Promoter-specific hypoacetylation of X-inactivated genes

Gilbert, Sandra L.; Sharp, Phillip A.

doi:10.1073/pnas.96.24.13825

Cited by 84 publications

(62 citation statements)

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“…Alternatively, it is likely that the heterogeneous nature of breast cancer and its microenvironment dictates the selective upregulation or down-regulation of certain X-linked genes at the gene level regardless of chromosomal copy number or activation status. Gilbert et al (28) also suggested that the promoter-specific hypoacetylation may be a key component of the X-inactivation machinery that operates at the level of individual genes. Nevertheless, we do not exclude the possibility of aneuploidy-associated aberrant gene expression.…”

Section: Discussionmentioning

confidence: 99%

Aberrant Expression of X-Linked Genes RbAp46, Rsk4, and Cldn2 in Breast Cancer

Thakur

Rahman

et al. 2007

Molecular Cancer Research

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The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes. (Mol Cancer Res 2007;5(2):171 -81)

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Section: Discussionmentioning

confidence: 99%

Aberrant Expression of X-Linked Genes RbAp46, Rsk4, and Cldn2 in Breast Cancer

Thakur

Rahman

et al. 2007

Molecular Cancer Research

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“…The specificity of the anti-acetylated antibodies was confirmed in quality control testing by the manufacturer, as treatment of cells with the deacetylase inhibitor sodium butyrate greatly enhanced detection of the acetylated forms of histones H3 and H4 in immunoblot analysis of acid-extracted proteins. Further demonstration that the antibodies are specific in the chromatin IP assay comes from our previous work, in which antibodies against acetylated histone H4 were specifically unable to immunoprecipitate detectable levels of promoter-specific sequences of X-inactivated genes, whereas they were able to immunoprecipitate many other X-linked sequences (8). The specificity of the anti-macroH2A1.2 antibodies has been previously demonstrated by immunoblot analysis from a variety of cell types, including mouse liver cells (10), undifferentiated and differentiated mouse embryonic stem cells (11), and mouse blastocysts (12).…”

Section: Methodsmentioning

confidence: 99%

“…Chromatin immunoprecipitations were then performed using various polyclonal antisera as described previously (8,9). Briefly, sonication of cross-linked nuclei was performed in a cup horn (Branson Sonifier 450) under conditions that gave a range in DNA fragments from 200 -1000 bps.…”

Section: Methodsmentioning

confidence: 99%

“…Histone hypoacetylation has been shown to feature prominently in X inactivation; microscopy studies have shown that histones H2A, H3, and H4 are underacetylated in the Xi as compared with other chromosomes (16 -18). Furthermore, we have recently shown by chromatin IP analysis from somatic cell hybrids containing a human Xi that histone H4 hypoacetylation is restricted to promoter regions of X-inactivated genes, whereas regions downstream of these promoters show a moderate level of acetylation (8). Antibodies capable of distinguishing between the acetylated and unacetylated isoforms of histones H3 and H4 were used to immunoprecipitate chromatin from 293 cells.…”

Section: Xist Rna Can Be Co-precipitated With Antibodies Againstmentioning

confidence: 99%

“…Histone macroH2A-To investigate whether XIST RNA directly associates with the chromatin of the Xi, we used the chromatin IP/RT-PCR (ChIP/RT-PCR) assay, which is a variation of the previously described chromatin immunoprecipitation (ChIP) assay (8,14). Chromatin fragments of 200 -1000 bp in length were generated by sonication and immunoprecipitated with antibodies against known components of the Xi chromatin.…”

Section: Xist Rna Can Be Co-precipitated With Antibodies Againstmentioning

confidence: 99%

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XIST RNA Associates with Specific Regions of the Inactive X Chromatin

Gilbert¹,

Pehrson²,

Sharp³

2000

Journal of Biological Chemistry

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Microscopy studies have shown that XIST RNA colocalizes with the inactive X chromosome (Xi). However, the molecular basis for this colocalization is unknown. Here we provide two lines of evidence from chromatin immunoprecipitation experiments that XIST RNA physically associates with the Xi chromatin. First, XIST RNA can be co-precipitated by antiserum against macroH2A, a histone H2A variant enriched in the Xi. Second, XIST RNA can be co-precipitated by antisera that recognize unacetylated, but not acetylated, isoforms of histones H3 and H4. The specificity of XIST RNA association with hypoacetylated chromatin, together with the previous finding that hypoacetylated histone H4 is enriched at promoters of X-inactivated genes, raises the possibility that XIST RNA may contribute to the hypoacetylation of specific regions of the Xi so as to alter the expression of X-linked genes.X-chromosome inactivation in mammals is controlled by the cis-acting XIST locus, which in humans encodes a 17-kilobase untranslated RNA that is expressed only from the inactive X (Xi) 1 (1). Mouse X chromosomes with targeted deletions of the Xist gene lose the ability to undergo X inactivation (2, 3). In addition, ectopic expression of Xist RNA from multicopy transgenes during embryonic stem cell differentiation causes autosomes to exhibit features typically associated with X inactivation, such as late replication, histone H4 hypoacetylation, and transcriptional down-regulation (4, 5). These studies indicate that the Xist locus is required for X inactivation, but they do not distinguish between several possible modes of Xist action; one model is that the Xist genomic locus serves as a chromatin organizing region that, when transcribed, initiates changes in chromatin structure that are propagated in cis along the length of the Xi chromosome (6). An alternative model is that the noncoding RNA expressed from the Xist gene is functional and directly participates in the silencing process (1). One observation suggesting that the XIST transcript may directly participate in silencing comes from fluorescence in situ hybridization experiments that show that XIST RNA colocalizes with the inactive X chromosome (1, 7). However, the limited resolution of microscopy analysis does not provide any information on the molecular basis or the consequences of this colocalization.To examine the nature of the colocalization between XIST RNA and the Xi chromatin, the chromatin IP assay was used to precipitate chromatin from human female cells and then RT-PCR was performed to determine whether XIST RNA was co-precipitated. The results indicate that XIST RNA is indeed co-precipitated with chromatin by antibodies directed against several histone species. Significantly, XIST RNA was co-precipitated with antibodies against unacetylated, but not acetylated, histones H3 and H4. Given the previous finding that histone H4 is hypoacetylated at promoters of X-inactivated genes (8), these results raise the possibility that XIST RNA may be associated with promoters of silenced gen...

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Histone acetylation and an epigenetic code

2000

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The enzyme-catalyzed acetylation of the N-terminal tail domains of core histones provides a rich potential source of epigenetic information. This may be used both to mediate transient changes in transcription, through modification of promoter-proximal nucleosomes, and for the longer-term maintenance and modulation of patterns of gene expression. The latter may be achieved by setting specific patterns of histone acetylation, perhaps involving acetylation of particular lysine residues, across relatively large chromatin domains. The histone acetylating and deacetylating enzymes (HATs and HDACs, respectively) can be targeted to specific regions of the genome and show varying degrees of substrate specificity, properties that are consistent with a role in maintaining a dynamic, acetylation-based epigenetic code. The code may be read (ie. exert a functional effect) either through non-histone proteins that bind in an acetylation-dependent manner, or through direct effects on chromatin structure. Recent evidence raises the interesting possibility that an acetylationbased code may operate through both mitosis and meiosis, providing a possible mechanism for germ-line transmission of epigenetic changes.

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Promoter-specific hypoacetylation of X-inactivated genes

Cited by 84 publications

References 47 publications

Aberrant Expression of X-Linked Genes RbAp46, Rsk4, and Cldn2 in Breast Cancer

Aberrant Expression of X-Linked Genes RbAp46, Rsk4, and Cldn2 in Breast Cancer

XIST RNA Associates with Specific Regions of the Inactive X Chromatin

Histone acetylation and an epigenetic code

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