult erythroid cells. We have shown a role for the methylcytosine binding domain protein 2 (MBD2) in the developmental silencing of the avian embryonic ρ-globin and human fetal γ-globin genes. To determine the roles of MBD2 and DNA methylation in human ε-globin gene silencing, transgenic mice containing all sequences extending from the 5' hypersensitive site 5 (HS5) of the β-globin locus LCR to the human γ-globin gene promoter were generated. These mice show correct developmental expression and autonomous silencing of the transgene. Either the absence of MBD2 or treatment with the DNA methyltransferase inhibitor 5-azacytidine increases ε-globin transgene expression by 15-20 fold in adult mice. Adult mice containing the entire human β-globin locus also show an increase in expression of both the ε-globin gene transgene and endogenous ε(Y) and β(H1) genes in the absence of MBD2. These results indicate that the human ε-globin gene is subject to multilayered silencing mediated in part by MBD2.
Project description:Haem-regulated eIF2alpha kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression.
Project description:The heterogeneous collection of nucleosome remodelling and deacetylation (NuRD) complexes can be grouped into the MBD2- or MBD3-containing complexes MBD2-NuRD and MBD3-NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here, we show that MBD2-NuRD, in contrast to MBD3-NuRD, converts open chromatin with euchromatic histone modifications into tightly compacted chromatin with repressive histone marks. Genome-wide, a strong enrichment for MBD2 at methylated CpG sequences is found, whereas CpGs bound by MBD3 are devoid of methylation. MBD2-bound genes are generally lower expressed as compared with MBD3-bound genes. When depleting cells for MBD2, the MBD2-bound genes increase their activity, whereas MBD2 plus MBD3-bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes.