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

  • ️Sun Jan 01 2006

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

Nele Gheldof et al. Proc Natl Acad Sci U S A. 2006.

Abstract

We have analyzed the effects of gene activation on chromatin conformation throughout an approximately 170-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 ( approximately 50 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.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.

3C analysis of the fragile X locus. (A) Map of the 170-kb fragile X locus located on the X chromosome. The location of the CGG repeat in FMR1 is indicated. Hatch marks, EcoRI sites; arrowheads, location of 3C primers; large arrows, transcription start sites; gray boxes, CpG islands. (B) RT-PCR analysis of USP22, HPRT1, FMR1, and FMR1NB transcription in normal and fragile X cells. The different sizes of the FMR1 transcripts are due to alternative splicing. (C) Analysis of interactions between the FMR1 promoter (primer 16) and fragments up- and downstream in normal cells (filled circles) and fragile X cells (open circles). Vertical bars indicate standard error of the mean (n = 3). (D) Analysis of interactions between an EcoRI fragment located 60 kb upstream of the FMR1 promoter (primer 5) and surrounding fragments. (Inset) The fold difference (average log ratio) of (normalized) interaction frequencies in fragile X cells compared with normal cells in the control locus (USP22) and the regions around fragments 16 and 5 in the fragile X locus. A 2.4-fold difference in interaction frequencies is observed in the region around the promoter (primer 16) (∗, P < 0.05), but no significant difference is observed in the upstream region (primer 5).

Fig. 2.
Fig. 2.

CCP of the fragile X locus. (A and B) Schematic representations of relative interaction frequencies (see Results) throughout the fragile X locus for sites separated by up to 50 kb in normal cells (A) and fragile X cells (B). Each relative interaction frequency was assigned a color depending on whether it was higher than (yellow hues), equal to (black), or lower than (blue hues) zero. Numbers above and left of the matrix refer to 3C primers (Fig. 1A). (C) Moving average of relative interaction frequencies determined with a 30-kb sliding window through the matrices in A and B (filled circles, normal cells; open circles, fragile X cells). A value of zero on the x axis corresponds to the transcriptional start site of the FMR1 gene. Vertical bars indicate standard error of the mean. (D) Fold change in relative interaction frequencies in fragile X cells versus normal cells along the fragile X locus. For each pair of fragments, the fold difference (log ratio) in interaction frequency in fragile X cells versus normal cells was calculated. The moving average of the fold difference is plotted by using a 30-kb sliding window. Horizontal dashed lines indicate the 95% confidence interval based on the error in data normalization (two times the standard error of the normalization factor). Points above the higher line or below the lower line are considered significantly different in normal cells versus fragile X cells.

Fig. 3.
Fig. 3.

Differences in interaction frequencies are not due to differential protein density. (A) Distribution of histone H3 across the fragile X locus in normal cells (filled bars) and fragile X cells (open bars) as determined by ChIP. (B) EcoRI digestion efficiency of cross-linked chromatin was calculated as the ratio of undigested to digested material in the three regions of the fragile X locus (indicated in A) in normal cells (filled bars) and fragile X cells (open bars). For each region, digestion efficiency of four EcoRI sites was determined in triplicate.

Fig. 4.
Fig. 4.

Histone modification patterns of the fragile X locus as determined by ChIP. (A) Distribution of H3 acetylation across the fragile X locus in normal cells (filled bars) and fragile X cells (open bars). (B) H4 acetylation. (C) H3K4 dimethylation. (D) H3K9 methylation.

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