The β-globinlocus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation

Abstract

To investigate the molecular basis of ␤-globin gene activation, we analyzed factor recruitment and histone modification at the adult ␤-globin gene in wild-type (WT)/locus control region knockout (⌬LCR) 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 ␤-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 ␤-globin promoters of the ⌬LCR 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 ␤-globin gene activation: (1) [Keywords: Allele-specific chromatin immunoprecipitation analysis; locus control region; ␤-globin locus; transcription elongation; NF-E2; CTD-phosphorylation] Supplemental material is available at http://www.genesdev.org. Received January 6, 2003; revised version accepted February 19, 2003. In higher eukaryotes, gene activation involves several events including chromatin opening, activator binding to regulatory regions, recruitment of basal transcription factors (TFIIA to TFIIJ) and RNA polymerase II (pol II) to the promoter [preinitiation complex (PIC) assembly], and transcription elongation. A general model has emerged in which activators function to stabilize or modulate transcription through interactions with histone acetyltransferases, as well as components of the basal transcription machinery, such as TATA binding protein (TBP), TBP-associated factors (TAFs), and TFIIB (for review, see Lemon and Tjian 2000). PIC assembly is followed by initiation and elongation, during which the Cterminal domain (CTD) of the RPB1 subunit of pol II is phosphorylated (for review, see Dahmus 1996). These steps in gene activation are often regulated by distal elements called enhancers (for review, see Blackwood and Kadonaga 1998;Martin 2001). Enhancers increase either the rate of transcription, the number of the templates engaged in transcription, or both. Studies using transgenes or in vitro templates have linked enhancer activities to various events such as PIC assembly (Kim et al. 1998;Yie et al. 1999), histone acetylation at the promoter (Madisen et al. 1998), and nuclear localization (Francastel et al. 1999). However, it is poorly understood which events in transactivation are the actual targets of long-range enhancer function at native gene loci.The murine ␤-globin gene locus is a model system for studying the molecular mechanisms of enhancer-dependent gene activation at a native locus. The locus contains embryonic and adult ␤-like globin genes that are ordered as they are expressed during development. Hig...

Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network

“…5C and data not shown). In addition, as shown (18,22,28), p45 recruitment to the LCR and ␤-globin promoter is not detectable in uninduced cells and peaks 4-5 days after DMSO treatment. These results indicate that the hememediated departure of Bach1 from the LCR is not sufficient for the activation of ␤-globin expression or the induction of MEL cell differentiation.…”

“…Gene expression can be regulated by recruitment of RNA polymerase II (27) and by events downstream of preinitiation complex formation, such as clearance of RNA polymerase from the promoter (28). To gain insight into the pathway of ho-1 gene activation, we performed ChIP assays with an anti-pol II antibody.…”

AIRE Recruits P-TEFb for Transcriptional Elongation of Target Genes in Medullary Thymic Epithelial Cells

“…Indeed, DNA looping and interactions of distal enhancers and locus control regions with promoters are important for the expression of MHC class II and ␤-globin genes (12,42), which are regulated at the level of transcriptional elongation. Moreover, one study found AIRE to be associated with such matrix attachment sites (44).…”

The Role of p22 NF-E4 in Human Globin Gene Switching

“…Recent data suggest that the primary role of the LCR is to enhance the transition from transcription initiation to elongation (9). However, the LCR does not provide the temporal specificity of globin gene expression as transgenic mice lacking this region still exhibit appropriate developmental switching (10).…”