Changes In Chromatin Architecture Research Articles

Chromatin-remodelling factors and the maintenance of transcriptional states through DNA replication

At the replication fork, nucleosomes, transcription factors and RNA polymerases are stripped off the DNA, the DNA double strands are unzipped and DNA methylation marks may be erased. Therefore DNA replication is both a 'curse' and 'bliss' for the epigenome, as it disrupts its stability by causing chromatin perturbations, yet it offers an opportunity to initiate changes in chromatin architecture and gene expression patterns, especially during development. Thus the DNA replication site is a critical point for regulation. It has become apparent that there is a close functional relationship between those factors that regulate transcriptional competence and the DNA replication programme. In this review we discuss novel insights into how chromatin-remodelling factors at replication sites are involved in both the maintenance and regulation of transcriptional states.

Development of 3D Chromatin Texture Analysis Using Confocal Laser Scanning Microscopy

Introduction: Analysis of nuclear texture features as a measure of nuclear chromatin changes has been proven to be useful when measured on thin (5–6 μm) tissue sections using conventional 2D bright field microscopy. The drawback of this approach is that most nuclei are not intact because of those thin sections. Confocal laser scanning microscopy (CLSM) allows measurements of texture in 3D reconstructed nuclei. The aim of this study was to develop 3D texture features that quantitatively describe changes in chromatin architecture associated with malignancy using CLSM images. Methods: Thirty-five features thoughtfully chosen from 4 categories of 3D texture features (discrete texture features, Markovian features, fractal features, grey value distribution features) were selected and tested for invariance properties (rotation and scaling) using artificial images with a known grey value distribution. The discriminative power of the 3D texture features was tested on artificially constructed benign and malignant 3D nuclei with increasing nucleolar size and advancing chromatin margination towards the periphery of the nucleus. As a clinical proof of principle, the discriminative power of the texture features was assessed on 10 benign and 10 malignant human prostate nuclei, evaluating also whether there was more texture information in 3D whole nuclei compared to a single 2D plane from the middle of the nucleus. Results: All texture features showed the expected invariance properties. Almost all features were sensitive to variations in the nucleolar size and to the degree of margination of chromatin. Fourteen texture features from different categories had high discriminative power for separating the benign and malignant nuclei. The discrete texture features performed less than expected. There was more information on nuclear texture in 3D than in 2D. Conclusion: A set of 35 3D nuclear texture features was used successfully to assess nuclear chromatin patterns in 3D images obtained by confocal laser scanning microscopy, and as a proof of principle we showed that these features may be clinically useful for analysis of prostate neoplasia.

Methylation-mediated Silencing of TMS1/ASC Is Accompanied by Histone Hypoacetylation and CpG Island-localized Changes in Chromatin Architecture

Aberrant methylation of CpG-dense islands in the promoter regions of genes is an acquired epigenetic alteration associated with the silencing of tumor suppressor genes in human cancers. In a screen for endogenous targets of methylation-mediated gene silencing, we identified a novel CpG island-associated gene, TMS1, which is aberrantly methylated and silenced in response to the ectopic expression of DNA methyltransferase-1. TMS1 functions in the regulation of apoptosis and is frequently methylated and silenced in human breast cancers. In this study, we characterized the methylation pattern and chromatin architecture of the TMS1 locus in normal fibroblasts and determined the changes associated with its progressive methylation. In normal fibroblasts expressing TMS1, the CpG island is defined by an unmethylated domain that is separated from densely methylated flanking DNA by distinct 5' and 3' boundaries. Analysis of the nucleoprotein architecture of the locus in intact nuclei revealed three DNase I-hypersensitive sites that map within the CpG island. Strikingly, two of these sites coincided with the 5'- and 3'-methylation boundaries. Methylation of the TMS1 CpG island was accompanied by loss of hypersensitive site formation, hypoacetylation of histones H3 and H4, and gene silencing. This altered chromatin structure was confined to the CpG island and occurred without significant changes in methylation, histone acetylation, or hypersensitive site formation at a fourth DNase I-hypersensitive site 2 kb downstream of the TMS1 CpG island. The data indicate that there are sites of protein binding and/or structural transitions that define the boundaries of the unmethylated CpG island in normal cells and that aberrant methylation overcomes these boundaries to direct a local change in chromatin structure, resulting in gene silencing.

Analyzing the contributions of chromatin structure in nuclear hormone receptor activated transcription in vivo.

The authors have described three approaches for analyzing the chromatin architecture of a steroid-responsive promoter. Mnase allows one to map the positions of nucleosomes on the target gene. The more sensitive restriction enzyme hypersensitivity procedure permits detection of changes in chromatin architecture upon hormonal stimulation. Additional insight into transcriptional regulation of a gene can be obtained by using the related ExoIII footprinting protocol, which provides complementary data on transcription factor binding to chromatin templates. The use of these in vivo chromatin analysis techniques have provided evidence for a role of chromatin structure in regulation of transcription of steroid-responsive promoters including MMTV (2,7,10,14), tyrosine aminotransferase (15), TR beta A (16-19), and retinoic acid receptor beta (RAR beta) (20).

SUV39H1 Drives Chromatin Structure

Modification of the histone proteins H4 and H3 occurs before changes in chromatin architecture. Whereas histone acetylation leads to greater DNA accessibility and transcription, histone methylation can also regulate the higher ordered structure of chromatin. Little is known of the mammalian proteins involved in the methylation of histones; however, Rea et al . have now identified a human protein termed SUV39H1 that methylates H3. In vitro methylation assays revealed that Lys 9 , located in the NH 2 -terminus of H3, is methylated in the presence of SUV39H1. Prior acetylation of Lys 9 blocked Lys 9 methylation, and phosphorylation of Ser 10 blocked the ability of SUV39H1 to methylate Lys 9 in vitro. Reciprocally, prior methylation of Lys 9 decreased the ability of aurora kinase (which can phosphorylate Ser 10 in H3) to use H3 as a substrate. However, prior acetylation of Lys 9 (compared to an unacetylated control) increased the ability of aurora kinase to phosphorylate Ser 10 . This suggests that steric hindrance is not the reason that methylation and phosphorylation were antagonistic, but that H3 NH 2 -terminal modification events are interdependent. Fluorescence assays indicated that SUV39H-deficient cells had increased amounts of phosphorylated H3, supporting the observation that methylation of Lys 9 blocked the phosphorylation of Ser 10 . Thus, having local regions of phosphorylated, acetylated, or methylated histones may contribute to epigenetic heritability of DNA states. A News & Views article by Paro discusses the role of catalytic domains in methyltransferases and the importance of histone modification. Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B.D., Sun, Z.-W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C.D., and Jenuwein, T. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406 : 593-599. [Online Journal] Paro, R. (2000) Chromatin regulation: Formatting genetic text. Nature 406 : 579-580. [Online Journal]

RPD3 (REC3) mutations affect mitotic recombination in Saccharomyces cerevisiae.

Prior research identified the recessive rec3-1ts mutation in Saccharomyces cerevisiae which, in homozygous diploid cells, confers a conditional phenotype resulting in reduced levels of spontaneous mitotic recombination and loss of sporulation at the restrictive temperature of 36 degrees C. We found that a 3.4-kb genomic fragment that complements the rec3-1ts/rec3-1ts mutation and which maps to chromosome XIV, is identical to RPD3, a gene encoding a histone de-acetylase. Sporulation is reduced in homozygous diploid strains containing the rec3-1ts allele at 24 degrees C, suggesting that this allele of RPD3 encodes a gene product with a reduced function. Sporulation is abolished in diploid strains homozygous for the rpd3Delta or rec3-1ts alleles, as well as in rpd3Delta/rec3-1ts heteroallelic diploids, at the non-permissive temperature. Acid-phosphatase expression has been shown to be RPD3 dependent. We found that acid-phosphatase activity is greater in diploid strains homozygous for the temperature-sensitive rec3-1ts allele than in RPD3/RPD3 strains and increased further when mutant strains are grown at 36 degrees C. We also tested the rpd3Delta/rpd3Delta strains for their effects on spontaneous mitotic recombination. By assaying a variety of intra- and inter-genic recombination events distributed over three chromosomes, we found that in the majority of cases spontaneous mitotic recombination was reduced in diploid rpd3Delta/rpd3Delta cells (relative to a RPD3/RPD3 control). Finally, although 90% of mitotic recombinant events are initiated in the G1 phase of the growth cycle (i.e., before DNA synthesis) we show that RPD3 is not regulated in a cell-cycle-dependent manner. These data suggest that mitotic recombination, in addition to gene expression, is affected by changes in chromatin architecture mediated by RPD3.