The active FMR1 promoter is associated with a large domain of altered chromatin conformation with embedded local histone modifications

Abstract

We have analyzed the effects of gene activation on chromatin conformation throughout an Ϸ170-kb region comprising the human fragile X locus, which includes a single expressed gene, FMR1 (fragile X mental retardation 1). We have applied three approaches: (i) chromosome conformation capture, which assesses relative interaction frequencies of chromatin segments; (ii) an extension of this approach that identifies domains whose conformation differs from the average, which we developed and named chromosome conformation profiling; and (iii) ChIP analysis of histone modifications. We find that, in normal cells where FMR1 is active, the FMR1 promoter is at the center of a large (Ϸ50 kb) domain of reduced intersegment interactions. In contrast, in fragile X cells where FMR1 is inactive, chromatin conformation is uniform across the entire region. We also find that histone modifications that are characteristic of active genes occur tightly localized around the FMR1 promoter in normal cells and are absent in fragile X cells. Therefore, the expression-correlated change in conformation affects a significantly larger domain than that marked by histone modifications. Domain-wide changes in interaction probability could reflect increased chromatin expansion and may also be related to an altered spatial disposition that results in increased intermingling with unrelated loci. The described approaches are widely applicable to the study of conformational changes of any locus of interest.chromatin domain ͉ chromosome conformation capture ͉ transcription ͉ fragile X locus G ene expression is associated with alterations in chromatin structure, both at the nucleosome level and at the level of larger chromatin domains. Whereas modification at the nucleosome level has been studied in detail, conformational changes of larger domains in relation to gene expression are much less understood, in part because of technical challenges to the study of chromatin at the level of tens of kilobases.Active genes are marked by acetylation of histones H3 and H4 and by methylation of H3 at lysine 4 (H3K4). Recent studies have shown that these modifications occur in a punctate fashion at or near transcription start sites (1, 2). Promoter regions of active genes are also characterized by changes in chromatin accessibility, as exemplified by formation of DNase I hypersensitive sites (3).In several cases, gene activation is accompanied by large-scale changes in chromatin conformation. First, activation or repression of genes can involve formation of chromatin loops through long-range interactions between regulatory elements (4-8). Second, activation of gene clusters can be accompanied by large-scale chromatin decondensation. For instance, upon activation of the ␤-globin locus, the entire Ϸ120-kb gene cluster becomes more accessible to nucleases, suggesting that chromatin throughout the locus is less condensed (9). Similarly, induction of arrays of multiple copies of reporter genes resulted in formation of extended chromatin fibers (10, 11). Decondensation is o...

Variable Requirements for DNA-Binding Proteins at Polycomb-Dependent Repressive Regions in Human HOX Clusters

“…The ChIP protocol provided by Agilent was followed. The following antibodies were used: rabbit anti-human BMI1 (Kingston lab), which was validated in references 18 and 26, SUZ12 (ab12073; Abcam) for ChIP (27,28) Fig. 5A and B).…”

Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements

“…These interactions likely reflect nonfunctional random collisions resulting from the intrinsic close proximity of neighboring restriction fragments (Dekker et al 2002;Dekker 2006). The frequent random interactions between adjacent genomic fragments are likely dependent on local physical properties of the chromatin fiber and limit the ability to detect specific looping interactions between elements separated by small genomic distances (2-5 kb) (Dekker 2006;Gheldof et al 2006). Importantly, random collisions are predicted to decrease progressively for sites separated by increasingly large genomic distances.…”

“…Generation of complete interaction matrices can be used as a discovery tool for unbiased detection of chromatin looping interactions between previously unannotated elements. Analysis of a matrix of interaction frequencies can also provide global information regarding the general spatial conformation of a genomic region (Dekker et al 2002;Gheldof et al 2006). …”

Integrating one-dimensional and three-dimensional maps of genomes

“…3C involves analyzing the interactions between pairs of loci one by one through quantitative PCR, using specific primers to detect and quantify the corresponding ligation products in the library of 3C-ligation products (Dekker et al, 2002). 3C is typically used to analyze relatively small genomic regions (up to several hundred Kb) (Gheldof et al, 2006;Miele et al, 2009;Tolhuis et al, 2002). 4C (3C-on-chip or Circular 3C) allows detection of a genome-wide interaction profile of a single locus of interest (Simonis et al, 2006;Zhao et al, 2006).…”