scholarly journals HEXIM1 Is Essential for the Establishment of Appropriate Patterns of Gene Expression and Chromatin Architecture during Terminal Erythroid Maturation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 284-284
Author(s):  
Zachary C. Murphy ◽  
Kristin Murphy ◽  
Michael Getman ◽  
Laurie A. Steiner
Keyword(s):  
Gene Expression ◽  
Critical Role ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Pol Ii ◽  
Chromatin Architecture ◽  
Cell Extracts ◽  
Active Transcription ◽  
Erythroid Maturation ◽  
In Erythroid Cells

Abstract Terminal erythroid maturation is associated with dramatic changes in gene expression in the setting of a cell that is undergoing rapid division and nuclear condensation. Disruption of this process is associated with inherited anemias and myelodysplastic syndromes. Recent work from our laboratory revealed that terminal erythroid maturation is associated with a dramatic decline in the level of total and elongation competent RNA polymerase II (Pol II), and that control of pol II activity is a critical step in the regulation of gene expression during terminal erythroid maturation. We further demonstrated that HEXIM1, which is highly expressed in early erythroid cells compared to most other cell types (biogps.org; bloodspot.eu), is essential for erythropoiesis (Murphy Blood 2021). The goal of our current study is to understand the mechanisms by which HEXIM1 regulates erythroid gene expression. HEXIM1 can impact gene expression though multiple mechanisms, most notably by associating with pTEFb, which is required for release of "paused" pol II into active transcription (reviewed in Michels, Transcription, 2018). HEXIM1 can inhibit transcription through sequestration of pTEFb in the 7SK ribonuclear complex, rendering it incapable of facilitating pause release. Alternatively, it can activate transcription by delivering pTEFb to target loci (McNamara Genome Data 2016). In erythroid cells, disruption of HEXIM1 impaired the expression of many erythroid specific genes, such as GYPA and many of the heme synthesis enzymes, while overexpression (OE) of HEXIM1 promoted their expression (Murphy, Blood, 2021). We therefore hypothesized that in maturing erythroblasts, HEXIM1 targets pTEFb to erythroid specific genes, promoting the establishment of appropriate patterns of gene expression and facilitating terminal erythroid maturation. To address this hypothesis, we generated novel HUDEP2 lines that OE HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb (Mbonye Proteomics 2015). Biotinylated 7SK pulldown confirmed that the Y271A mutation maintains the ability to bind the 7SK complex in erythroid cell extracts and RNA immunoprecipitation confirmed that the Y271A mutation increases the affinity of HEXIM1 for the 7SK complex in HUDEP2 cells. The Y271A mutation has significant functional consequences in erythroid cells. OE of wild type (WT) HEXIM1 in HUDEP2 cells resulted in enhanced proliferation in both expansion and maturation conditions, which was accompanied by increased cell and nuclear size, and a dramatic increase in the level of CD235a. Similar to our previously published HEXIM1 mutant with tyrosine to phenylalanine mutations at residues 271 and 274, the Y271A HEXIM1 mutation abrogated the enhanced proliferation seen with HEXIM1 OE in both expansion and maturation conditions. The Y271A mutation also rescued the larger cell and nuclear area associated with HEXIM1 OE, as well as the dramatic increase in the level of CD235a. Conversely, disruption of HEXIM1 via genome editing resulted in poor expansion and viability of HUDEP2 cells, which was rescued by expression of WT but not Y271A mutated HEXIM1, highlighting the importance of HEXIM1-pTEFb interactions for erythroid proliferation and survival. Further, OE of WT HEXIM1, but not the Y271A mutant, promoted erythroid gene expression while facilitating repression of genes that are normally silenced during terminal maturation, such as RPS19. In cells expressing WT HEXIM1 these gene expression changes were accompanied by increases in the global levels of ser2 and ser5 phosphorylated Pol II, as well as genome wide changes in their distribution. In contrast, the Y271A mutant decreased the global level of ser2 and ser5 pol II, consistent with its reduced ability to release pTEFb at target genes. Intriguingly, levels of H3K79me2, a histone mark reflective of active transcription through gene bodies, were decreased with OE of both WT and Y271A mutant HEXIM1, suggesting that the ability of HEXIM1 to promote transcriptional activation or repression is context dependent. Together, these data demonstrate a critical role for HEXIM1 and its interaction with pTEFb and the 7SK complex in the establishment of appropriate patterns of gene expression and chromatin architecture in maturing erythroblasts. Disclosures No relevant conflicts of interest to declare.

scholarly journals Antagonistic Regulation of β-Globin Gene Expression by Helix-Loop-Helix Proteins USF and TFII-I

2006 ◽  
Vol 26 (18) ◽  
pp. 6832-6843 ◽  
Author(s):  
Valerie J. Crusselle-Davis ◽  
Karen F. Vieira ◽  
Zhuo Zhou ◽  
Archana Anantharaman ◽  
Jörg Bungert
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Control Region ◽  
Adult Stage ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Globin Gene Expression ◽  
In Erythroid Cells

ABSTRACT The human β-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the β-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased β-globin gene expression, while diminishing TFII-I activity increased β-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult β-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the β-globin gene locus and play important roles in restricting β-globin gene expression to the adult stage of erythropoiesis.

scholarly journals Histone Methyltransferase Setd8 Represses Gata2 Expression and Regulates Erythroid Maturation

2015 ◽  
Vol 35 (12) ◽  
pp. 2059-2072 ◽  
Author(s):  
Jeffrey Malik ◽  
Michael Getman ◽  
Laurie A. Steiner
Keyword(s):  
Global Gene Expression ◽  
Regulatory Elements ◽  
Histone Methyltransferase ◽  
Tissue Type ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Histone H4 ◽  
Nuclear Condensation ◽  
Erythroid Maturation ◽  
In Erythroid Cells

Setd8 is the sole histone methyltransferase in mammals capable of monomethylating histone H4 lysine 20 (H4K20me1). Setd8 is expressed at significantly higher levels in erythroid cells than any other cell or tissue type, suggesting that Setd8 has an erythroid-cell-specific function. To test this hypothesis, stable Setd8 knockdown was established in extensively self-renewing erythroblasts (ESREs), a well-characterized, nontransformed model of erythroid maturation. Knockdown of Setd8 resulted in impaired erythroid maturation characterized by a delay in hemoglobin accumulation, larger mean cell area, persistent ckit expression, incomplete nuclear condensation, and lower rates of enucleation. Setd8 knockdown did not alter ESRE proliferation or viability or result in accumulation of DNA damage. Global gene expression analyses following Setd8 knockdown demonstrated that in erythroid cells, Setd8 functions primarily as a repressor. Most notably, Gata2 expression was significantly higher in knockdown cells than in control cells and Gata2 knockdown rescued some of the maturation impairments associated with Setd8 disruption. Setd8 occupies critical regulatory elements in the Gata2 locus, and knockdown of Setd8 resulted in loss of H4K20me1 and gain of H4 acetylation at the Gata2 1S promoter. These results suggest that Setd8 is an important regulator of erythroid maturation that works in part through repression of Gata2 expression.

scholarly journals Terminal Erythroid Maturation Is Associated with Dynamic Changes in the Abundance of Histone Marks Associated with Active Transcription Elongation and RNA Polymerase II Pausing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 154-154 ◽  
Author(s):  
Zachary C. Murphy ◽  
Tyler A Couch ◽  
Jacquelyn Lillis ◽  
Michael Getman ◽  
Kimberly Lezon-Geyda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Posttranslational Modifications ◽  
Histone H3 ◽  
Histone H4 ◽  
Pol Ii ◽  
Active Transcription ◽  
Pol Ii Pausing ◽  
Erythroid Maturation

Maturation of erythroid progenitors is associated with significant changes in gene expression in the context of a nucleus that dramatically decreases in size in preparation for enucleation, and is regulated by the coordinated action of transcriptional regulators and epigenetic modifiers. In eukaryotes, all DNA is bound by histone proteins into chromatin. Posttranslational modifications of the N-terminal "tails" of these proteins are key regulators of chromatin structure and gene expression. We hypothesized that terminal erythroid maturation is associated with changes in the abundance of specific histone posttranslational modifications. To address this hypothesis, we utilized mass spectrometry to perform an unbiased assessment of the abundance histone post translational modifications in maturing erythroblasts. We cultured peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) down the erythroid lineage using a semi-synchronous culture system (as outlined in Gautier et al. Cell Reports 2016), and sent cells for mass spectrometry on day 7 of erythroid maturation, when the cells are predominately basophilic erythroblasts, and on day 12 of erythroid maturation, when they are predominately poly- and ortho- chromatic erythroblasts. The maturation stage of the cells was confirmed by both cytospins and imaging flow cytometric analyses. Two independent replicates were performed and key results confirmed by western blotting. Terminal erythroid maturation was associated with a dramatic decline in the abundance of multiple histone marks associated with active transcription elongation, including Histone H3 lysine 36 di- and tri-methylation (H3K36me2, H3K36me3), and Histone H3 Lysine 79 di-methylation (H3K79me2). Surprisingly, this was not accompanied by an increase in the abundance of repressive heterochromatin marks (H3K27me3, H3K9me3, and H4K20me3) or a global decline in histone acetylation. Histone H4 lysine 16 acetylation (H4K16Ac), associated with RNA polymerase II pause release (Kapoor-Vazirani MCB 2011) significantly declined, but multiple acetylation marks including H3K36Ac and H3K23Ac increased in abundance. As expected, the abundance histone H4 lysine 20 mono-methylation (H4K20me1), which is implicated both in erythroblast chromatin condensation (Malik Cell Reports 2017) and the regulation of RNA Polymerase II pausing (Kapoor-Vazirani MCB 2011) also significantly increased. Consistent with these data, integration of RNA-seq and ChIP-seq data identified 3,058 genes whose expression decreased from basophilic erythroblast to orthochromatic erythroblasts, which lost enrichment for H3K36me3 (mark of active elongation) without accumulating H3K27me3 (heterochromatin mark). Based on these data, we hypothesized that RNA polymerase II pausing is a critical regulator of gene expression in maturing erythroblasts. RNA Polymerase II (Pol II) pausing is a highly regulated mechanism of transcriptional regulation, whereby transcription is initiated, but pauses 30-60bp downstream of the transcription start site. For paused Pol II to be released into active elongation, pTEFb must hyper-phosphorylate Serine 2 of the Pol II c-terminal domain (CTD). Importantly, pTEFb can be directed to specific loci through interaction with transcription factors, including GATA1 (Elagib Blood 2008; Bottardi NAR 2011). Hexim1 is a key regulator of Pol II pausing that sequesters pTEFb and inhibits its action. Consistent with a central role for Pol II pausing dynamics in the regulation of terminal erythroid maturation, Hexim1 is highly expressed in erythroid cells compared to most other cell types and its expression increases during terminal erythroid maturation. Conversely, the expression of CCNT1 and CKD9, the components of pTEFb, decline during terminal maturation, and the level of elongation competent (Ser2 and Ser2/Ser5 CTD phosphorylated) Pol II also decreases dramatically. To gain insights into the function of Pol II pausing in maturing erythroblasts, we induced Hexim1 expression in HUDEP2 cells (Kurita PLoS One 2013) using hexamethane bisacetamide (HMBA). HMBA treatment increased Hexim1 levels a dose dependent manner and was associated with gene expression and phenotypic changes suggestive of accelerated erythroid maturation. Together, these data suggest that RNA Pol II pausing dynamics are an important regulator of terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Dynamic CO-Localization of GATA1, NFE2, and EKLF and Changes in Gene Expression During Hematopoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 741-741 ◽  
Author(s):  
Laurie A. Steiner ◽  
Vincent P Schulz ◽  
Yelena Maksimova ◽  
Milind Mahajan ◽  
David M. Bodine ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Mrna Expression ◽  
Erythroid Cell ◽  
Common Finding ◽  
P Value ◽  
Erythroid Cells ◽  
Three Factor ◽  
In Erythroid Cells ◽  
Proximal Promoters

Abstract Abstract 741 Regulation of lineage choice during the development and differentiation of erythroid cells in hematopoiesis is a complex process. GATA1, NFE2, and EKLF are transcription factors critical for erythropoiesis. Focused studies, including detailed analyses of the human beta globin gene locus and a select group of erythrocyte membrane protein genes, have revealed that these three transcription factors may co-localize at common regulatory sites in erythroid-expressed genes. To address the hypothesis that GATA1, NFE2, and EKLF frequently co-localize on critical regulatory elements responsible for cell-type specific gene expression during erythropoiesis, chromatin immunoprecipitation coupled with ultrahigh throughput sequencing (ChIP-seq) was used to identify sites of GATA1, NFE2, and EKLF occupancy in human primary hematopoietic stem and progenitor cells (HSPCs) and human primary erythroid cells. ChIP was done using CD34+ HSPCs prepared by immunomagnetic bead selection and cultured CD71+/GPA+ erythroid cells (R3/R4 population) using antibodies against GATA1, NF-E2, and EKLF. The MACS algorithm (Zhang et al. Genome Biol, 2008) was used to identify regions of DNA-protein interaction, with a p-value ≤10e-5. Sites identified by MACS were ordered by p-value, and the 7000 sites with the most stringent p-values were selected for further analysis. Sites which occurred within 200bp of each other were treated as a single site. Unexpectedly, sites of GATA1, NFE2, and EKLF occupancy were common in HSPCs, with 6643 GATA1, 6657 NFE2, and 6579 EKLF sites identified, respectively. Sites identified in HSPCs were primarily in enhancers (>1kb from a RefSeq gene; 44% of GATA1, 49% of NFE2, and 51% of EKLF sites) and in introns (32% of GATA1, 34% of NFE2, and 34% of EKLF sites), with only a few sites at proximal promoters (within 1kb of a TSS; 7% of GATA1, 6% of NFE2, and 7% EKLF sites.) In erythroid cells, 6895 GATA1, 6907 NF-E2, and 6874 EKLF sites were identified. For all 3 factors, binding site occupancy varied greatly from that observed in HSPCs. Proximal promoter binding was much more common in erythroid cells than in HSPCs, with 19% of GATA1, 28% of NFE2 and 38% of EKLF sites found at promoters. Binding was frequently found at enhancers (41% of GATA, 38% NFE2, and 32% EKLF sites) and in introns (29% of GATA1, 26% of NFE2, and 21% of EKLF). To gain insight into three factor co-occupancy on a genome-wide scale, GATA1, EKLF, and NFE2 binding sites were compared using the Active Region Comparer (http://dart.gersteinlab. org/). Surprisingly, co-localization of all three factors was common in HSPCs, occurring at 2666 sites (40%, 40% and 45% of GATA1, NFE2, and EKLF sites). Sites of GATA1-NFE2-EKLF co-localization in HSPCs were located primarily at enhancers (51% of sites), in introns (32% of sites), and rarely at proximal promoters (6% of sites). In erythroid cells, co-localization of all three transcription factors was also common, occurring at 2445 sites (35%, 35%, and 36% of GATA1, NFE2, and EKLF sites, respectively). In contrast to HSPCs, sites of GATA1-NFE2-EKLF co-localization in erythroid cells were located primarily at proximal promoters (35% of sites) and enhancers (34% of sites), with co-localization in introns accounting for 20% of sites. A limited subset of sites, 1429 GATA1, 921 NFE2, and 1038 EKLF sites, were present in both HSPC and erythroid cells. Throughout the genome, there were only 233 sites of three factor co-localization in common in both HSPC and erythroid cells. Gene expression in HSPC and erythroid cell was analyzed via RNA hybridization to Illumina HumanHT-12 v3 Expression BeadChip arrays. In erythroid cells, genes with GATA1-NFE2-EKLF co-localization from 5kb upstream to 2kb downstream had significantly higher levels of mRNA expression than genes without GATA1-NFE2-EKLF co-localization (p<2.2e-16). The reverse was observed in HSPCs, where genes with GATA1-NFE2-EKLF co-localization had significantly lower levels of mRNA expression than genes without GATA1-NFE2-EKLF co-localization (p<7.3e-05). These data support the hypothesis that co-localization of GATA1, NFE2, and EKLF is a common finding in hematopoietic cells. Significant differences in factor co-localization and gene expression in HSPC and erythroid cells suggest that this coordinated binding orchestrates different patterns of gene expression during hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Linking Transcription Factor Pathways to Disease-Causing GATA1 Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2371-2371
Author(s):  
Amy E Campbell ◽  
Gerd A. Blobel
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Erythroid Cells ◽  
Amino Terminal ◽  
Erythroid Maturation ◽  
Cofactor Recruitment ◽  
In Erythroid Cells

Abstract Abstract 2371 Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. Seven such mutations are reported. All of these give rise to amino acid substitutions within the amino terminal zinc finger (NF) of GATA1, producing a range of clinical phenotypes. Thus, V205M, G208R, and D218Y cause severe anemia and thrombocytopenia; G208S, R216Q, and D218G cause thrombocytopenia with minimal anemia; R216W gives rise to thrombocytopenia and congenital erythropoietic porphyria. One of these mutations, R216Q, occurs at the DNA binding interface and alters the ability of GATA1 to recognize a subset of cis motifs in vitro. Other mutations, including V205M, G208S, D218G, and D218Y, occur outside the DNA binding domain of the NF and inhibit interactions with the GATA1 cofactor FOG1 as determined by in vitro binding assays. However, these two mechanisms do not easily explain the broad spectrum of phenotypes associated with the mutations. For example, how do two substitutions of the same residue bring about disparate phenotypes? We examined the effects of each mutation on erythroid maturation, lineage-specific gene expression, in vivo target gene occupancy, and cofactor recruitment by introducing altered forms of GATA1 into murine GATA1-null proerythroblasts. The V205M, G208R, and D218Y mutations severely impaired erythroid maturation, recapitulating patient phenotypes. The G208S mutation also severely impaired erythroid maturation, causing a more pronounced defect than that expected from the clinical presentation. In contrast, R216Q and D218G produced mild effects in erythroid cells consistent with patient phenotypes. The porphyria-associated mutation R216W also produced relatively subtle effects in erythroid cells. We note that among the mutants, failure to activate gene expression strongly correlated with failure to repress gene expression. ChIP assays revealed that the V205M, G208R, and D218Y mutations impaired GATA1 target site occupancy. This indicates that despite normal DNA binding in vitro, the association with cofactor complexes is required for stable binding to chromatinized target sites in vivo. In contrast, the G208S mutant exhibited relatively normal chromatin occupancy, but reduced recruitment of FOG1 and SCL/Tal1 to GATA1-bound sites at erythroid genes. D218G also perturbed cofactor recruitment without greatly affecting GATA1 binding to its target genes. Notably, this mutation diminished SCL/Tal1 recruitment without significantly altering FOG1 occupancy. This implicates the SCL/Tal1 transcription complex in the pathogenesis of disorders caused by certain GATA1 mutations. Moreover, by uncoupling GATA1 chromatin occupancy and cofactor recruitment, G208S and D218G offer potentially useful tools for unraveling site-specific mechanisms of GATA1-regulated gene expression. Finally, both the R216Q and R216W mutants displayed relatively normal GATA1 chromatin occupancy and FOG1 and SCL/Tal1 recruitment at most sites. R216W presents as porphyria, and selective defects in the regulation of heme biosynthetic genes have yet be uncovered. Given that R216Q presents as thrombocytopenia, defects caused by this mutation may be revealed only in the context of megakaryocytes. Studies using similar rescue assays of a GATA1-null megakaryocyte-erythroid progenitor line are underway and will be discussed. In concert, our results reveal that in vivo analysis of GATA1 in its native environment provides mechanistic insights not obtainable from in vitro studies. Moreover, they demonstrate the usefulness of gene complementation assays for the dissection of transcription pathways surrounding normal and altered GATA1 to improve our understanding of disease. Disclosures: No relevant conflicts of interest to declare.

Enhancers and Super Enhancers Are Associated With Genes That Control Phenotypic Traits In Primary Human Erythroid Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1200-1200
Author(s):  
Vincent P Schulz ◽  
Kimberly Lezon-Geyda ◽  
Yelena Maksimova ◽  
Patrick G Gallagher
Keyword(s):  
Gene Expression ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Cell Type ◽  
Biologically Relevant ◽  
Alpha Globin ◽  
Development And Differentiation ◽  
Cell Type Specific ◽  
Gwas Catalog ◽  
In Erythroid Cells

Abstract Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Recent studies have shown that enhancers are frequently associated with biologically relevant and disease-associated genetic variants. We hypothesized that unique sets of enhancers and super enhancers regulate gene expression in erythroid cells, a specialized cell type evolved to carry oxygen, and associated variants influence erythroid phenotypic variability. Active enhancers are part of a chromatin landscape marked by histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27Ac). A subset of enhancers, called super enhancers, important for regulating genes critical for cell-type specific identify, have been described. Super enhancers span large regions of chromatin, have domains of transcription factors (TF), significant amounts of H3K4me1 and H3K27Ac modification, and significant amounts of Mediator (MED1) occupancy, frequently with the transcriptional activator BRD4. Using ChIP-seq, genome wide maps of enhancers were constructed for H3K4me1, H3K27Ac, MED1, and BRD4 using primary human erythroid cell chromatin. These data were combined with parallel gene expression analyses determined via RNA-seq and enhancers and super enhancers identified. Cell and tissue-type specific enhancers act over distances of tens to hundreds of kilobases, thus bona fide erythroid enhancers are expected to be enriched in the genomic vicinity of genes expressed and functional in erythroid cells. Sites of occupancy of H3K4me1 were correlated with levels of gene expression in erythroid cells. To exclude gene promoters, H3K4me1 within 1kb of annotated transcriptional start sites (TSS) were excluded from analyses. Consistent with their predicted function, there was significantly higher levels of erythroid transcription for genes with H3K4me1 occupancy within 1-50kb of the TSS of genes cf. genes with H3K4me1 occupancy >50kb of a TSS (p value<2.2e-16). There was also significantly higher expression of genes with H3K4me1 occupancy within 1-50kb of the TSS in erythroid cells cf. non-erythroid cells (T lymphocyte). The top over represented TF motifs at sites of H3K4me1 were GATA1, AP1/NFE2, and KLF1. To explore whether candidate erythroid enhancers are enriched in regions associated with biologically relevant erythroid cell traits, candidate enhancers were mapped to a data set of erythroid-associated SNPs from the NHGRI GWAS catalog. 32 enhancers mapped to sites previously associated with biologically relevant erythroid traits. SNPs changed TF binding motifs at numerous enhancers including GATA1 motifs in the BCL11A, TFRC and ATP24 loci, an NFE2 motif in the ATP2B4 locus, and a TAL1 motif in the BCL11A locus. Super enhancers were identified as described (Cell 153:307, 2013) by finding regions with the highest levels of clustered chromatin modification/occupancy. Super enhancers defined by H3K4me1 and H3K27Ac modifications yielded 231 regions, BRD4 occupancy yielded 166 regions, and MED1 occupancy yielded 52 regions. H3K4me1/H3K27Ac-marked SE regions were found near the FOXO3, GATA2, STAT5A, TAL1, and ZFPM1 gene loci. BRD4- and MED1-marked super enhancers were found near the critical erythroid volume regulatory gene PIEZO1. The top over represented TF motifs at super enhancer sites defined by H3K4me1 were TAL1/RUNX1, GATA1, KLF1, defined by BRD4 were TAL1, KLF1, and MYC, and defined by MED1 were GATA1, MYC and CTCF. Mapping of super enhancers to erythroid-associated SNPs from the GWAS catalog of the NHGRI revealed many super enhancers mapped to regions associated with biologically relevant erythroid cell traits. For example, super enhancers identified by H3K4me1 mapped to loci for BCL11A, TFRC, KIT, HBS1L, MYB, ANK1, HK1, and the alpha-globin gene cluster; super enhancers identified by BRD4 localized to the alpha-globin cluster and the PIEZO1 gene locus. Perturbation of enhancer function during erythroid development and differentiation may lead to dysregulation of gene expression with concomitant phenotypic consequences. Insights into regulation of programs of gene expression in obtained from study of erythroid enhancers will provide insights into the functional significance of sequence variation associated with quantitative traits and inherited and acquired hematologic disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High-Level Setd8 Expression Is Important for Establishment of Normal Patterns of Erythroid Gene Expression and Is Essential for Stress Erythropoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 499-499
Author(s):  
Tyler A Couch ◽  
Zachary C. Murphy ◽  
Jacquelyn Lillis ◽  
Michael Getman ◽  
Paul D. Kingsley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Differentially Expressed ◽  
Erythroid Cells ◽  
Protein Levels ◽  
Stress Erythropoiesis ◽  
Erythroid Maturation ◽  
High Level ◽  
Putative Enhancer ◽  
In Erythroid Cells

Abstract Setd8 is the sole histone methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 is expressed at basal levels in most cell types and is important for many basic cellular functions, including cell cycle progression, transcriptional regulation, and mitotic chromatin condensation. Setd8 is expressed ~10-fold higher in erythroblasts than any other cell type and during erythroid maturation of human CD34+ HSPC, Setd8 protein levels increase in parallel with Gata1 levels, suggesting that Setd8 may have an erythroid-specific function(s). Consistent with this hypothesis, erythroid-specific deletion of Setd8 was embryonic lethal, resulting in profound anemia. Setd8-null erythroblasts had cell cycle abnormalities, failure of transcriptional repression, and defective terminal erythroid maturation. (Malik et al., Cell Reports, 2017). These studies provided important insights into the function of Setd8 in erythroid cells, but were not able to clearly delineate the "housekeeping" functions of Setd8 from its specific functions in erythropoiesis. To identify the erythroid-specific functions of Setd8, we sought to identify and disrupt the enhancer that drives high level Setd8 expression in erythroid cells. Using publically available ChIP-seq data sets, we identified a putative enhancer located in intron 1 of the SETD8 gene that was occupied by Gata1, Tal1, and H3K4me1 in human erythroblasts derived from culture of CD43+ HSPCs. This putative enhancer was able to drive luciferase expression in a reporter gene assay, and deletion of the Gata1:Tal1 site at the center of this region was sufficient to abrogate reporter gene activity. Based on these data, we hypothesized that this was the enhancer that drives high level expression of Setd8 in erythroid cells. To test this hypothesis, we used CRISPR/Cas9 genome editing to delete this region in HUDEP-2 cells. Briefly, Cas9 and guide RNA ribonucleoprotein complexes targeting the enhancer were delivered into the cells using electroporation (Gundry et al., Cell Reports, 2015). PCR and sequencing were used to confirm genome editing in monoclonal cell lines. Homozygous deletion of the enhancer (Δ/Δ) reduced SETD8 expression to 27.8% of WT (+/+) controls by RT-qPCR (n=3 for each genotype; p=0.0018). Decreased Setd8 protein levels and H4K20 mono-methylation was confirmed by Western blot. Further supporting an important function of Setd8 in erythropoiesis, deletion of the enhancer and exon 7 in CD34+ HSPCs resulted in a decreased efficiency of erythroid colony formation to 49.6% of control (n=5, p=0.0359). To gain insights into Setd8 gene regulation in erythroid cells, we performed RNA-seq, comparing the Δ/Δ and +/+ enhancer lines. In total, there were 603 genes differentially expressed (p<0.05; fold change >1.5), including SETD8, FAS, and CDKN1A (p21Cip1). Pathway analyses identified numerous genes associated with apoptosis and cell death to be up-regulated. Intriguingly, multiple genes in important for stress erythropoiesis were differentially expressed in the Setd8 Δ/Δ and +/+ enhancer lines and were also differentially expressed in Setd8-null murine erythroblasts (Malik et al., Cell Reports, 2017). Most notably, both the Δ/Δ enhancer lines and the Setd8-null erythroblasts had significantly higher levels of Fas death receptor transcript than control cells. Down-regulation of Fas is essential for stress erythropoiesis (Liu et al., Blood, 2006). We therefore hypothesized that Setd8 is important for the stress erythropoiesis response. To test this hypothesis, we subjected EpoR-Cre+/-;Setd8fl/+ (Setd8Δ/+) and EpoRCre+/-;Setd8+/+ (Setd8+/+) mice to anemic stress by retro-orbital bleeding. Setd8Δ/+ and Setd8+/+ mice had similar hematocrit after anemic stress (26.6 vs 29.4%; p=0.216), but the Setd8Δ/+ had an impaired ability to mount a stress response, with a lower MCV (43.0 vs 45.1 fL, p=0.003) and reticulocyte count (8.05 vs 2.14%, p=0.031) Consistent with the transcriptomic data, Setd8Δ/+ mice had higher levels of Fas transcript in splenic erythroblasts than Setd8+/+ controls. Together, these data suggest that high level Setd8 expression is important for normal erythroid maturation and gene expression, and for regulating the stress erythropoiesis response. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparison of Fetal and Adult Erythroid Chromosomal Architectures Identifies a Novel Fetal Hemoglobin Regulatory Region

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 774-774
Author(s):  
Peng Huang ◽  
Cheryl A. Keller ◽  
Belinda Giardine ◽  
James O.J. Davies ◽  
Jim R. Hughes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Globin Gene ◽  
Cell Lineage ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Chromatin Architecture

Abstract Chromatin structure is tightly intertwined with transcription regulation. The extent to which global chromatin architecture is subjected to alterations at different developmental stages within the same cell lineage has not been examined in great depth. Erythropoiesis offers an ideal model system to study the molecular mechanisms of gene regulation within the same cell lineage during development. Here, we comparatively defined via RNA-seq the transcriptomes, and via Hi-C and Capture-C the chromosome architectures of primary human fetal and adult erythroid cells. Overall, fetal and adult chromosomal conformations displayed a high degree of similarity. This includes the maintenance of A and B compartments representing active and inactive chromatin regions, respectively. Only ~5% of the genome switched compartments from A to B or vice versa, in agreement with the highly similar gene expression profiles. Moreover, topologically associating domains (TADs) were extensively preserved from fetal to adult stages. The developmentally regulated β-globin gene cluster is contained within one topologically associating domain (TAD) but folds into a three sub-TADs structure, the central one of which encompasses the β-globin locus. Notably, although the three sub-TAD structures are flanked by tissue invariant CTCF bound sites, they engage in looped contacts only in erythroid cells, indicating that erythroid specific transcription factors are required for CTCF mediated boundary contacts. At a finer scale, Capture-C detected distinct folding patterns at the developmentally controlled β-globin locus, including the expected stage-specific interactions between the enhancer (LCR) and the fetal γ-globin and adult β-globin genes. Importantly, we identified new developmental stage-specific chromatin contacts involving a region compassing a pseudogene (HBBP1) that resides between the fetal and adult globin genes. Specifically, HBBP1 engages in fetal stage-specific contacts with DNase hypersensitive sites HS5 and 3'HS1 while contacting the embryonic ε-globin gene at the adult stage. Deletion of a 2.3kb fragment encompassing HBBP1 (but not its transcriptional silencing) leads to strong reactivation of γ-globin gene expression in an adult erythroid cell line. This is accompanied by an architecturally restructured locus, including increased LCR-γ-globin chromatin interactions. Notably, the effects of HBBP1 deletion on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the HBBP1 region. In sum, our results identify a new segment, distinct from previously described regions linked to hereditary persistence of fetal hemoglobin, which engages in functionally important chromatin contacts. Since the HBBP1 region resides quite distantly from the structural globin genes, it might be a useful target for therapeutic genome editing without risking damage to the globin genes. Finally, our study highlights the power of high resolution chromosome architectural analysis to identify new regulatory regions. Disclosures No relevant conflicts of interest to declare.

Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells

1994 ◽  
Vol 14 (5) ◽  
pp. 3108-3114
Author(s):  
M H Baron ◽  
S M Farrington
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Targeted Mutagenesis ◽  
Erythroid Cell ◽  
Erythroid Cells

The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.

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