GATA-1 Modulates the Chromatin Structure and Activity of the Chicken α-Globin 3′ Enhancer

Abstract

Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken ␣-globin silencer-enhancer elements located 3 of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken ␣-globin enhancer plays a central role in its capacity to differentially regulate ␣-globin gene expression during erythroid differentiation and development.In recent years, the relevance of promoter elements has been outlined based on the varied promoter architectures required for regulatory specificity (28). A large amount of data has been generated describing how the basal transcriptional machinery is incorporated into promoters and how proximal elements are required for full and specific activity (17). However, a subset of genes requires long-distance regulatory elements for their developmental timing and tissue-specific gene expression (9, 39). Locus control regions (LCRs), enhancers, silencers, and insulators represent some of the elements exerting remote regulation (5, 10, 26). Such regulatory elements require characteristic chromatin structures, and the great majority of these regulatory elements and chromatin components are usually associated with nuclease hypersensitive sites (HSs) (6, 39). Despite progress in our understanding of promoters and long-distance regulatory elements, the mechanisms by which enhancers control gene expression are poorly understood, particularly in terms of how their own chromatin structure modulates their activity.In early work, we identified a silencer-enhancer element located at the chicken 3Ј side of the ␣-globin domain, around 400 bp downstream of the adult ␣ A gene (Fig. 1A) (16, 32, 33). We have adopted this enhancer element as a model system to investigate the differential regulation of chicken ␣-globin gene expression during erythroid differentiation and development. Based on recent data, we have proposed that the 3Ј-side enhancer is able to modulate its own function through the binding of GATA-1 and YY1 (Fig. 1B) (33) and the associated chromatin remodeling machinery. Much less is known about the molecular features of the chicken ␣-globin silencer. What we have found until now is that the silencer is located side by side with th...

Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes

“…7a). Chicken red blood cells (RBC) CTCF ChIP experiments were performed as previously described 44,45 . We evaluated the CTCF-ChIP quality with several positive and negative PCR controls (Supplementary Figure 7b).…”

Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation

“…2Bii) networks. Judged by the number of connections, GATA-1 appears to be an influential TF with respect to both hemin- and PMA-mediated differentiation and is known to recruit many proteins to mediate chromatin remodeling (Escamilla-Del-Arenal and Recillas-Targa, 2008; Grass et al, 2003; Im et al, 2005). According to the model, GATA-1 is a target of most TFs that were screened.…”

Epigenetic regulation of the human p53 gene promoter by the CTCF transcription factor in transformed cell lines

“…We found that the histone H3K4me2 modification and the polyacetylation of histone H3 and H4 are drastically enriched in SW480 cells (Figure 1c). To corroborate the in situ chromatin status of the promoter we performed a restriction enzyme accessibility assay (Escamilla-Del-Arenal and Recillas-Targa, 2008) (Figure 1d). The results of these experiments are in accordance with the previous assays, and they show that the p53 gene promoter has an open chromatin conformation in the SW480 cell line, whereas the HeLa and Kb cell lines show a more compacted chromatin structure (Figure 1d).…”