MBD2a-NuRD Binds to the Methylated γ-Globin Gene Promoter and Uniquely Forms a Complex Required for Silencing of HbF Expression
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ABSTRACT:
PROVIDER: PRJNA932758 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23372466.fastq.gz | Fastqsanger.gz | |||
| SRR23372467.fastq.gz | Fastqsanger.gz | |||
| SRR23372468.fastq.gz | Fastqsanger.gz | |||
| SRR23372469.fastq.gz | Fastqsanger.gz | |||
| SRR23372470.fastq.gz | Fastqsanger.gz |
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Project description:During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report we present direct evidence that MBD2- NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine binding domain of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings 2 support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5 and DNA methylation in HbF silencing.
2023-06-28 | GSE224834 | GEO
Project description:During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report, we present direct evidence that MBD2-NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine-binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5, and DNA methylation in HbF silencing.
| S-EPMC10288633 | biostudies-literature
Project description:As high fetal hemoglobin (HbF) levels ameliorate the underlying pathophysiologic defects in sickle cell anemia and β-thalassemia, understanding the mechanisms that enforce silencing of HbF postnatally offers the promise of effective molecular therapy. Depletion of Methyl cytosine Binding Domain protein 2 (MBD2) causes a 10-20 fold increase in γ-globin gene expression in adult β-YAC transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, CRISPR-Cas9 mediated knockout (KO) was carried out in Human Umbilical cord Derived Erythroid Progenitor-2 (HUDEP-2) erythroid cells resulting in γ/γ+β mRNA levels of ~50% and ~40% HbF by HPLC. In contrast, MBD3 KO had no appreciable effect on γ-globin mRNA. Knockdown (Kd) of MBD2 in primary adult erythroid cells consistently increased γ/γ+β mRNA ratios by ~10-fold resulting in ~30-40% γ/γ+β mRNA levels and a corresponding increase in γ-globin protein. MBD2 exerts its repressive effects through recruitment of the CHD4 chromatin remodeling component of NuRD through a coiled-coil (CC) domain, and the histone deacetylase (HDCC) component via an intrinsically disordered region (IDR). Enforced expression of wild-type MBD2 in MBD2KO HUDEP-2 cells caused a 5-fold decrease in γ-globin mRNA while neither the CC mutant nor the IDR mutant MBD2 proteins had an inhibitory effect. Consistently, co-immunoprecipitation assays showed that the CC and IDR domain mutations disrupt NuRD complex formation by dissociating the CHD4 and HDCC domains respectively. These results establish MBD2-NuRD as a major silencer of HbF in human erythroid cells and point to the CC and IDR domains of MBD2-NuRD as potential therapeutic targets.
2020-01-27 | GSE121992 | GEO
Project description:Hemoglobinopathies, including sickle cell disease and _-thalassemia, are global public health concerns. Induction of fetal-type hemoglobin (HbF) is a promising means to treat these disorders; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Here, we show that the LRF/ZBTB7A transcription factor is a potent repressor of HbF production. LRF inactivation derepresses embryonic/fetal _-globin expression in mouse and human adult erythroid cells. We employed genome-wide analysis of the transcriptome, chromatin accessibility and LRF occupancy sites, and demonstrate that LRF occupies the _-globin loci and maintains nucleosome density necessary for _-globin silencing. LRF confers its repressive activity through a unique NuRD repressor complex independent of BCL11A. Strikingly, human erythroid lines lacking both LRF and BCL11A exhibited almost a complete switch in expression from adult- to fetal-type globin, suggesting that these two factors cumulatively represent the near entirety of _-globin repressive activity in adult erythroid cells. RNA-seq, LRF ChIP-seq and ATAC-seq assays were used to investigtae LRF binding, effect of LRF depletion on transcription and chromatin landscape in mouse and human cells.
2016-01-15 | E-GEOD-74977 | biostudies-arrayexpress
Project description:ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression
| PRJNA648059 | ENA
Project description:Longstanding observations that fetal hemoglobin (HbF, a2g2) expression is reactivated in postnatal red blood cells (RBCs) during hypoxia or recovery from anemia remain unexplained. We identified the hypoxia inducible factor (HIF) signaling pathway as a direct regulator of HbF expression in a CRISPR/Cas9 genetic screen. In RBC precursors, depletion of the von Hippel–Lindau (VHL) E3 ligase stabilized its ubiquitination target HIF1a to induce g-globin gene transcription. Mechanistically, HIF1a-HIF1bheterodimers bound cognate DNA elements in BGLT3, a long-noncoding RNA gene located 2.7 kb downstream of the tandem g-globin genes. This was followed by recruitment of transcriptional activators, chromatin opening, and increased long-range interactions between the g-globin genes and their upstream enhancer. These effects were recapitulated by hypoxia or inhibition of prolyl hydroxylase domain (PHD) enzymes that target HIF1a for ubiquitination. Our findings link globin gene regulation with canonical hypoxia adaptation, elucidate a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies.
2022-07-14 | GSE204899 | GEO
Project description:Longstanding observations that fetal hemoglobin (HbF, a2g2) expression is reactivated in postnatal red blood cells (RBCs) during hypoxia or recovery from anemia remain unexplained. We identified the hypoxia inducible factor (HIF) signaling pathway as a direct regulator of HbF expression in a CRISPR/Cas9 genetic screen. In RBC precursors, depletion of the von Hippel–Lindau (VHL) E3 ligase stabilized its ubiquitination target HIF1a to induce g-globin gene transcription. Mechanistically, HIF1a-HIF1bheterodimers bound cognate DNA elements in BGLT3, a long-noncoding RNA gene located 2.7 kb downstream of the tandem g-globin genes. This was followed by recruitment of transcriptional activators, chromatin opening, and increased long-range interactions between the g-globin genes and their upstream enhancer. These effects were recapitulated by hypoxia or inhibition of prolyl hydroxylase domain (PHD) enzymes that target HIF1a for ubiquitination. Our findings link globin gene regulation with canonical hypoxia adaptation, elucidate a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies.
2022-07-14 | GSE204897 | GEO
Project description:Longstanding observations that fetal hemoglobin (HbF, a2g2) expression is reactivated in postnatal red blood cells (RBCs) during hypoxia or recovery from anemia remain unexplained. We identified the hypoxia inducible factor (HIF) signaling pathway as a direct regulator of HbF expression in a CRISPR/Cas9 genetic screen. In RBC precursors, depletion of the von Hippel–Lindau (VHL) E3 ligase stabilized its ubiquitination target HIF1a to induce g-globin gene transcription. Mechanistically, HIF1a-HIF1bheterodimers bound cognate DNA elements in BGLT3, a long-noncoding RNA gene located 2.7 kb downstream of the tandem g-globin genes. This was followed by recruitment of transcriptional activators, chromatin opening, and increased long-range interactions between the g-globin genes and their upstream enhancer. These effects were recapitulated by hypoxia or inhibition of prolyl hydroxylase domain (PHD) enzymes that target HIF1a for ubiquitination. Our findings link globin gene regulation with canonical hypoxia adaptation, elucidate a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies.
2022-07-14 | GSE184459 | GEO
Project description:Longstanding observations that fetal hemoglobin (HbF, a2g2) expression is reactivated in postnatal red blood cells (RBCs) during hypoxia or recovery from anemia remain unexplained. We identified the hypoxia inducible factor (HIF) signaling pathway as a direct regulator of HbF expression in a CRISPR/Cas9 genetic screen. In RBC precursors, depletion of the von Hippel–Lindau (VHL) E3 ligase stabilized its ubiquitination target HIF1a to induce g-globin gene transcription. Mechanistically, HIF1a-HIF1bheterodimers bound cognate DNA elements in BGLT3, a long-noncoding RNA gene located 2.7 kb downstream of the tandem g-globin genes. This was followed by recruitment of transcriptional activators, chromatin opening, and increased long-range interactions between the g-globin genes and their upstream enhancer. These effects were recapitulated by hypoxia or inhibition of prolyl hydroxylase domain (PHD) enzymes that target HIF1a for ubiquitination. Our findings link globin gene regulation with canonical hypoxia adaptation, elucidate a mechanism for HbF induction during stress erythropoiesis, and identify a novel therapeutic approach for β-hemoglobinopathies.
2022-07-14 | GSE204900 | GEO
Project description:Hemoglobinopathies, including sickle cell disease and _-thalassemia, are global public health concerns. Induction of fetal-type hemoglobin (HbF) is a promising means to treat these disorders; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Here, we show that the LRF/ZBTB7A transcription factor is a potent repressor of HbF production. LRF inactivation derepresses embryonic/fetal _-globin expression in mouse and human adult erythroid cells. We employed genome-wide analysis of the transcriptome, chromatin accessibility and LRF occupancy sites, and demonstrate that LRF occupies the _-globin loci and maintains nucleosome density necessary for _-globin silencing. LRF confers its repressive activity through a unique NuRD repressor complex independent of BCL11A. Strikingly, human erythroid lines lacking both LRF and BCL11A exhibited almost a complete switch in expression from adult- to fetal-type globin, suggesting that these two factors cumulatively represent the near entirety of _-globin repressive activity in adult erythroid cells.
2016-01-15 | GSE74977 | GEO