HIC2 Controls Developmental Hemoglobin Switching By Repressing BCL11A Transcription

Abstract One of the oldest and most deeply studied problems in developmental gene expression is the switch from fetal to adult type hemoglobin production in red blood cell precursors. Interest in this question has been fueled by its relevance to genetic blood disorders such as sickle cell disease (SCD) and thalassemia. BCL11A is a transcriptional repressor that is thought to directly silence the fetal β-type globin (HBG1/2) genes in adult erythroid cells. Transcriptome and RNA polymerase II profiling indicate that the BCL11A gene is transcribed considerably more highly in adult erythroblasts compared to fetal cells, accounting in large part for corresponding changes in BCL11A protein levels. Yet, the mechanism governing BCL11A developmental regulation is still unclear. To identify novel regulators of the fetal-to-adult globin switch, we interrogated our recent CRISPR based genetic screens that employed single guide RNAs (sgRNAs) targeting transcription factors (Huang et al., Blood, 2020) and uncovered HIC2, a penta-dactyl zinc finger DNA binding protein bearing a BTB/POZ domain as a novel regulator of hemoglobin switching. HIC2 is expressed more highly in fetal erythroblasts compared to adult cells, a pattern inverse to that of BCL11A. Overexpression (OE) of HIC2 in the adult type erythroid HUDEP2 cell line stimulated the expression of 322 genes while impairing that of 224 genes (FDR < 0.01 and fold change ≥ 2). The most highly upregulated genes (>150-fold) were HBG1/2. Upregulation was accompanied by gains in chromatin accessibility and histone H3K27acetylation of HBG1/2, and increased chromatin contacts between the distal globin gene enhancer (LCR) and the HBG1/2 genes. Overexpression of HIC2 in primary human erythroblasts also significantly increased HBG1/2 mRNA and protein levels, sufficient to reduce cell sickling in SCD patient-derived erythroid cells. HIC2 OE lowered BCL11A mature and pre-mRNA production, indicating that HIC2 attenuates BCL11A transcription. Forced expression of BCL11A restored HBG1/2 silencing in HIC2 OE HUDEP2 cells, suggesting that BCL11A repression accounts for the effects of HIC2 on fetal globin genes. ChIP-seq revealed a strong HIC2 binding peak at the erythroid BCL11A +55 enhancer. HIC2 OE reduced chromatin accessibility and H3K27acetylation of the +55 enhancer, as well as the enhancer-promoter contacts, suggesting that HIC2 directly decommissions the enhancer to attenuate BCL11A transcription. The BCL11A +55 enhancer contains two consensus HIC2 binding motifs under the HIC2 peak adjacent to GATA:E-box and GATA motifs. CRISPR-mediated mutagenesis of both HIC2 motifs raised BCL11A basal level transcription and diminished the ability of overexpressed HIC2 to repress BCL11A transcription. Notably, HIC2 OE impaired binding of transcription factor GATA1 at the +55 enhancer, suggesting that this enhancer is under developmental control. Indeed, GATA1 binding and chromatin accessibility of +55 enhancer were virtually undetectable in HUDEP1 cells, which represent a more fetal-like state. CRISPR-mediated depletion of HIC2 in HUDEP1 cells reversed this pattern with gains in GATA1 binding, chromatin accessibility, and BCL11A transcription. In sum, HIC2 emerges as a critical regulator of hemoglobin switching that operates by imposing developmental stage-specific control onto a BCL11A transcriptional enhancer. Disclosures Blobel: Fulcrum therapeutics: Consultancy; Pfizer: Research Funding.

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Controlling Long-Range Genomic Interactions to Reprogram the β-Globin Locus

Blood ◽

10.1182/blood.v120.21.280.280 ◽

2012 ◽

Vol 120 (21) ◽

pp. 280-280

Author(s):

Wulan Deng ◽

Jeremy W Rupon ◽

Hongxin Wang ◽

Andreas Reik ◽

Philip D. Gregory ◽

...

Keyword(s):

Long Range ◽

Globin Gene ◽

Chromatin Interaction ◽

Globin Genes ◽

Erythroid Cells ◽

Distal Enhancer ◽

Adult Type ◽

High Level ◽

Target Promoters

Abstract Abstract 280 Distal enhancers physically contact target promoters to confer high level transcription. At the mammalian β-globin loci long-range chromosomal interactions between a distal enhancer, called the locus control region (LCR), and the globin genes are developmentally dynamic such that the LCR contacts the embryonic, fetal and adult globin genes in a stage-appropriate fashion. LCR-globin gene interactions require the nuclear factor Ldb1. Recently, we employed artificial zinc finger (ZF) proteins to target Ldb1 to the endogenous β-globin locus to force an LCR-promoter interaction. This led to substantial activation of β-globin transcription and suggested that forced chromatin looping could be employed as a powerful tool to manipulate gene expression in vivo (Deng et al., Cell 2012). Reactivation of the fetal globin genes in adult erythroid cells has been a long-standing goal in the treatment of patients with sickle cell anemia. Therefore, building on our findings, we investigated whether the developmentally silenced embryonic globin gene βh1 can be re-activated in adult murine erythroblasts by re-directing the LCR away from the adult type globin gene and towards its embryonic counterpart. To this end, Ldb1 was fused to artificial ZF proteins (ZF-Ldb1) designed to bind to the βh1 promoter. ZF-Ldb1 was introduced into definitive erythroid cells in which only the adult but not the embryonic β-like globin gene is expressed. In vivo binding of the ZF-Ldb1 to its intended target was verified by chromatin immunoprecipitation assay. Strikingly, expression of ZF-Ldb1 re-activated βh1 transcription up to approximately ∼15% of total cellular β-globin production. This suggests that forced tethering of a looping factor to a select promoter can be employed to override a pre-existing developmental long-range chromatin interaction to reprogram a developmentally controlled gene locus. We are now in the process of testing whether our approach might be suitable to reactivate the silent fetal globin genes in adult human erythroid cells. These studies are underway and the results will be discussed at the meeting. Disclosures: Reik: Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment.

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Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes.

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1116 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1116-1125 ◽

Cited By ~ 19

Author(s):

C Perez-Stable ◽

F Costantini

Keyword(s):

Blood Cells ◽

Developmental Regulation ◽

Globin Gene ◽

Upstream Region ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Cis Acting ◽

Beta Globin Gene ◽

Gamma Globin

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.

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ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression

10.1101/2020.08.31.274324 ◽

2020 ◽

Author(s):

Xianjiang Lan ◽

Ren Ren ◽

Ruopeng Feng ◽

Lana C. Ly ◽

Yemin Lan ◽

...

Keyword(s):

Dna Binding ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

List Type ◽

Globin Genes ◽

Erythroid Cells ◽

Adult Type ◽

In Erythroid Cells

SummaryMetazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA binding protein that in human erythroid cells directly and measurably activates only one gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplant settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells whose perturbation might offer new therapeutic opportunities in the treatment of hemoglobinopathies.HighlightsA CRISPR screen implicates ZNF410 in fetal globin gene repressionThe CHD4 gene is the singular direct ZNF410 target in erythroid cellsThe fetal globin genes are exquisitely sensitive to CHD4 levelsFive C2H2 zinc fingers of ZNF410 recognize the major groove of a 14 base pair sequence

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Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1116-1125.1990 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1116-1125

Author(s):

C Perez-Stable ◽

F Costantini

Keyword(s):

Blood Cells ◽

Developmental Regulation ◽

Globin Gene ◽

Upstream Region ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Cis Acting ◽

Beta Globin Gene ◽

Gamma Globin

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Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7457-7468.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7457-7468

Author(s):

W L Trepicchio ◽

M A Dyer ◽

M H Baron

Keyword(s):

Developmental Regulation ◽

Regulatory Element ◽

Globin Gene ◽

Negative Control ◽

Erythroid Cell ◽

Globin Genes ◽

Erythroid Cells ◽

Specific Expression ◽

Systematic Analysis ◽

Specific Regulation

The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene.

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Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells

Blood ◽

10.1182/blood.v72.5.1536.bloodjournal7251536 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1536-1542

Author(s):

LJ Burns ◽

JG Glauber ◽

GD Ginder

Keyword(s):

Sodium Butyrate ◽

Transcriptional Activation ◽

Globin Gene ◽

Gene Activation ◽

Globin Genes ◽

Erythroid Cells ◽

Hemoglobin Switching ◽

High Level

An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis- regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.

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Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells

Blood ◽

10.1182/blood.v72.5.1536.1536 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1536-1542 ◽

Cited By ~ 32

Author(s):

LJ Burns ◽

JG Glauber ◽

GD Ginder

Keyword(s):

Sodium Butyrate ◽

Transcriptional Activation ◽

Globin Gene ◽

Gene Activation ◽

Globin Genes ◽

Erythroid Cells ◽

Hemoglobin Switching ◽

High Level

Abstract An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis- regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.

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Control of Human Hemoglobin Switching By LIN28B-Mediated Regulation of BCL11A Translation

Blood ◽

10.1182/blood-2018-99-117440 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 412-412

Author(s):

Anindita Basak ◽

Mathias Munschauer ◽

Jacob C. Ulirsch ◽

Kara E. Montbleau ◽

Christina R. Hartigan ◽

...

Keyword(s):

Sickle Cell Disease ◽

Protein Expression ◽

Sickle Cell ◽

Developmental Regulation ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Human Hemoglobin ◽

Erythroid Cells ◽

Cell Disease ◽

Hemoglobin Switching

Abstract Both sickle cell disease and β-thalassemia are major sources of morbidity and mortality worldwide. Continued production of the β-like γ-globin genes that form fetal hemoglobin after infancy has been shown to ameliorate the severity of these disorders. As a result, there has been considerable interest in understanding the underlying regulation of the physiologic fetal-to-adult hemoglobin switch in humans to be able to better manipulate this process for therapeutic purposes. To date, only a single factor, BCL11A, has been identified as being involved in the developmental regulation of human hemoglobin switching. BCL11A is a direct transcriptional repressor of the γ-globin genes. Moreover, BCL11A is expressed in a developmental stage-specific manner to regulate human hemoglobin switching. However, despite extensive studies, the mechanisms that act upstream to regulate BCL11A expression and thereby control hemoglobin switching have remained elusive. To gain further insights, we have directly explored the developmental regulation of BCL11A expression at various stages of human erythropoiesis. We find that BCL11A is regulated at the level of mRNA translation during development. While BCL11A mRNA is comparably expressed in a similar manner at all developmental stages in erythroid cells, robust protein expression only occurs in adult erythroid cells. Importantly, we demonstrate that at the earlier stages of development, the observed reduction in protein expression is attributable to decreased synthesis and not increased degradation of BCL11A through direct assessment of both protein synthesis and degradation rates in primary erythroid cells from various stages of human development. Interestingly, while BCL11A protein is not well synthesized at these earlier stages of development, we find that its mRNA curiously continues to be associated with ribosomes in a comparable manner between newborn and adult erythroid precursors using polysome fractionation of stage-matched erythroid cells. Through use of an unbiased proteomic analysis approach involving RNA affinity purification of the 18S ribosomal RNA in erythroid cells, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a reciprocal pattern to BCL11A, directly interacts with ribosomes. We additionally show that the observed suppression of BCL11A protein translation is mediated by LIN28B. Using a newly developed RNA isolation and crosslinking immunoprecipitation approach coupled to massively parallel sequencing we mapped the direct interaction of LIN28B with BCL11A mRNA at nucleotide resolution. Through a number of functional assays in both newborn and adult erythroid cells, we additionally demonstrate that this alteration of BCL11A translation is entirely independent of the role of LIN28B in the biogenesis of let-7 microRNAs. Finally, we show that BCL11A is the major functional target in LIN28B-mediated fetal hemoglobin induction through functional complementation experiments in primary human erythroid cells. Our results reveal a previously unappreciated regulatory mechanism underlying human hemoglobin switching. Moreover, our findings highlight opportunities for developing improved treatments for sickle cell disease and β-thalassemia by understanding the upstream regulation of human hemoglobin switching. Disclosures No relevant conflicts of interest to declare.

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In Human Beta-Globin Gene Locus, ERV-9 LTR Retrotransposon Interacts with and Activates Beta- but Not Gamma-Globin Gene

Blood ◽

10.1182/blood.v124.21.2686.2686 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2686-2686

Author(s):

Dorothy Tuan ◽

Wenhu Pi

Keyword(s):

Rna Polymerase Ii ◽

Gene Locus ◽

Conflicts Of Interest ◽

Globin Gene ◽

Endogenous Retroviruses ◽

Ltr Retrotransposon ◽

Globin Genes ◽

Erythroid Cells ◽

Primary Target ◽

Bac Clone

Abstract Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) comprise over 40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions was not known. In the human b-globin gene locus, an ERV-9 LTR retrtransposon is located near the 5’ end of the locus control region (LCR) at 40-70 kb upstream of the human fetal g- and adult b-globin genes. To address the functional significance of the intergenic ERV-9 LTR, we generated transgenic (Tg) mice carrying a 100 Kb BAC clone spanning the entire human b-globin gene locus from the ERV-9 LTR to b-globin gene and showed that the LTR retrotransposon serves long-range, beneficial host function (Pi et al., PNAS 2010): The ERV-9 LTR containing multiple CCAAT and GATA motifs competitively recruits high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. Deletion of the ERV-9 LTR by Cre-loxP mediated in situ recombination in the BAC Tg mice suppresses transcription of b-globin gene but activates transcription of g-globin gene. The results indicate that the ERV-9 LTR activates transcription of b-globin gene in erythroid cells during development. Therefore, LTR deletion drastically suppressed b-globin gene and re-activated g-globin gene through a competitive mechanism of globin gene switching. Alternatively, the primary effect of the ERV-9 LTR could be to suppress g-globin gene during development. Therefore, LTR deletion re-activated g-globin gene, which then suppressed transcription of b-globin gene. To differentiate between these two possibilities, we utilized Mx1-Cre mice to conditionally delete the ERV-9 LTR in the erythroid cells of the adult BAC transgene mice, in which g-globin gene was already silenced and b-globin gene fully activated. If the primary target of the ERV-9 LTR enhancer complex was g-globin gene, deletion of the ERV-9 LTR should not be able to activate the already silenced g-globin gene nor to suppress the fully active b-globin gene. However, the same effect on transcriptional suppression of b-globin gene and re-activation of g-globin gene was observed. These results indicate that the primary target of the ERV-9 LTR is the b-globin gene and not the g-globin gene. The molecular factors underlying the preferential interaction between the ERV-9 LTR and the b-globin gene are under investigation and will be presented Disclosures No relevant conflicts of interest to declare.

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“Maturational” Globin Switching in Primary Primitive Erythroid Cells.

Blood ◽

10.1182/blood.v106.11.3634.3634 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3634-3634

Author(s):

James Palis ◽

Jeff Malik ◽

Rachael L. Emerson ◽

Tim P. Bushnell ◽

Kathleen E. McGrath ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Globin Gene ◽

Developmental Time ◽

Yolk Sac ◽

Transcriptional Level ◽

Globin Genes ◽

Erythroid Cells ◽

Chromosomal Arrangement ◽

Predominant Cell Type

Abstract Mammals have two distinct erythroid lineages. The “definitive” erythroid lineage generates small, enucleated erythrocytes that constitute the predominant cell type in the fetal and postnatal circulation. It is preceded by the “primitive” erythroid lineage, which originates in the yolk sac and generates a semi-synchronous wave of large erythroblasts that terminally differentiate in the bloodstream. This feature provides a unique opportunity to investigate changes in gene expression during erythroid maturation. Here, we have examined expression of the various α- and β-globin genes in purified populations of primary primitive erythroid cells isolated from progressive developmental time-points of mouse embryogenesis. Our studies, using both in situ hybridization and quantitative PCR, indicate that βH1 is the predominant β-globin transcript in the early yolk sac. Thus, unlike the human, the murine β-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a βH1- to εy-globin switch, up-regulate low levels of the adult β1- and β2-globins, and down-regulate ζ-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions as assayed by ChIP assays. Furthermore, we found that the εy- and βH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. Taken together, these results are consistent with the notion that this βH1- to εy-globin “maturational” switch is dynamically regulated at the transcriptional level. We conclude that the embryonic to adult globin switch that occurs during murine ontogeny is due not only to the sequential appearance of primitive and definitive erythroid lineages but also to striking changes in globin gene expression that occur as primitive erythroblasts mature in the bloodstream.

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