Abstract
Abstract 3868
Insulators are DNA sequences and associated binding proteins that establish and/or maintain boundaries between regions of active and silenced chromatin domains. In higher organisms, there are 2 types of insulators, enhancer-blocking insulators, which establish chromatin domains to separate enhancers and promoters, and barrier insulators, which create a barrier to protect against heterochromatin-mediated gene silencing. Despite their role as critical regulators of tissue-specific gene expression, barrier insulators are poorly understood in mammalian cells, with much of our knowledge from studies of the barrier insulator in the chicken β-globin locus, cHS4. The DNA region of cHS4 that functions as a barrier binds upstream stimulatory factor (USF) proteins that recruit histone methyltransferase (HMT) activity, and histone acetyltransferase (HAT) activity, supporting a model that recruitment of enzymes and other proteins associated with activating histone modifications block the mechanism(s) that lead to spreading of gene-silencing. Our goal is to identify a regulatory signature associated with barrier insulators in erythroid cells. We utilized chromatin immunoprecipitation coupled with ultrahigh throughput Solexa sequencing (ChIP-seq) to generate genome-wide maps of regulatory and chromatin modifying proteins in erythroid cells. To generate cells for ChIP, human CD34+ cells were cultured in serum free media with erythropoietin to yield a population of CD71+/GPA+ erythroid cells (R3/R4 population). First, a genome-wide map of USF1 and USF2 occupancy in primary erythroid cells was created. A total of 19213 USF1 and 20115 USF2 sites of occupancy were identified. USF1 and USF2 frequently heterodimerize; co-localization was present at 15882 sites (83% of USF1 and 79% of USF2 sites). USF heterodimers were commonly located near proximal promoters (within 1KB of TSS, 48% of sites) and enhancers (>1kb from RefSeq gene, 30% of sites). To analyze co-localizing barrier-associated arginine methyltransferases from erythroid cells, ChIP-seq was performed with PRMT1 and PRMT4/CARM1. A total of 7062 PRMT1 sites and 15900 PRMT4 sites were identified. PRMT1 and PRMT4 were commonly found at sites of USF occupancy, with 6120 sites demonstrating occupancy of all four factors, consistent with the hypothesis that the USF proteins frequently recruit HMT's in mammalian cells. Sites of PRMT/USF co-occupancy were more likely to be at proximal promoters (68%) than sites of USF occupancy alone. Genome-wide occupancy of four acetyltransferases commonly found in erythroid cells, CBP, p300, PCAF, and SRC1, was also studied using ChIP-seq. 6804, 46932, 25688, and 25833 sites of occupancy were found for CBP, p300, PCAF, and SRC1 respectively. Co-localization with the p300, PCAF, and SRC1 with the USF/PRMT binding sites was common, occurring in 3825 sites. These sites were most commonly located near proximal promoters (71%) and enhancers (17%). In contrast, CBP co-localized with the USF/PRMT/p300/PCAF/SRC regions in only 10 locations and sites of CBP occupancy were more commonly found at enhancers (64%) and introns (29%) than at promoters (0.4%). Detection of barrier insulators near gene promoters is not surprising. Recent studies have revealed many similarities between barriers and promoters, including binding of specific transcription factors, and have led to the suggestion that barrier insulators have evolved as specialized derivatives of gene promoters, each with specific, yet discrete function. The regulatory protein CTCF mediates enhancer-blocking insulator activity. ChIP-seq was utilized to create a genome-wide map of CTCF binding in erythroid cells. 38503 sites of CTCF occupancy were identified. These sites were located at enhancers (41%), introns, (28%) and proximal promoters (18%). 4459 CTCF sites (12%) co-localized with regions of USF/PRMT/p300/PCAF/SRC binding. These sites most commonly occurred at promoters (65%) and enhancers (19%). The role of CTCF in barrier insulator function is controversial; our data are consistent with recent data demonstrating its mark at chromatin boundaries. The signature composed of USF/PRMT/p300/PCAF/SRC/CTCF was found in the well characterized functional erythroid barrier located in the ankyrin-1 gene proximal promoter region. These data indicate that a common regulatory signature is likely associated with barrier elements in erythroid cells.
Disclosures:
No relevant conflicts of interest to declare.
Pollen Specificity Elements Reside in 30 bp of the Proximal Promoters of Two Pollen-Expressed Genes