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| Status |
Public on Aug 19, 2012 |
| Title |
Transcriptional environment and chromatin modifications interplay decides globin expression patterns. |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
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.
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| Overall design |
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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| Contributor(s) |
Beyer RP, Chang K, Papayannopoulou T, Huang A, Han H, Fang X, Song C, Wang H, Li Q, Stamatoyannopoulos J, Stamatoyannopoulos G |
| Citation(s) |
23993951 |
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| Submission date |
Aug 18, 2011 |
| Last update date |
Mar 04, 2019 |
| Contact name |
James William MacDonald |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Department |
Environmental and Occupational Health Sciences
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| Street address |
4225 Roosevelt Way NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98105-6099 |
| Country |
USA |
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| Platforms (2) |
| GPL6244 |
[HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version] |
| GPL6246 |
[MoGene-1_0-st] Affymetrix Mouse Gene 1.0 ST Array [transcript (gene) version] |
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| Samples (20)
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| Relations |
| BioProject |
PRJNA145515 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE31523_RAW.tar |
81.1 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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