Globin Genes In Cells Research Articles

HEXIM1 Overexpression Alters GATA1 Activity and Promotes Fetal Globin Expression

Erythropoiesis is a highly organized process whereby multipotent HSCs become committed to the erythroid linage and mature into enucleate erythrocytes. This process is associated with significant changes in gene expression as non-erythroid genes are switched off while expression of erythroid genes is maintained. HEXIM1 is a key regulator of RNA Polymerase II (RNPII) activity that is essential for erythropoiesis. Following transcription initiation, RNPII transcribes 30-50 base pairs before pausing. Phosphorylation of the c-terminal domain of RNPII by the dimer pTEFb (positive transcription elongation factor beta) is necessary for RNPII to resume active transcription elongation. HEXIM1 associates with pTEFb in the 7SK complex and renders it inactive. Phosphorylation of HEXIM1 releases pTEFb from the 7SK complex, allowing it to act on RNPII. As 7SK can be targeted to specific sequences, HEXIM1/7SK mediated pTEFb delivery is a crucial mechanism to facilitate on-site pTEFb activation and RNPII pause release. Recent work from our lab demonstrated that HEXIM1 is essential in the regulation of erythroid proliferation, viability and gene expression, and also promotes the expression of GATA1 target genes (Murphy Blood 2021). New analyses of HEXIM1 overexpression (OE) in HUDEP2 cells revealed a significant increase of gamma globin production, with a higher proportion of cells expressing fetal hemoglobin. These changes were accompanied by downregulation of beta globin and multiple key repressors of fetal hemoglobin, including BCL11A, MYB, and SOX6. These phenotypes were dependent on pTEFb activity, as the pTEFb (CDK9) inhibitor NVP-2 rescued the proliferative advantage granted by HEXIM1 OE and greatly decreased both gamma globin levels and the portion of fetal hemoglobin expressing cells. Further suggesting the effects of HEXIM1 OE are pTEFb-dependent, overexpression of HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb cannot enhance proliferation or gamma globin production. To gain insights into the mechanisms underlying the increased fetal globin expression, we performed CUT&RUN assays for Ser5 phosphorylated and total RNPII in HEXIM1 OE and control cells. Surprisingly, OE of HEXIM1 had only modest impacts on RNPII pausing, but instead lead to changes in the genes occupied by RNPII. Notably, the gene that lost the most RNPII was HBB and the gene that gained the most RNPII was HBG. Of note, GATA1 motifs were highly enriched in regions that gained RNPII occupancy. CUT&RUN identified peaks of GATA1 in the HEXIM1 OE cells at regions of increased RNPII occupancy that were not present in the EV or Y271A controls. These novel GATA1 sites occurred in regions of previously closed chromatin, suggesting that HEXIM1 OE enhanced the pioneer ability of GATA1. Similar to RNPII, GATA1 occupancy shifted from the beta globin gene in cells transduced with EV or HEXIM Y271A to the gamma globin genes in cells transduced with WT HEXIM1. Novel RNPII and GATA1 peaks were also identified in other genes associated with a fetal erythroid phenotype, most notably ARID3A and LIN28B. Together, these results support a model where elevated HEXIM1 levels promote the pioneer activity of GATA1, and facilitate the expression of a fetal transcriptional program in adult cells in a pTEFb-dependent manner.

Statistical Evidence for the Existence of a Regulatory Mechanism That Balances α-Globin and AHSP Expression in Erythroid Cells.

Alpha haemoglobin stabilizing protein (AHSP) is encoded by a gene abundantly expressed in erythroid cells whose function is to chaperone α-chains in the process of haemoglobin assembly (Yu et al, JCI 2007; 117:1856). The central role of the excess of α-chains in the pathogenesis of β-thalassemia and the AHSP ability to limit the toxicity of excessive α-globin suggest that increases of AHSP expression might ameliorate the clinical phenotype of β-thalassemia. To clarify the relationship between AHSP and globin gene expression, we measured the levels of mRNA for these genes in erythroblasts generated in vitro from the blood of 30 normal donors and 8 β-thalassemic patients. Normal erythroblasts presented a marked donor-to-donor variability in the expression levels of all the genes analysed. Inter-quartile range (IQR) analyses indicates that the gene whose expression has the highest variability is α-globin (IQR=31.5), followed by β-globin (IQR=8.74), AHSP (IQR=2.82) and γ-globin (IQR=0.86). The IQR value for the α/non-α globin ratio (1.91) is higher than that of the γ/γ+β ratio (0.11), an indication of the existence of donor variegation in the levels of unbalance between expression of α- and non-α globin genes in cells from different donors. The extent of this variegation is even more apparent by the high IQR level of the α-(non-α) expression difference (IQR=38.6). β-thalassemic erythroblasts expressed normal levels of α- and γ-globin, significantly (P<.05) lower levels of β-globin mRNA and, surprisingly, high levels (by 10-fold) of AHSP mRNA. Subject-variability in gene expression was also observed for β-thalassemic erythroblasts. In this case, the gene whose expression had the highest variability is AHSP (IQR=42.8), followed by α-globin (IQR=11.75), β-globin (IQR=3.32), and γ-globin (IQR=1.74). The IQR for the α/non-α globin ratio (7.2) is higher than that of the γ/γ+β ratio (0.67) also for β-thalassemic erythroblasts. The difference between the variances of the excess of α-expression [α-(non-α)] in β-thalassemic and normal erythroblasts is significant by F test (P=.0023). Statistical analyses of these results indicates that, as expected, the levels of α-globin mRNA are positively correlated to those of the non-α globin genes in normal erythroblasts (R2=.93, P<.001) but not in β-thalassemic cells (R2=.22, P<.24). In contrast, the levels of α-globin mRNA are positively correlated with those of AHSP both in normal (R2=.86, P<.0001) and β-thalassemic (R2=.66, P<.05) erythroblasts. Moreover, in spite of the fact that expression of α-globin is correlated, at least in normal erythroblasts, with that of γ+β mRNA, no correlation is found between levels of AHSP mRNA and those of γ+β mRNA. No correlation is also observed between levels of AHSP mRNA and the α/non-α ratio. In contrast, the levels of AHSP mRNA are correlated with the levels of excess of α-globin mRNA in normal erythroblasts (R2=0.86, P<.0001) and the fact that are not correlated in β-thalassemic cells (R2=.45, P=.066) might be due to the limited experimental points available for analyses. In conclusion, this statistical analyses provides evidence for the existence of a regulatory mechanism that balances expression of AHSP with that of excess of α-globin mRNA in erythroid cells. It is suggested that this regulatory mechanism may represent a target for eventual gene modifiers of the β-thalassemic trait. MEF is the recipient of a Marie Curie training Network Fellowship from EU.

Regulation of rat brain natriuretic peptide transcription. A potential role for GATA-related transcription factors in myocardial cell gene expression.

GATA-binding proteins are transcription factors that regulate the stage- and tissue-specific expression of globin genes in cells of the erythroid lineage. Recently, a cardiac GATA-binding protein was found to be the earliest gene expressed during cardiogenesis; however, the target genes of this transcription factor in the heart are unknown. Since brain natriuretic peptide (BNP) is activated early in cardiac growth and development, we evaluated whether it could serve as a target gene for GATA-binding protein-mediated induction. Upon isolating and sequencing 2.5 kilobases of the rat BNP 5'-flanking sequence (FS), a variety of putative transcriptional enhancer sites were identified, including several GATA consensus sequences (WGATAR), one of which apparently serves as the major promoter site. Primary myocardial cells were transfected with BNP/luciferase fusion genes; reporter expression was strongly induced by typical growth factors such as phorbol esters, serum, or alpha 1-adrenergic agonists, as well as by GATA-4 overexpression. Truncation analyses showed that inducibility mapped primarily to the proximal -116 base pair of the rat BNP 5'-FS, where there are two consensus GATA sites in addition to the GATA sequence at the TATA box. Point mutation analyses showed that at least one of the GATA sites was required to confer full GATA-4-inducible transcription. These results demonstrate that a proximal region of the rat BNP 5'-FS is required for growth factor- and GATA-inducible transcription, supporting the view that the BNP gene could serve as a target for GATA-binding proteins during early cardiac development.

Murine erythroleukemia cell differentiation: DNase I hypersensitivity and DNA methylation near the globin genes.

The sensitivity to digestion by DNase I of chromatin containing the alpha- and beta(major)-globin genes and the pattern of DNA methylation near these genes was examined during hexamethylenebisacetamide (HMBA)-mediated erythroid differentiation of murine erythroleukemia cells (MELC). In uninduced and induced cells, the chromatin regions containing the alpha- and beta-(major)-globin genes are more sensitive to digestion by DNase I than is the region containing an immunoglobulin gene (Igalpha) not expressed during erythroid differentiation. However, at low concentrations of DNase I, a 6- to 10-fold increase in site-specific cleavages was generated in chromatin regions near both the alpha- and beta(major)-globin genes in cells induced to differentiate by HMBA. The DNase I hypersensitive site near the beta(major)-globin gene maps to a small region near the 5' terminus of the gene. No detectable change in the pattern of DNA methylation around either the alpha- or beta-globin genes was observed during HMBA-mediated erythroid differentiation. Of the potentially methylated sites assayed and mapped near the beta(major)-globin gene, one site is fully methylated, one is partially methylated, and one is unmethylated both in uninduced and induced cells. Many (but not all) sites assayed near the alpha-globin genes are unmethylated in both uninduced and induced cells. These results show that specific alterations of chromatin structure occur during MELC differentiation and suggest that these changes may not involve alterations in the pattern of DNA methylation.

Lineage-dependent transcription of globin genes

Hb first appears in first generation erythroblasts of the chicken embryo at about 32 hr of incubation. These cells are the daughters of hematocytoblasts which themselves do not synthesize Hb. Employing a 3H-DNA complementary to purified adult chicken globin mRNA, we have assayed the transcription of the globin genes in cells in different compartments of the erythrogenic lineage. In addition, we have attempted to determine whether the globin genes are transcribed in cells not committed to Hb synthesis. Our results indicate that if the hematocytoblast contains any globin RNA, it would contain such molecules at a concentration less than 10 −5 times that of the erythroblast. Embryonic fibroblast and muscle cells lack globin RNA molecules to the same extent as the hematocytoblast. Comparison of nuclear RNA globin sequence content reveals that fibroblasts and muscle cells do not contain primary transcripts of the globin genes, and that primitive erythroblasts do not transcribe all the adult globin sequences in detectable quantities.