Journal
GENES & DEVELOPMENT
Volume 17, Issue 8, Pages 1009-1018Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gad.1072303
Keywords
allele-specific chromatin immunoprecipitation analysis; locus control region; beta-globin locus; transcription elongation; NF-E2; CTD-phosphorylation
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Funding
- NHLBI NIH HHS [HL57620] Funding Source: Medline
- NIDDK NIH HHS [DK44746, R37 DK044746] Funding Source: Medline
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To investigate the molecular basis of P-globin gene activation, we analyzed factor recruitment and histone modification at the adult p-globin gene in wild-type (WT)/locus control region knockout (ALCR) heterozygous mice and in murine erythroleukemia (MEL) cells. Although histone acetylation and methylation (Lys 4) are high before and after MEL differentiation, recruitment of the erythroid-specific activator NF-E2 to the promoter and preinitiation complex (PIC) assembly occur only after differentiation. We reported previously that targeted deletion of the LCR reduces beta-globin gene expression to 1%-4% of WT without affecting promoter histone acetylation. Here, we report that NF-E2 is recruited equally efficiently to the adult P-globin promoters of the ALCR and WT alleles. Moreover, the LCR deletion reduces PIC assembly only twofold, but has a dramatic effect on Ser 5 phosphorylation of RNA polymerase II and transcriptional elongation. Our results suggest at least three distinct stages in beta-globin gene activation: (1) an LCR-independent chromatin opening stage prior to NF-E2 recruitment to the promoter and PIC assembly; (2) an intermediate stage in which NF-E2 binding (LCR-independent) and PIC assembly (partially LCR-dependent) occur; and (3) an LCR-dependent fully active stage characterized by efficient pol II elongation. Thus, in its native location the LCR functions primarily downstream of activator recruitment and PIC assembly.
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