Purification of proteins associated with specific genomic Loci.
TL;DR: Proteomics of isolated chromatin segments (PICh) is described, identifying and validated several proteins that specifically bind to ALT telomeres, establishing PICh as a useful tool for characterizing chromatin composition.
About: This article is published in Cell.The article was published on 2009-01-09 and is currently open access. It has received 498 citations till now. The article focuses on the topics: Chromatin & Non-histone protein.
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TL;DR: This work has shown how plants are able to integrate the multitude of abiotic and biotic stresses in their natural habitat into their phenotypic plasticity.
Abstract: Plants constantly face a plethora of abiotic and biotic stresses in their natural habitat. Adapting to such changes requires a great degree of phenotypic plasticity that is mainly determined by the plant's genome. We currently do not know how plants are able to integrate the multitude of partly
1,034 citations
Cites background from "Purification of proteins associated..."
...…such as proteomics of isolated chromatin segments and stable isotope labeling with amino acids, are starting to demonstrate that identification of TFs and associated proteins in vivo at given promoters may become feasible in the near future (Dèjardin and Kingston, 2009; Mittler et al., 2009)....
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TL;DR: The TTAGGG repeat arrays of mammalian telomeres pose a challenge to the DNA replication machinery, giving rise to replication-dependent defects that resemble those of aphidicolin-induced common fragile sites.
887 citations
Cites background from "Purification of proteins associated..."
...Neither RTEL1 nor BLM were observed in an exhaustive PIChbased analysis of proteins associated with HeLa cell telomeres, although BLM was found at ALT telomeres (Dejardin and Kingston, 2009)....
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TL;DR: There are nine protein arginine methyltransferases (PRMTs) encoded in mammalian genomes, the protein products of which catalyse three types of ARG modifications: monomethylation and two types of dimethylation as discussed by the authors.
Abstract: There are nine protein arginine methyltransferases (PRMTs) encoded in mammalian genomes, the protein products of which catalyse three types of arginine methylation--monomethylation and two types of dimethylation. Protein arginine methylation is an abundant modification that has been implicated in signal transduction, gene transcription, DNA repair and mRNA splicing, among others. Studies have only recently linked this modification to carcinogenesis and metastasis. Sequencing studies have not generally found alterations to the PRMTs; however, overexpression of these enzymes is often associated with various cancers, which might make some of them viable targets for therapeutic strategies.
853 citations
TL;DR: This work has shown that the sequestration of the telomeric sequence into a protective nucleoprotein cap that masks the ends from constitutive exposure to the DNA damage response machinery reduces the risk of genome instability.
Abstract: The natural ends of linear chromosomes require unique genetic and structural adaptations to facilitate the protection of genetic material. This is achieved by the sequestration of the telomeric sequence into a protective nucleoprotein cap that masks the ends from constitutive exposure to the DNA damage response machinery. When telomeres are unmasked, genome instability arises. Balancing capping requirements with telomere replication and the enzymatic processing steps that are obligatory for telomere function is a complex problem. Telomeric proteins and their interacting factors create an environment at chromosome ends that inhibits DNA repair; however, the repair machinery is essential for proper telomere function.
842 citations
Cites background from "Purification of proteins associated..."
...In a recent study, the biochemical purification of the telomeric proteome produced a list of 210 proteins that interacted with and might influence telomeric structur...
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TL;DR: These studies identify points in the transcription cycle where RNA polymerase II accumulates after encountering a rate-limiting step and identify key regulatory steps and factors and provide an understanding of the mechanistic generalities, as well as the rich diversities, of gene regulation.
Abstract: In the eukaryotic genome, the thousands of genes that encode messenger RNA are transcribed by a molecular machine called RNA polymerase II. Analysing the distribution and status of RNA polymerase II across a genome has provided crucial insights into the long-standing mysteries of transcription and its regulation. These studies identify points in the transcription cycle where RNA polymerase II accumulates after encountering a rate-limiting step. When coupled with genome-wide mapping of transcription factors, these approaches identify key regulatory steps and factors and, importantly, provide an understanding of the mechanistic generalities, as well as the rich diversities, of gene regulation.
575 citations
Cites background from "Purification of proteins associated..."
...There has already been some success in such an examination of a repetitive region of the genom...
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References
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TL;DR: This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor, by using tandem affinity columns containing different protein binding sites.
Abstract: We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.
1,058 citations
"Purification of proteins associated..." refers background in this paper
...These include yeast one-hybrid (Li and Herskowitz, 1993) and nucleic acid affinity capture (Kadonaga and Tjian, 1986), which identify sequence-specific DNA-binding proteins....
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TL;DR: It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom.
Abstract: Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes We have now identified homologous proteins in fission yeast and in humans These Pot1 (protection of telomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends Deletion of the fission yeast pot1+ gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom
1,037 citations
"Purification of proteins associated..." refers background in this paper
...Telomere lanes (5-10) represent 12 and 6% of PICh purified telomeres....
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TL;DR: Tankyrase, a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP), was identified and localized to human telomeres.
Abstract: Tankyrase, a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP), was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1), a negative regulator of telomere length maintenance. Like ankyrins, tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro, with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro, suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation.
1,035 citations
TL;DR: The human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin and contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function.
Abstract: The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.
995 citations
"Purification of proteins associated..." refers background in this paper
...…Tankyrase 1 poly-ADP ribose polymerase (Smith et al., 1998); the Rad51D helicase (Tarsounas et al., 2004); the Werner Syndrome helicase WRN (Crabbe et al., 2004), although WRN interactors such as the WHIP ATPase and the TERA protein were found; and the SIRT6 deacetylase (Michishita et al., 2008)....
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..., 2004), although WRN interactors such as the WHIP ATPase and the TERA protein were found; and the SIRT6 deacetylase (Michishita et al., 2008)....
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TL;DR: These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.
Abstract: Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.
753 citations
"Purification of proteins associated..." refers methods in this paper
...Chromatin Immunoprecipitations ChIP assays have been performed essentially as described (Lee et al., 2006)....
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