The beta -globin locus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation.
To investigate the molecular basis of beta-globin gene activation, we analyzed factor recruitment and histone modification at the adult beta-globin gene in wild-type (WT)/locus control region knockout (DeltaLCR) 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 beta-globin promoters of the DeltaLCR 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.
Sawado
Tomoyuki
T
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Halow
Jessica
J
Bender
M A
MA
Groudine
Mark
M
D000818:Animals; D002454:Cell Differentiation; D002843:Chromatin / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D017353:Gene Deletion; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000378:metabolism; D019901:Locus Control Region / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D002843:Chromatin; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D006657:Histones; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494693:Nfe2 protein, mouse; D014157:Transcription Factors; D005914:Globins; D012319:RNA Polymerase II