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Complex developmental patterns of histone modifications associated with the human beta-globin switch in primary cells.

ABSTRACT: The regulation of the beta-globin switch remains undetermined, and understanding this mechanism has important benefits for clinical and basic science. Histone modifications regulate gene expression and this study determines the presence of three important histone modifications across the beta-globin locus in erythroblasts with different beta-like globin-expression profiles. Understanding the chromatin associated with weak gamma gene expression in bone marrow cells is an important objective, with the goal of ultimately inducing postnatal expression of weak gamma-globin to cure beta-hemoglobinopathies.These studies use uncultured primary fetal and bone marrow erythroblasts and human embryonic stem cell-derived primitive-like erythroblasts. Chromatin immunoprecipitation with antibodies against modified histones reveals DNA associated with such histones. Precipitated DNA is quantitated by real-time polymerase chain reaction for 40 sites across the locus.Distribution of histone modifications differs at each developmental stage. The most highly expressed genes at each stage are embedded within large domains of modifications associated with expression (acetylated histone H3 [H3ac] and dimethyl lysine 4 of histone H3 [H3K4me2]). Moderately expressed genes have H3ac and H3K4me2 in the immediate area around the gene. Dimethyl lysine 9 of histone H3 (H3K9me2), a mark associated with gene suppression, is present at the epsilon and gamma genes in bone marrow cells, suggesting active suppression of these genes.This study reveals complex patterns of histone modifications associated with highly expressed, moderately expressed, and unexpressed genes. Activation of gamma postnatally will likely require extensive modification of the histones in a large domain around the gamma genes.

SUBMITTER: Hsu M

PROVIDER: S-EPMC2748252 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Complex developmental patterns of histone modifications associated with the human beta-globin switch in primary cells.

Hsu Mei M Richardson Christine A CA Olivier Emmanuel E Qiu Caihong C Bouhassira Eric E EE Lowrey Christopher H CH Fiering Steven S

Experimental hematology 20090519 7

<h4>Objective</h4>The regulation of the beta-globin switch remains undetermined, and understanding this mechanism has important benefits for clinical and basic science. Histone modifications regulate gene expression and this study determines the presence of three important histone modifications across the beta-globin locus in erythroblasts with different beta-like globin-expression profiles. Understanding the chromatin associated with weak gamma gene expression in bone marrow cells is an importa ...[more]

PMID: 19460472

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Project description:Background: Developmental stage-specific globin expression is a complex phenomenon that involves both trans- and cis-acting elements. While functional analyses ensuing recent genome-wide association studies have highlighted the important roles of trans-factors in regulating hemoglobin expression, these factors can not exert their functions without permissive chromatin domains. By transferring thoroughly profiled beta globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroid cells into an adult erythroid transcriptional environment, we studied the influences of histone modifications on the globin expression decision within a fixed transcriptional environment. Shortly after the locus transfer, embryonic epsilon globin was not expressed regardless of original chromatin states, whereas fetal gamma globin was either expressed or not activated depending on original chromatin configurations, and the originally silent adult beta globin either remained silent or became activated depending on the expression status of gamma globin. These data suggest the interplay between transcriptional environment and the chromatin modifications determine the outcome of globin expression. As the ultimate silencing of gamma globin from hESC-derived erythroid cells in the adult transcriptional environment occurred after months-long cell proliferation, our work also has implications on attempts to generate beta globin expressing erythroid cells from hESCs or induced pluripotent stem cells. hESC line H1 (NIH code WA01, WiCell, Madison, WI) and adeno-associated virus (AAV)-targeted lines, were maintained and differentiated as previously described.12 (Details can be found in the supplemental information.) The expression of stem cell markers including SSEA-3, SSEA-4, TRA-1-60, and TRA-1-61 was detected by flow cytometry. To determine whether AAV-targeted lines retained their hematopoietic differentiation potential, confluent hESCs were harvested off the feeder layers and transferred to ultra-low attachment plates to allow for the formation of embryoid bodies (EBs). Day-14 EBs were dissociated into single cells and the expression of surface markers including CD34, CD71, CD45, CD31, CD41 and glycophorin-A was determined by flow cytometry. To induce erythroid differentiation, day-7 EBs were made into single cell suspension and cultured in erythroid-inducing medium for 14 days. Primers for real time PCR analysis of beta locus globin mRNA expression are provided in the supplemental information.

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Complex developmental patterns of histone modifications associated with the human beta-globin switch in primary cells.

Publications

Complex developmental patterns of histone modifications associated with the human beta-globin switch in primary cells.

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