Abstract
The human beta-globin gene is expressed at high levels in erythroid cells and regulated by proximal and distal cis-acting DNA elements, including promoter, enhancer, and a locus control region (LCR). Transcription complexes are recruited not only to the globin gene promoters but also to the LCR. Previous studies have implicated the ubiquitously expressed transcription factor USF and the tissue-restricted activator NF-E2 in the recruitment of transcription complexes to the beta-globin gene locus. Here we demonstrate that although USF is required for the efficient association of RNA polymerase II (Pol II) with immobilized LCR templates, USF and NF-E2 together regulate the association of Pol II with the adult beta-globin gene promoter. Recruitment of Pol II to the LCR occurs in undifferentiated murine erythroleukemia cells, but phosphorylation of LCR-associated Pol II at serine 5 of the C-terminal domain is mediated by erythroid differentiation and requires the activity of NF-E2. Furthermore, we provide evidence showing that USF interacts with NF-E2 in erythroid cells. The data provide mechanistic insight into how ubiquitous and tissue-restricted transcription factors cooperate to regulate the recruitment and activity of transcription complexes in a tissue-specific chromatin domain.
Highlights
Eukaryotic transcription is regulated at many different levels, including the modification and remodeling of chromatin structure and perhaps the recruitment of genes to transcription factories [1,2,3]
We show here that polymerase II (Pol II), NF-E2, USF1, USF2, TFIIB, and the coactivator CBP are efficiently recruited to locus control region (LCR) element HS2, whereas only TFIIB and USF2 are recruited to the adult majglobin gene promoter in undifferentiated murine erythroleukemia (MEL) cells
Previous studies have shown that -globin LCR HS sites recruit transcription complexes and that the LCR is required for the association of the -globin gene locus with transcription factories [45, 50, 53, 54]
Summary
Cell Culture and Protein Extracts—MEL cells were grown in Dulbecco’s modified Eagle’s medium (Cellgro) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin antibiotic mixture. The protein complexes were captured by incubating the antibody containing cell nuclear extracts with anti-rabbit IgG beads for 2 h. The beads were washed 3–5 times with 1 ml of pull-down binding buffer (without BSA), collected, boiled with Laemmli sample buffer (BioRad), and loaded onto 4 –20% Ready gels (Bio-Rad), followed by a Western blotting assay with antibody against NF-E2 After incubating beads-LCR-protein complexes with -globin gene constructs for 30 min at 30 °C, the tubes were placed on a magnetic device, and the supernatant was collected from each tube, and subjected to Western blotting analysis. All samples were dialyzed against ChIP dilution buffer and subjected to IP analysis using the ChIP assay protocol described previously with antibodies against Pol II (clone CTD4H8, 05-623, Upstate) and rabbit IgG (Bethyl Laboratories). Precipitation of the -globin gene template was monitored by quantitative real-time PCR using primers located downstream of the -globin gene promoter: US, 5Ј-ATTGCATCAGTGTGGAAGTC-3Ј; DS, 5Ј-ATTGCCCTGAAAGAAAGAGATTAG-3Ј
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