Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene

Abstract

The 85-kb breast cancer-associated gene BRCA1 is an established tumor suppressor gene, but its regulation is poorly understood. We demonstrate by gene conformation analysis in both human cell lines and mouse mammary tissue that gene loops are imposed on BRCA1 between the promoter, introns, and terminator region. Significantly, association between the BRCA1 promoter and terminator regions change upon estrogen stimulation and during lactational development. Loop formation is transcription-dependent, suggesting that transcriptional elongation plays an active role in BRCA1 loop formation. We show that the BRCA1 terminator region can suppress estrogen-induced transcription and so may regulate BRCA1 expression. Significantly, BRCA1 promoter and terminator interactions vary in different breast cancer cell lines, indicating that defects in BRCA1 chromatin structure may contribute to dysregulated expression of BRCA1 seen in breast tumors.transcriptional regulation ͉ chromatin conformation ͉ gene repression ͉ mammary gland ͉ breast cancer E xpression of the tumor suppressor gene BRCA1 is reduced in a significant proportion of human breast tumors (1-3). Although up to one-third of these cases can be explained by promoter hypermethylation (4, 5) for most cases the cause is unknown. Understanding the underlying mechanisms of BRCA1 gene repression is critical for generating effective strategies for re-establishing BRCA1 expression and thus restoring its tumor suppressor function.Transcriptional initiation of protein-coding genes depends on a coordinated interplay of protein-DNA and protein-protein interactions (6). In addition to the assembly of RNA polymerase II (Pol II) with basal transcription machinery on the gene promoter, numerous transcription factors are recruited to either activate or repress transcription. As many of these factors associate with DNA sequences distant to the promoter, transcriptional regulation often involves long-range DNA associations, possibly mediated by the formation of chromatin loops (7). Chromatin loops can be detected by the chromosome conformation capture (3C) technique (8), which involves formaldehyde cross-linking of chromatin in live cells, digesting DNA with restriction enzymes, and then religating DNA in dilute solution to favor intramolecular ligation. PCR is then used to detect the presence of such ligation products. 3C has been used to study the normal regulation of genes in multiple eukaryotic species and supports a looping model for gene activation and repression. For example, transcriptional activation of the ␤-globin gene in mouse is associated with interactions between multiple hypersensitive sites spanning Ͼ50 kb of DNA (9), whereas repression of the maternal IGF2 gene is linked to a long-range association between IGF2 and H19 loci, restricting access to an IGF2 enhancer (10).Several human diseases are associated with mutations in long-range control elements (11). Examples include Campomelic dysplasia, which can be caused by deletion of critical regulatory elements Ϸ50 kb upst...

Gene Looping Is Conferred by Activator-dependent Interaction of Transcription Initiation and Termination Machineries

“…Gene Looping Accompanies Activated Transcription-Several yeast, mammalian, and viral genes have been shown to adopt a looped configuration during transcription (12)(13)(14)(15)(16)(17). We further demonstrated that the looping of galactose-inducible genes accompanied activated transcription and not the repressed or transcriptionally poised state of the gene (13).…”

“…Accordingly, the termination factor-mediated loop at the mitochondrial rDNA transcription unit brought about efficient translocation of RNA polymerase from the terminator to the promoter, resulting in a 15-fold stimulation of transcription. The existence of similar gene loops has been recently reported for RNAP II-transcribed genes in yeast and mammalian cells (12)(13)(14)(15)(16). It has been shown that RNAP II-dependent gene looping is the consequence of the physical interaction of the terminator with the promoter of the same gene and is dependent on ongoing transcription.…”

“…The terminator-facilitated reinitiation may also result in an economic utilization of the limited amount of transcription factors and RNAP II molecules in the cell. It has been recently demonstrated that gene looping juxtaposes an inhibitory regulatory element located at the 3Ј end of BRCA1 gene near its promoter region, leading to transcriptional repression of the gene in breast tumor cell lines (16). In this case, gene looping represses rather than activates transcription.…”

Transcription termination by nuclear RNA polymerases

“…Moreover, these studies showed that gene looping is dependent on components of the cleavage/polyadenylation machinery and the general transcription factor TFIIB. In mammals, gene looping has been proposed to regulate transcription of the human BRCA1 gene (Tan-Wong et al 2008). Additionally, Perkins et al (2008) showed the existence of gene looping at an integrated HIV-1 provirus, which required a functional poly(A) signal.…”

CCCTC-binding Factor Acts Upstream of FOXA1 and Demarcates the Genomic Response to Estrogen

“…For this purpose, 3C assays were performed in estrogen-responsive MCF-7 cells and estrogen non-responsive MDA-MB-231 cells. But first the DNA looping of the GAPDH locus was evaluated to exclude a possible variation resulting from potentially differential enzymatic activity on different cellular DNAs (40). As evident in Fig.…”