Epigenetic modifications and chromosome conformations of the beta globin locus throughout development
ABSTRACT: Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic epsilon and fetal gamma globin by the hESC-derived erythroblasts allows the interrogation of epsilon globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the beta globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34+ cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the beta globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the epsilon globin promoter, as compared to the gamma or beta globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active epsilon globin promoter, similar to the loop that forms between the gamma or beta globin promoters and the LCR, in contrary to the previously proposed tracking mechanism. Human embryonic stem cells, hESC-, FL-, and PB derived erythroblasts were studied. The enrichment of H3K4me3 and AcH3 acrossed the beta globin locus was studied using ChIP-seq.
SUBMITTER: Andy Huang John A StamatoyannopoulosYadong YangKai-Hsin ChangThalia PapayannopoulouXiangdong FangHao WangHua CaoHalvard Bonig
Project description:In primitive erythroid cells of human beta-globin locus transgenic mice (TgM), the locus control region (LCR)-proximal epsilon- and gamma-globin genes are transcribed, whereas the distal delta- and beta-globin genes are silent. It is generally accepted that the beta-globin gene is competitively suppressed by gamma-globin gene expression at this developmental stage. Previously, however, we observed that epsilon-globin gene expression was severely attenuated when its distance from the LCR was extended, implying that beta-globin gene might also be silenced because of its great distance from the LCR. Here, to clarify the beta-globin gene silencing mechanism, we established TgM lines carrying either gamma- or epsilon- plus gamma-globin promoter deletions, without significantly altering the distance between the beta-globin gene and the LCR. Precocious expression of delta- and beta-globin genes was observed in primitive erythroid cells of mutant, but not wild-type TgM, which was most evident when both the epsilon and gamma promoters were deleted. Thus, we clearly demonstrated that the repression of the delta- and beta-globin genes in primitive erythroid cells is dominated by competitive silencing by the epsilon- and gamma-globin gene promoters, and that epsilon- and the other beta-like globin genes might be activated by two distinct mechanisms by the LCR.
Project description:To test the role of gene order in globin gene expression, mutant human beta-globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a "marked" transcript (epsilon(m), gamma(m), or beta(m)) integrated at different locations within the locus. When a beta(m)-globin gene was placed between the locus control region (LCR) and the epsilon-globin gene, beta(m)-globin expression dominated primitive and definitive erythropoiesis; only beta(m)-globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an (A)gamma(m)-globin gene was placed at the same location, (A)gamma(m)-globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type gamma-globin genes were not expressed. When an epsilon(m)-globin gene was placed between the delta- and beta-globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type epsilon-globin gene was expressed. Placement of a beta(m)-globin gene upstream of the (G)gamma-globin gene resulted in expression of beta(m)-globin in embryonic cells and in a significant decrease in expression of the downstream wild-type beta-globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development.
Project description:To delineate the relationship between epigenetic modifications and hemoglobin switching, we compared the pattern of histone acetylation and pol II binding across the beta-globin locus at fetal and adult stages of human development. To make this comparison possible, we introduced an external control into experimental samples in chromatin immunoprecipitation (ChIP) assays. Using this common standard, we found that the locus control region (LCR) was acetylated to the same level at all stages, whereas acetylation levels at the individual gene regions correlated with the state of transcription. In the active genes, the promoters were less acetylated compared with the coding regions. Furthermore, all globin promoters were acetylated to a similar level irrespective of the state of transcription. However, after correction for the loss of nucleosomes, the level of acetylation per histone at the active gamma and beta promoters was 5- to 7-fold greater than that at the inactive epsilon promoter. Although the histone acetylation level within the LCR was developmentally stable, pol II binding in fetal erythroblasts was 2- to 3-fold greater than that in adult erythroblasts. These results demonstrate that dynamic changes in histone acetylation and pol II take place as the human beta-globin gene region undergoes its developmental switches.
Project description:Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic ? and fetal ? globin by the hESC-derived erythroblasts allows the interrogation of ? globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the ? globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34(+) cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the ? globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the ? globin promoter, as compared to the ? or ? globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active ? globin promoter, similar to the loop that forms between the ? or ? globin promoters and the LCR, in contrary to the previously proposed tracking mechanism.
Project description:A 213 kb human beta-globin locus yeast artificial chromosome (beta-YAC) was modified by homologous recombination to delete 2.9 kb of cross-species conserved sequence similarity encompassing the LCR 5' hypersensitive site (HS) 4 (Delta5'HS4 beta-YAC). In three transgenic mouse lines, completion of the gamma- to beta-globin switch during definitive erythropoiesis was delayed relative to wild-type beta-YAC mice. In addition, quantitative per-copy human beta-like globin mRNA levels were similar to wild-type beta-YAC transgenic lines, although beta-globin gene expression was slightly decreased in the day 12 fetal liver of Delta5'HS4 beta-YAC mice. A 0.8 kb 5'HS1 fragment was similarly deleted in the YAC. Three Delta5'HS1 beta-YAC transgenic lines were established. epsilon-globin gene expression was markedly reduced, approximately 16 fold, during primitive erythropoiesis compared to wild-type beta-YAC mice, but gamma-globin expression levels were unaffected. However, during the fetal stage of definitive erythropoiesis, gamma-globin gene expression was decreased approximately 4 fold at day 12 and approximately 5 fold at day 14. Temporal developmental expression profiles of the beta-like globin genes were unaffected by deletion of 5'HS1. Decreased expression of the epsilon- and gamma-globin genes is the first phenotype ascribed to a 5'HS1 mutation in the human beta-globin locus, suggesting that this HS does indeed have a role in LCR function beyond simply a combined synergism with the other LCR HSs.
Project description:Phylogenetic inferences drawn from comparative data on mammalian beta-globin gene clusters indicate that the ancestral primate cluster contained a locus control region (LCR) and five paralogously related beta-type globin loci (5'-LCR-epsilon-gamma-psieta-delta-beta-3'), with epsilon and gamma expressed solely during embryonic life. A gamma locus tandem duplication (5'-gamma(1)-gamma(2)-3') triggered gamma's evolution toward fetal expression but by a different trajectory in platyrrhines (New World monkeys) than in catarrhines (Old World monkeys and apes, including humans). In platyrrhine (e.g., Cebus) fetuses, gamma(1) at the ancestral distance from epsilon is down-regulated, whereas gamma(2) at increased distance is up-regulated. Catarrhine gamma(1) and gamma(2) acquired longer distances from epsilon (14 and 19 kb, respectively), and both are up-regulated throughout fetal life with gamma(1)'s expression predominating over gamma(2)'s. On enlarging the platyrrhine expression data, we find Aotus gamma is embryonic, Alouatta gamma is inactive at term, and in Callithrix, gamma(1) is down-regulated fetally, whereas gamma(2) is up-regulated. Of eight mammalian taxa now represented per taxon by embryonic, fetal, and postnatal beta-type globin gene expression data, four taxa are primates, and data for three of these primates are from this laboratory. Our results support a model in which a short distance (<10 kb) between epsilon and the adjacent gamma is a plesiomorphic character that allows the LCR to drive embryonic expression of both genes, whereas a longer distance (>10 kb) impedes embryonic activation of the downstream gene.
Project description:During erythroid development, the embryonic ε-globin gene becomes silenced as erythropoiesis shifts from the yolk sac to the fetal liver where γ-globin gene expression predominates. Previous studies have shown that the ε-globin gene is autonomously silenced through promoter proximal cis-acting sequences in adult erythroid cells. We have shown a role for the methylcytosine binding domain protein 2 (MBD2) in the developmental silencing of the avian embryonic ρ-globin and human fetal γ-globin genes. To determine the roles of MBD2 and DNA methylation in human ε-globin gene silencing, transgenic mice containing all sequences extending from the 5' hypersensitive site 5 (HS5) of the β-globin locus LCR to the human γ-globin gene promoter were generated. These mice show correct developmental expression and autonomous silencing of the transgene. Either the absence of MBD2 or treatment with the DNA methyltransferase inhibitor 5-azacytidine increases ε-globin transgene expression by 15-20 fold in adult mice. Adult mice containing the entire human β-globin locus also show an increase in expression of both the ε-globin gene transgene and endogenous ε(Y) and β(H1) genes in the absence of MBD2. These results indicate that the human ε-globin gene is subject to multilayered silencing mediated in part by MBD2.
Project description:High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.
Project description:We have identified novel nuclear transcripts in the human beta-globin locus using nuclear run-on analysis in erythroid cell lines and in situ hybridization analysis of erythroid tissue. These transcripts extend across the LCR and intergenic regions but are undetectable in nonerythroid cells. Surprisingly, transient transfection of a beta-globin gene (epsilon, gamma, or beta) induces transcription of the LCR and intergenic regions from the chromosomal beta-globin locus in nonerythroid cell lines. The beta-globin genes themselves, however, remain transcriptionally silent. Induction is dependent on transcription of the globin gene in the transfected plasmid but does not require protein expression. Using in situ hybridization analysis, we show that the plasmid colocalizes with the endogenous beta-globin locus providing insight into the mechanism of transinduction.
Project description:Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human beta-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the beta-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual beta-like globin genes. Moreover, in transgenic mice expressing the entire human beta-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter beta-like globin gene expression. Here, we show that abnormal expression of human beta-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human epsilon- and gamma-promoters, but not at the beta-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human gamma-promoters. We also find that HS2 is dispensable for high-level human beta-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.