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Intramural Papers of the Month

By Robin Arnette and Laura Hall
September 2009

RAP80, Part of an Autoregulatory Loop with HDM2 and p53, Enhances p53 Degradation

NIEHS researchers have discovered a new role for receptor-associated protein 80 (RAP80) as a mediator of E3 ubiquitin ligase HDM2 (HDM2) ubiquitination of p53 and as a direct transcriptional target of p53 following DNA damage.

RAP80 together with HDM2 and p53 form an autoregulatory loop. Higher RAP80 levels lead to destabilization of p53, making blocking of the p53-RAP80 interaction a possible therapeutic strategy against human cancers with low p53 expression.

The transcriptional factor p53 is a master regulator in DNA damage, cell cycle control and cell death. It is involved in preventing cancer by functioning as a tumor suppressor. The physiological level of p53 is normally kept low through ubiquitination by HDM2 and then degradation by the proteosome. p53 can activate the transcription of HDM2 gene, initiating p53 destabilization and creating an autoregulatory loop.

RAP80 gene was shown to also be a direct transcriptional target of p53 through a noncanonical response element in RAP80. The p53 DNA-binding domain bound RAP80. The effect of RAP80 on p53 ubiquitination was dependent on HDM2 expression - the ubiquitin interaction motifs of RAP80 were not involved.

Citation: Yan J, Menendez D, Yang X-P, Resnick M, Jetten AM.(http://www.ncbi.nlm.nih.gov/pubmed/19433585?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2009. A regulatory loop composed of RAP80-HDM2-p53 provides RAP80-enhanced p53 degradation by HDM2 in response to DNA damage. J Biol Chem 284(29):19280-19289.

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Alpers Disease Mutations In Polymerase Gamma Impair Mitochondrial DNA Replication

Structure function analysis of the catalytic subunit polymerase subdomain of the human mitochondrial DNA polymerase (polγ) showed that four Alpers syndrome mutations greatly reduced biochemical activity. The 99 percent activity loss in purified, recombinant forms of polγ compared to the wild type polymerase (wt) would severely impair mitochondrial DNA replication.

The four disease point mutations are in highly conserved regions of the thumb subdomain of the polymerase domain of POLG, the gene encoding polγ. Recombinants of two other Alpers mutations in less conserved regions of the adjacent palm subdomain retained 50-70 percent wild-type activity.

All replication and repair of mitochondrial DNA is done by polγ. Mutations in POLG can impair the polymerase leading to mitochondrial diseases such as Alpers syndrome, an autosomal recessive disease with early onset mitochondrial DNA depletion. Mitochondria make over 90 percent of the energy needed by cells to function. Seriously impaired mitochondrial function can be fatal.

The NIEHS researchers showed that the mutants retained the wild type physical and functional relationships in the interaction with p55, a polγ accessory subunit, had no misinsertion fidelity defects, and were properly folded with secondary structure similar to the wild-type enzyme. Differences in mutant protein DNA binding suggest a possible orientation of the polymerase to DNA during catalysis.

Citation: Kasiviswanathan R, Longley MJ, Chan SS, Copeland WC.(http://www.ncbi.nlm.nih.gov/pubmed/19478085?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2009. Disease mutations in the human mitochondrial DNA polymerase thumb subdomain impart severe defects in mitochondrial DNA replication. J Biol Chem 284(29): 19501-19510.

Article (http://www.niehs.nih.gov/news/newsletter/2009/august/science-investigators.cfm) in August Environmental Factor

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Mi-2/NuRD Is Part of a Novel Chromatin Assembly Pathway

The chromatin remodeling complex, Mi-2/NuRD, mobilizes nucleosomes through the Mi-2 ATPase subunit and deacetylates histones through the histone deacetylase (HDAC) subunit, but recent work has determined that the complex is also involved in the maturation and maintenance of heterochromatin in rapidly proliferating lymphoid cells. Investigators at NIEHS, Duke University and Emory University School of Medicine have shown that Mi-2/NuRD localizes to pericentromeric heterochromatin on human chromosomes 1, 9 and 16 during late S phase. The association occurs in some but not all cell types.

The research team used immunofluorescence and fluorescence in situ hybridization to visualize where Mi-2/NuRD localized within lymphoid cells. The NuRD bodies were present in a variety of B lymphocyte-derived cell lines as well as primary human cells. NuRD localized to heterochromatin that contained HP1 proteins and histone H3 trimethylated at lysine 9. In contrast, heterochromatin that lacked NuRD bodies differed in composition and instead assembled Polycomb proteins.

The research suggests the NuRD bodies are cytologic markers of a novel chromatin assembly pathway used by lymphoid cells at pericentromeric heterochromatin.

Citation: Helbling Chadwick L, Chadwick BP, Jaye DL, Wade PA.(http://www.ncbi.nlm.nih.gov/pubmed/19296121?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2009. The Mi-2/NuRD complex associates with pericentromeric hetrochromatin during S phase in rapidly proliferating lymphoid cells. Chromosoma 118(4): 445-457.

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Web-Based SNP Tools Offer Unique Usability Features

NIEHS researchers have developed a set of single nucleotide polymorphism (SNP) selection tools that allow investigators to select SNPs using results from genome-wide associated studies (GWAS), linkage disequilibrium (LD) and predicted functional characteristics of both coding and non-coding SNPs. The tools are web based and may be used for small or large-scale SNP selection.

The authors designed the application, SNPinfo, to have three pipelines for SNP selection based on candidate genes, whole genome and linkage regions, with options to combine all three pipelines. In addition, the tools incorporate functional predictions of protein structure, gene regulation, splicing and miRNA binding and the ability to consider whether the alternative alleles of a SNP are likely to have differential effects on function.

As a validation step the authors show that a very small panel of SNPs chosen with the application captures most of the SNPs subsequently shown to be associated with disease. A variety of tools on the web site allows investigators to easily see all of the SNPs in a gene, their frequency in different populations, linkage disequilibrium, predicted functional consequences and sequence context. Public access to the site is at www.niehs.nih.gov/snpinfo(http://www.niehs.nih.gov/snpinfo), and help files on the site provide a useful introduction to the different tools.

Citation: Xu Z, Taylor JA.(http://www.ncbi.nlm.nih.gov/pubmed/19417063?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2009. SNPinfo: integrating GWAS and candidate gene information into functional SNP selection for genetic association studies. Nucl Acids Res 37: W600-W605; doi:10.1093/nar/gkp290 (Web Server Issue).

(Laura Hall is a biologist in the NIEHS Laboratory of Pharmacology currently on detail as a writer for the Environmental Factor.)



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