Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

Pfeifer, Gerd P.; Riggs, Arthur D.

doi:10.1101/gad.5.6.1102

Cited by 189 publications

(135 citation statements)

References 54 publications

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“…Therefore, it is possible that the three critical methylated sites in the HPRT promoter region may function on the inactive X chromosome by binding MeCP2 and recruiting histone deacetylases that, in turn, maintain the repressive chromatin structure of the promoter. However, in vivo footprinting studies of both the HPRT gene (20) and the human PGK-1 gene (PGK-1) on the active and inactive X chromosomes (29,30) have not detected any evidence for stable binding of MeCP2 at methylated CpG dinucleotides on the inactive X chromosome (i.e. no in vivo footprints have been detected over methylated CpGs).…”

Section: De Novo Methylation Of the Human Hprt 5јmentioning

confidence: 99%

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

Chen¹,

Yang²,

Yang³

2001

Journal of Biological Chemistry

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The strong correlation between promoter hypermethylation and gene silencing suggests that promoter methylation represses transcription. To identify methylation sites that may be critical for maintaining repression of the human HPRT gene, we treated human/hamster hybrid cells containing an inactive human X chromosome with the DNA demethylating agent 5-azadeoxycytidine (5aCdr), and we then examined the high resolution methylation pattern of the HPRT promoter in single cell-derived lines. Reactivation of HPRT correlated with complete promoter demethylation. In contrast, the 61 5aCdr-treated clones that failed to reactivate HPRT exhibited sporadic promoter demethylation. However, three specific CpG sites remained methylated in all unreactivated clones, suggesting these sites may be critical for maintaining transcriptional silencing of the HPRT gene. Re-treatment of partially demethylated (and unreactivated) clones with a second round of 5aCdr did not increase the frequency of HPRT reactivation. This is consistent with mechanisms of methylation-mediated repression requiring methylation at specific critical sites and argues against models invoking overall levels or a threshold of promoter methylation. Treatment of cells with the histone deacetylase inhibitor, trichostatin A, failed to reactivate HPRT on the inactive X chromosome, even when the promoter was partially demethylated by 5aCdr treatment, suggesting that transcriptional repression by DNA methylation is unlikely to depend upon a trichostatin A-sensitive histone deacetylase.In mammals, DNA methylation at CpG dinucleotides in the 5Ј region of genes is frequently associated with transcriptional silencing (1), particularly in housekeeping genes on the inactive X chromosome. Numerous studies suggest that this association between promoter hypermethylation and transcriptional repression has a functional basis. For instance, individual loci on the inactive human X chromosome in human/ hamster hybrid cell lines may be reactivated using DNA-demethylating agents such as 5-azacytidine (2, 3) and 5-azadeoxycytidine, which inhibit the maintenance methyltransferase and incorporate into newly synthesized DNA (4), resulting in a failure to maintain methylation patterns during growth. Likewise, in vitro methylation of various promoter constructs results in inhibition of transcription in transient expression assays (5-9). However, despite significant evidence that DNA methylation represses transcription, specific mechanisms of this repression are only now becoming apparent.Recent reports suggest that DNA methylation mediates transcriptional repression indirectly, via binding of the methylated DNA-binding protein MeCP2, which in turn recruits histone deacetylases that modify the local chromatin structure (10, 11). However, additional mechanisms may also act to repress transcription by methylation, such as direct inhibition of transcription factor binding to its cognate site in DNA. Indeed, methylation of the binding sites of several transcription factors has been shown to alter ...

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Section: De Novo Methylation Of the Human Hprt 5јmentioning

confidence: 99%

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

Chen¹,

Yang²,

Yang³

2001

Journal of Biological Chemistry

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“…BALB/3T3 cells were permeabilized as described elsewhere (21). BALB/ 3T3 cells were used because NIH3T3 cells have a polymorphism in the MCP-1 promoter region that prevents a discernible footprint pattern (9,16).…”

Section: Micrococcal Nuclease (Mnase) Sensitivity Assaysmentioning

confidence: 99%

Communication Between NF-κB and Sp1 Controls Histone Acetylation Within the Proximal Promoter of the Monocyte Chemoattractant Protein 1 Gene

Boekhoudt

Guo

Beresford

et al. 2003

The Journal of Immunology

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The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-κB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-κB and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-κB p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-κB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.

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“…Due to the lack of footprints demonstrated using DMS, another method of footprinting was employed, DNaseI genomic footprinting (Pfeifer and Riggs, 1991). DNaseI is less base selective than DMS, is more sensitive to minor groove protein-DNA contacts, provides information on chromatin structure, and exhibits larger and clearer footprints than DMS.…”

Section: Genomic Footprinting In Ml-1 Cells With Dnaseimentioning

confidence: 99%

In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation

1997

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The tumor suppressor protein p53 has a transcriptional activation activity thought to mediate its biologic function including G 1 arrest and perhaps apoptosis. To learn more about p53's transactivator function in vivo, we performed genomic footprinting experiments examining p53-DNA interactions in the regulatory regions of the p53-regulated genes p21, GADD45, and MDM2. Using ionizing radiation to induce DNA damage in human ML-1 myeloblastic leukemia cells, the promoter and intronic regions of these genes containing p53-consensus binding sites were examined for in vivo footprints. There was a uniform and sustained expression of p53 protein as well as a strong induction of p21, GADD45, and MDM2 mRNA following irradiation. At the two p53 consensus binding sites in the p21 promoter, reduced DNaseI cleavage was observed in irradiated cells beginning 1 to 2 h after irradiation, being most pronounced after 2 h and diminishing after 8 h. A partial in vivo footprint was also observed in the third intron of the GADD45 gene beginning 2 h after irradiation. No in vivo footprints were seen at the two p53 binding sites in the MDM2 gene. Our study provides direct evidence that the DNA damage-induced activity of p53 is mediated by its consensus DNA binding sites in the p21 and GADD45 genes. We suggest that the transient nature and relative instability of p53-DNA interactions in vivo may make the p53 protein more accessible to a rapid turnover pathway which might be impaired under conditions when the protein is stably bound to DNA.

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Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

Cited by 189 publications

References 54 publications

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

Communication Between NF-κB and Sp1 Controls Histone Acetylation Within the Proximal Promoter of the Monocyte Chemoattractant Protein 1 Gene

In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation

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