Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional environment and chromatin modifications interplay decides globin expression patterns.

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Richard Beyer

PROVIDER: E-GEOD-31523 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The Krüppel-like factors, KLF1 and KLF2, positively regulate embryonic β-globin expression, and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1-/-KLF2-/- double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1-/- and KLF1-/-KLF2-/-. Among these, c-myc emerged as a central node in the most significant gene network. c-myc expression is synergistically regulated by KLF1 and KLF2, and both factors bind the c-myc promoters. To characterize the role of c-myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia analogous to KLF1-/-KLF2-/-. In the absence of c-myc, circulating erythroid cells do not show the normal increase in α- and β-like globin expression, but interestingly, have accelerated erythroid maturation, between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate c-myc, to control the primitive erythropoietic program. Timed-pregnant KLF1+/-, KLF1+/- KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 8 different wild-type, 3 KLF1-/-, 3 KLF1-/- KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays.

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Transcriptional environment and chromatin modifications interplay decides globin expression patterns.

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