Transcription In Erythroid Cells Research Articles

Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching

AbstractThe switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (β-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A–mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA–mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

A Zinc Finger Transcription Factor Faithfully Dedicated to Only a Single Target Gene in Erythroid Cells

Lan etal. carry out a CRISPR-mediated genetic screen and discover that ZNF410 uniquely regulates theNuRD component CHD4 to repress γ-globin transcription in erythroid cells, establishing a novel fetal hemoglobin regulatory mechanism.

Erythropoietin Regulates Transcription and YY1 Dynamics in a Pre-established Chromatin Architecture.

SummaryThe three-dimensional architecture of the genome plays an essential role in establishing and maintaining cell identity. However, the magnitude and temporal kinetics of changes in chromatin structure that arise during cell differentiation remain poorly understood. Here, we leverage a murine model of erythropoiesis to study the relationship between chromatin conformation, the epigenome, and transcription in erythroid cells. We discover that acute transcriptional responses induced by erythropoietin (EPO), the hormone necessary for erythroid differentiation, occur within an invariant chromatin topology. Within this pre-established landscape, Yin Yang 1 (YY1) occupancy dynamically redistributes to sites in proximity of EPO-regulated genes. Using HiChIP, we identify chromatin contacts mediated by H3K27ac and YY1 that are enriched for enhancer-promoter interactions of EPO-responsive genes. Taken together, these data are consistent with an emerging model that rapid, signal-dependent transcription occurs in the context of a pre-established chromatin architecture.

Erythropoietin Regulates Transcription and YY1 Dynamics in a Pre-Established Chromatin Architecture

The three-dimensional architecture of the genome plays an essential role in establishing and maintaining cell identity. By contrast, the magnitude and temporal kinetics of changes in chromatin structure that arise during cell differentiation remain poorly understood. Here, we leverage a murine model of erythropoiesis to study the relationship between chromatin conformation, the epigenome, and transcription in erythroid cells. We discover that acute transcriptional responses induced by erythropoietin (EPO), the hormone necessary for erythroid differentiation, occur within an invariant chromatin topology. Within this pre-established landscape, Yin Yang 1 (YY1) occupancy dynamically redistributes to sites in proximity to EPO-regulated genes. Using HiChIP, we identify chromatin contacts mediated by H3K27ac and YY1 that are enriched for enhancer-promoter interactions of EPO-responsive genes. Taken together, these data are consistent with an emerging model that rapid, signal-dependent transcription occurs in the context of a pre-established chromatin architecture.

Development and characterization of cellular biosensors for HTS of erythroid differentiation inducers targeting the transcriptional activity of γ-globin and β-globin gene promoters.

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of β-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and β-globin genes by FACSwith efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the β-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from β-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of β-thalassemia and sickle cell anemia. Graphical abstract.

A CTCF-Dependent Silencer Located in the Differentially Methylated Area May Regulate Expression of a Housekeeping Gene Overlapping a Tissue-Specific Gene Domain

The tissue-specific chicken alpha-globin gene domain represents one of the paradigms, in terms of its constitutively open chromatin conformation and the location of several regulatory elements within the neighboring housekeeping gene. Here, we show that an 0.2-kb DNA fragment located approximately 4 kb upstream to the chicken alpha-globin gene cluster contains a binding site for the multifunctional protein factor CTCF and possesses silencer activity which depends on CTCF binding, as demonstrated by site-directed mutagenesis of the CTCF recognition sequence. CTCF was found to be associated with this recognition site in erythroid cells but not in lymphoid cells where the site is methylated. A functional promoter directing the transcription of the apparently housekeeping ggPRX gene was found 120 bp from the CTCF-dependent silencer. The data are discussed in terms of the hypothesis that the CTCF-dependent silencer stabilizes the level of ggPRX gene transcription in erythroid cells where the promoter of this gene may be influenced by positive cis-regulatory signals activating alpha-globin gene transcription.

A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo.

The GATA-1haematopoietic enhancer (G1HE), located between 3.9 and 2.6 kb 5' to the haematopoietic first exon, is essential for GATA-1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA-1 gene in vivo, indicating that additional regulatory sequences are necessary. We demonstrate here that two other upstream promoter elements containing a double GATA motif or two CACCC boxes are also indispensable for reporter gene expression in erythroid cells in the transgenic mouse. The combination of these three cis-acting regions was sufficient for reporter expression in primitive erythroid cells, as demonstrated by linking the elements together into a 659 bp artificial (GdC) minigene. The minigene activated the transcription of a reporter gene from either the endogenous or an exogenous thymidine kinase promoter, retaining cell type-specificity. The addition of a 320 bp fragment in the first intron to the GdC minigene sustained reporter expression in the definitive stage. Moreover, a line of transgenic mouse that expressed GATA-1 cDNA under the control of the complete 979 bp minigene rescued GATA-1 germ line mutant mice from embryonic lethality. A combination of four distinct sequence motifs co-operatively serve as a fundamental functional unit for GATA-1 erythroid transcription in vivo.

The Consequence of Nucleotide Substitutions in the Triosephosphate Isomerase (TPI) Gene Promoter

ABSTRACT: Mutations at −5A→G, −8→GA within the cap proximal element (CPE), and −24T→G within the TATA box of the triosephosphate isomerase (TPI) gene promoter have been identified in populations with a wide geographical distribution. These mutations lie within, or in close proximity to, known cis-active elements in the TPI gene promoter. To determine the functional significance of mutation at these sites, which remains controversial, their effect on the expression of erythrocyte TPI enzyme activity was studied in 110 healthy unrelated subjects. The −5G mutation did not alter erythrocyte TPI level, whereas the −8A mutation was accompanied by a significant reduction in enzyme activity to around 90% and 76% of normal erythrocyte TPI activity in heterozygotes and homozygotes, respectively. The −8A−24G genotype was associated with 75% of normal TPI activity in a heterozygote studied, implying that substitution of G at position −24 within the canonical TATA motif causes an additive decrease in TPI gene transcription in erythroid cells. A DNA–protein complex of 125kDa which was competitively blocked by specific unlabelled oligomers was demonstrated at the CPE and TATA box by electrophoretic mobility shift analysis. These findings provide direct evidence that TPI promoter mutations are linked to a reduction of TPI enzyme activity in vivo.

The Erythroid Krüppel-Like Factor Transactivation Domain is a Critical Component for Cell-Specific Inducibility of a β-Globin Promoter

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.

Enhancer-dependent transcription of the epsilon-globin promoter requires promoter-bound GATA-1 and enhancer-bound AP-1/NF-E2.

We analyzed epsilon-globin transcription in erythroid cells and in erythroid extracts to determine the requirements for enhancer-dependent expression of this gene. Mutations that abolished GATA-1 binding at a single position in the promoter prevented interaction with enhancers, whereas elimination of a second more distal promoter GATA-1 site had no effect. Deletion or mutation of the GATA-1 sites in either the human beta-globin locus control region DNase-hypersensitive site II enhancer or the chicken beta A/epsilon-globin enhancer did not diminish the ability of the enhancers to interact with the promoter. In contrast, mutation of the AP-1/NF-E2 sites in these enhancers resulted in elimination of enhancement. In vitro transcription of these constructs was promoter dependent and was not sensitive to abolition of GATA-1 binding in the promoter, consistent with the role of GATA-1 solely as a mediator of the enhancer effect. Thus, GATA-1 regulates the response of the epsilon-globin gene to enhancers through a specific site in the promoter and requires enhancer AP-1/NF-E2 binding to transduce the enhancer effect on transcription.

Enhancer-dependent transcription of the epsilon-globin promoter requires promoter-bound GATA-1 and enhancer-bound AP-1/NF-E2.

We analyzed epsilon-globin transcription in erythroid cells and in erythroid extracts to determine the requirements for enhancer-dependent expression of this gene. Mutations that abolished GATA-1 binding at a single position in the promoter prevented interaction with enhancers, whereas elimination of a second more distal promoter GATA-1 site had no effect. Deletion or mutation of the GATA-1 sites in either the human beta-globin locus control region DNase-hypersensitive site II enhancer or the chicken beta A/epsilon-globin enhancer did not diminish the ability of the enhancers to interact with the promoter. In contrast, mutation of the AP-1/NF-E2 sites in these enhancers resulted in elimination of enhancement. In vitro transcription of these constructs was promoter dependent and was not sensitive to abolition of GATA-1 binding in the promoter, consistent with the role of GATA-1 solely as a mediator of the enhancer effect. Thus, GATA-1 regulates the response of the epsilon-globin gene to enhancers through a specific site in the promoter and requires enhancer AP-1/NF-E2 binding to transduce the enhancer effect on transcription.

Regulation of globin gene expression in human K562 cells by recombinant activin A

Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot- blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.

Regulation of Globin Gene Expression in Human K562 Cells by Recombinant Activin A

AbstractRecent studies indicate that a purified protein, activin A, belongs to the transforming growth factor β(TGF-β) superfamily. Similar to TGF-β, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of a, 0, γ, and e globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for γ and є globins (209 nucleotides) as well as a (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of β globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic β-like and a globin transcripts also confirmed earlier studies of Northern and slot-blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an € globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells. © 1992 by The American Society of Hematology. 0006–4971/92/7903-0009S3.00/0

Enhancer dependent expression of the chicken β-hatching globin gene during erythroid differentiation

The activity of the chicken β H globin gene promoter has been analysed in functional assays in both chicken and murine erythroleukaemia cells. Sequences between −251 and −146 bp, in the presence or absence of the chicken β globin locus enhancer, strongly repress transcription in erythroid cells before and after the induction of terminal differentiation. A 50 bp sequence (−98 to −146 bp), which contains adjacent cGATA-1 and NF1 protein binding sites in vitro, and which is bound by non-histone protein in vivo, is essential for full promoter activity. Mutagenesis studies indicate that both protein binding sites are required. During terminal differentiation, both the absence of repressor and the presence of the erythroid enhancer are required for maximal promoter activity, suggesting that the β A, β ε and β H globin gene promoters compete for the enhancer during development.

The human beta-globin promoter; nuclear protein factors and erythroid specific induction of transcription.

We have shown that the promoter of the human beta-globin gene contains three regions in addition to the known CAC, CAAT and TATA box regions that are important for the induction of transcription in erythroid cells. By using DNaseI footprinting and gel mobility shift assays we were able to show that two of these regions bind the erythroid specific nuclear factor NF-E1 (and ubiquitous factors). The third region binds a ubiquitous CAAT-box factor (CP1). Deletion experiments suggest that only the combination of NF-E1 and CP1 binding sites, but not each of the sites alone, are capable of mediating the induction of transcription of a minimal (CAC, CAAT, TATA box) beta-globin promoter in mouse erythroleukaemia (MEL) cells.