Chromatin chronicles

While not Oscar-worthy, cinematography makes up for lack of plot twists and compelling drama in September Science paper…

In a dazzling visual display, Janssen et al. demonstrate a link a between chromosome missegregation and DNA-level structural defects, such as deletions and translocations, in human cells. The experimenters connect segregation errors, examining both induced missegregation in immortalized cell lines and endogenous errors in tumor cell lines, with structural damage using DNA damage markers such as γH2AX and 53BP1. Analysis of DNA damage marker immunofluorescence in real time suggested that DNA damage occurred at cytokinesis due to mechanical stress at the cleavage furrow. Janssen and peers confirmed these observations by inhibiting cytokinesis (DNA damage was reduced) and by observing the spatial and temporal occurrence of damage in relation to cytokinesis (damage occurred mainly at cytokinesis and when missegregating chromosomes became “trapped” in the cleavage furrow). Reasoning that chromosome breakage should activate double strand break repair, the experimenters demonstrated that chemically induced missegregations resulted in autophosphorylation of ATM and phosphorylation of Chk2 (two components of double strand break repair). Missegregation errors were also shown to cause p53 activation, demonstrating a connection between segregation and errors DNA damage checkpoint activation. The investigators also examined overall chromosome morphology under the induced segregation error conditions and observed an increase in the number of structural [...]

Chromatin Chronicles has a new look!

I’m happy to announce that I have a new lab in the Department of Microbiology at the University of Georgia! In honor of the new digs, chromatin chronicles has adopted a new “Red and Black” look. 2011 has been a busy year, but after a winter of job interviews and a big move from Oregon to Georgia, things are finally settling down. I hope to resume making regular posts to highlight recent breakthroughs in epigenetics and chromatin structure. As the lab grows, you may even see some new contributors here on the site. I hope you’ll forgive the recent lack of activity and check back in soon.    

CENP-B regulates genome stability

Controlling transposable elements is critical for preserving genome integrity. In a study published several years ago, Cam et al. demonstrated that in fission yeast, homologs of the transposase-derived protein CENP-B function to control the activity of Tf2 retrotransposons. Fission yeast CENP-B homologs associate with long-terminal repeats (LTRs) of Tf1 and Tf2 elements and direct their transcriptional silencing by recruiting histone deacetylases. In a new study published in Nature, Robert Martienssen’s group reports that CENP-B homologs also function to promote replication fork progression through transposons (Zaratiegui et al. CENP-B preserves genome integrity at replication forks stalled by retrotransposon LTR). Martienssen and colleagues found that CENP-B double mutants exhibit high rates of double strand breaks, which apparently result from replication fork collapse at Tf2 elements. Interestingly, they identified a mutation in the Sap-1 gene that suppresses both the growth and genome stability defects of CENP-B mutants. Sap-1 is a DNA binding protein that blocks DNA replication in a direction-dependent manner. The authors found that like the CENP-B proteins, Sap-1 binds to Tf1 and Tf2 LTRs. Interestingly, de novo Tf1 insertion events frequently occur upstream of Sap-1 binding sites, suggesting that Sap-1 influences the site and orientation of transposon insertion. Two-dimensional gel electrophoresis [...]

The power of Neurospora unleashed: the orgin of aberrant RNA and a shameless plug

In the spirit of shameless self-promotion, check out my latest paper in the November 4th issue of PLoS Genetics. Also, check out this exciting Neurospora study published by Heng-Chi Lee and colleagues in PLoS Biology. Lee et al. report a major breakthrough in understanding how quelling (RNA interference; RNAi) is initiated. In Neurospora, integration of multi-copy transgenes frequently leads to quelling of the transforming DNA. This process depends on the typical RNA-interference machinery, including a presumed RNA-dependent RNA polymerase (QDE-1), an Argonaute protein (QDE-2), a Dicer protein (DCL-1 or DCL-2), and a DNA helicase (QDE-3). The model for quelling in Neurospora is similar to models for RNAi in other organisms: 1) An aberrant RNA is recognized and converted into double strand RNA (dsRNA) by an RNA-dependent RNA polymerase. 2) The dsRNA is degraded into 20-25 nucleotide interfering RNAs by Dicer. 3) These small RNAs are then bound by the Argonaute-containing RNA-induced silencing complex (RISC) to direct cleavage of homologous RNAs by Argonaute. A major question is, what is the source of aberrant RNA? Lee et al. show that recombinant QDE-1 exhibits RNA polymerase activity using both DNA and RNA templates. The DNA-dependent activity requires single stranded DNA (ssDNA) and is [...]

Noncoding RNAs flip the switch to turn off X.

To achieve dosage compensation, mammalian females inactivate one of their two X chromosomes. This process is extremely complicated; cells must choose an X to inactivate, initiate inactivation at the appropriate developmental stage, and then maintain transcriptional silencing of more than 1000 genes across multiple cell divisions. X chromosome inactivation has been the subject of intense investigation for decades, and intricate details of this fascinating process continue to emerge. One of the critical steps in X inactivation is mono-allelic expression of the noncoding RNA Xist, which spreads in cis along the X destined to be silenced. Xist itself is regulated by another noncoding RNA, Tsix. Tsix also acts in cis, inhibiting expression of the overlapping Xist transcript from the opposite DNA strand, thereby designating the active X. Previous studies suggested that positive regulators of Xist must also be important for X inactivation, however, such factors had  yet to be identified. In the October 29th issue of Cell, Jennie Lee’s group reports the identification of one such factor. The paper by Tien et al. describes the investigation of Jpx, yet another noncoding gene that resides in the X inactivation center. Using a cultured embryonic stem (ES) cell model for X inactivation, the authors [...]

A non-coding heterochromatin locus causes hybrid lethality in Drosophila. Not bad for junk DNA, eh?

Do rapidly evolving repeated DNAs drive speciation? In some cases they can, according to a recent paper by Ferree and Barbash published in PLoS Biology . The report, entitled “Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila“, describes the characterization of a previously mapped locus, named Zhr (Zygotic hybrid rescue), which causes female lethality in crosses of Drosophila simulans females with D. melanogaster males. Previous studies had shown that the Zhr1 allele suppressed lethality in these interspecific crosses and that the Zhr+ locus resides within the pericentric heterochromatin of the X chromosome. Zhr consists largely of satellite DNA composed of a tandemly repeated 359bp sequence. Thus, Zhr could be a gene embedded in the pericentric heterochromatin or could be non-coding repeated DNA itself. Since pericentric heterochromatin is known to influence chromosome segregation, the authors examined mitotic chromosome segregation in developing hybrid female embryos. They observed high rates of failed mitoses and lagging chromosomes, suggesting that these hybrid embryos were defective for chromosome segregation. FISH experiments revealed that segregation defects were restricted to the D. melanogaster X chromosome and the 359bp sattelite block, which exhibited an abnormal, decondensed chromatin structure and was retained at the metaphase plate. [...]

Viral protein hijacks heterochromatin to silence the p53 tumor suppressor pathway.

In a recent article published in Nature, Soria et al. report the identification of a novel viral protein that suppresses the p53 tumor suppressor pathway. The article, entitled “Heterochromatin silencing of p53 target genes by a small viral protein,” describes an elegant and comprehensive study that led to the identification of E4-ORF3, a protein that works in concert with the E1B-55k pathway to disrupt p53 signaling in virus-infected cells. Whereas E1B-55k triggers degradation of p53, E4-ORF3 acts to silence p53 target genes by directing a repressive chromatin modification to these loci. Only viruses deleted for both E1B-55k and E4-ORF3 induce a full p53-mediated response upon viral infection. Interestingly, E4-ORF3 forms a complex scaffold structure within the nucleus that co-localizes with histone H3 methylated on K9 and two H3 K9 methyltransferases, SUV39H1 and SUV39H2. Significantly, microarray analysis revealed that E4-ORF3 represses a very specific group of target genes, which are highly enriched for p53 targets, but also for other genes that mediate anti-viral responses.

gamma-H2A marks fragile sites during S-phase

In a report published in last month’s issue of PLoS Genetics, Rozenzhak et al. describe the S-phase distribution of gamma-H2A in budding yeast. In their article, entitled “Rad3/ATR Decorates Critical Chromosomal Domains with gamma-H2A to Protect Genome Integrity during S-Phase in Fission Yeast“, the authors report that gamma-H2A accumulates at fragile genomic sites within the fission yeast genome during DNA replication. Notably, these sites include natural replication fork barriers as well as pericentric and telomeric heterochromatin domains. Accumulation of gamma-H2A at heterochromatin requires the Clr4 H3 K9 methyltrasferase and functions to recruit Brc1, a protein that stabilizes replication forks. Importantly, this function is critical in the absence of the RecQ helicase, Rqh1. This comprehensive study has revealed yet another link between H3 methylated on K9 and maintenance of genome stability, reminiscent of recent reports using Drosophila (Peng et al.) and mammalian (Sun et al.) systems. It seems increasingly clear that heterochromatin is an important regulator of genome stability, however, further studies are required to determine the precise molecular mechanisms at work.

Fun with microscopes – a model for higher-order centromere structure.

Centromeres are model epigenetic loci – their function depends on a specific chromatin structure rather than the associated DNA sequence. Specifically, centromeres are enriched in CENP-A, a variant of H3, which is found interspersed with H3-containing nucleosomes across centromere domains. These mixed CENP-A/H3 domains assemble into higher-order chromatin structures, which ultimately direct formation of the kinetichore. Given the complexity of chromatin and the massive kinetochore, it is not surprising that the precise structure of kinetichore-associated, centromeric chromatin is not well understood. One proposed model invokes a helical structure in which CENP-A nucleosomes form the outer kinetochore, while cannonical H3 nucleosomes make up the interior.  However, an interesting study of higher-order centromere structure in chicken cells led the authors to propose an alternative model for centromere structure (Ribeiro et al. PNAS). Individual chromatin fibers were resolved by incubating cells with low-salt buffer to unravel kinetichore-associated chromatin. Fluorescence microscopy revealed that CENP-A containing nucleosomes were interspersed with H3 nucleosomes methylated at K9, while lower levels of di-methyl H3K4 methylation were detected. Kinetichore-associated chromatin was more stable in mitotic cells compared to interphase cells, as measured by the level of unfolding in low-salt buffer. Significantly, depletion of the centromere protein CENP-C, but not [...]

No-crossing zone: A mechanism for suppression of recombination at centromeres.

Ellermeier et al. report in the early edition of PNAS that in the fission yeast, S. pombe, RNAi and heterochromatin repress meiotic recombination at the centromere. The idea that centromeres prevent meiotic recombination is certainly not a new one (just check out this paper by George Beadle published in 1932), but the molecular mechanisms responsible have remained a mystery. Ellermeier and colleagues examined recombination frequency across fission yeast centromere 3 in wildtype and various mutant strains. These analyses revealed a recombination rate of < 0.1% in wildtype cells, whereas mutants lacking components of the RNAi machinery (i.e. Dicer, RNA dependent RNA polymerase or argonaute) exhibited significantly increased recombination rates across the centromere. RNAi is required to direct methylation of H3 on lysine-9, and consistent with this, mutation of Clr4 or Rik1, members of the H3 lysine-9 methyltransferase complex, also resulted in dramatic increases in centromeric recombination. Heterochromatin protein-1/Swi6 is known to bind H3 methylated at lysine-9 and serves as an adaptor that recruits effector proteins to heterochromatin. Swi6 is required for transcriptional repression at fission yeast centromeres, but surprisingly, the authors found that Swi6 is dispensable for suppression of recombination. Furthermore, additional proteins that act to repress transcription at centromeres, [...]

It helps to be in the right place at the right time.

In the latest issue of Genes and Development, a report from Susan Gasser’s laboratory (Meister et al.) examines the relationship between a gene’s developmental regulation and its nuclear location. The authors visualized the sub-nuclear localization of several developmentally-regulated genes during differentiation of the nematode, C. elegans. Meister and colleagues found that the localization of these genes reflects their transcriptional activity. For example, the muscle-specific myo-3 gene was found near the nuclear periphery in gut, hypodermal and seam cells, but was found in the nuclear lumen in muscle cells. Similarly, an intenstinal-specific gene was localized to the nuclear core in the gut, but in cells where this gene is silent, it is found near the nuclear periphery. Internalization of the tissue-specific transgenes occurred independently of the chromatin context; high-copy transgene arrays were associated with heterochromatic marks, but were still actively transcribed and internalized in a tissue-specific manner. Using a large array containing an intestine-specific transgene, the authors determined the temporal sequence of transcriptional activation and relocation. During differentiation of the gut, transcription activation occurred first at the nuclear periphery and was followed by decondensation of the large transgene array. The array was then relocated to the nuclear lumen, where it formed [...]

Cfp1 – working to keep your CpG islands methylation free.

In mammals, CpG dinucleotides are largely depleted from the genome and most CpGs are methylated. However, many promoters buck this trend – over 13,000 promoters are associated with CpG-rich islands that are typically unmethylated. Keeping CpG islands free of methylation is critically important, as inappropriate promoter methylation can lead to gene silencing and disease (e.g. cancer). Consequently, there is significant interest in identifying the mechanisms that regulate chromatin structure at these sites. Previous studies suggested that tri-methylation of histone H3 at lysine 4 (H3K4me3) can antagonize DNA methylation at CpG islands; however, the mechanisms that direct H3K4me3 to these islands are not well-understood. A report published by Adrian Bird and colleagues in the journal Nature suggests that the DNA binding protein Cfp1 plays a role in this process. Previous work demonstrated that the CXXC domain of Cfp1 binds CpG-rich DNA if the CpGs are unmethylated. In the current paper, Dr. Bird’s group shows that in vivo, Cfp1 interacts specifically with over 80% of the unmethylated CpG islands in the genome. Furthermore, they show that Cfp1 exhibits extensive co-localization with with H3K4me3 at these genomic loci. RNA knockdown of Cfp1 resulted in reduced H3K4me3 at CpG islands, suggesting that Cfp1 may [...]

Pick of the week: Twin study uncovers a link between DNA methylation and lupus.

With so many interesting papers out this week, it was difficult to pick a favorite; however, the very cool twin study published in Genome Reseach by Javierre et al. (Genome Res. 2010. 20: 170-179 ) is definitely worth checking out. The authors examined DNA methylation at 807 CpG containing promoters from sets of monozygotic twins discordant for three autoimmune diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) or dermatomyositis (DM). No differences were observed for RA or DM, but 49 genes showed decreased DNA methylation in SLE twins compared to their healthy siblings. Methylation changes at these genes were reproducibly detected in multiple pairs of discordant twins, indicating that the observed changes in DNA methylation may be a general feature of the autoimmune disease. Significantly, this group of genes was enriched for modulators of immune function, and a subset of these genes exhibited increased gene expression in SLE siblings. SLE patients showed global decreases in DNA methylation, however, repeated sequences that are typically methylated were unaffected (e.g. Alu elements, LINE-1 elements, or the Satellite-2 repeats). In contrast, methylation at the rDNA repeats was reduced in SLE individuals. Currently, it isn’t clear if these changes in methylation are directly responsible for disease [...]

Chromatin dynamics in cancer cells – Dr. Siavash Kurdistani presents his work in the Institute of Molecular Biology seminar series

Members of the Institute of Molecular Biology here at the University of Oregon were treated to a great talk yesterday by Dr. Siavash Kurdistani from the Department of Biological Chemistry at UCLA. Work by the Kurdistani lab revealed that loss of histone H3 lysine-18 aceylation (H3K18ac) is correlated with a poor prognosis in cancer patients, suggesting that loss of this modification is an important event in tumor progression. Consistent with this observation, the Kurdistani lab demonstrated that oncogenic transformation by the adenovirus protein e1a is accompanied by dramatic changes in the genomic location of H3K18 acetylation. To achieve transformation, e1a binds temporally to three classes of promoters, orchestrating a cascade of gene expression changes. First, e1a represses anti-viral response genes. The protein then directs H3K18 acetylation to induce expression of cell cycle genes, thereby promoting cell division. Finally, e1a binds to a third set of genes, repressing their expression to inhibit differentiation. Gene regulation by e1a appears to involve direct interactions with the histone acetyltransferases p300 and CBP, which are required for normal levels of H3K18ac, and Rb family proteins. Check out these papers to learn more about this work (Science. 2008 Aug 22;321(5892):1086-8; Science. 2008 Aug 22;321(5892):1084-5). Dr. Kurdistani [...]

Semiconservative inheritance of nucleosomes?

The term “epigenetic” is used to describe phenotypic differences that don’t result from changes in the underlying DNA sequence, but rather, are the result of specific chromatin modifications associated with a gene sequence (e.g cytosine methylation or covalent histone modfications). Because these associated chromatin modifications ultimately determine whether a gene is inherited in the “silent” or “active” state, understanding the mechanism of their inheritance is of great interest.  The mechanisms responsible for faithful inheritance of DNA methylation are well-understood, but those responsible for faithful propagation of histone modification patterns are less clear. Xu et al. report in the April 2nd issue of Science that the histone variant H3.3 can be inherited semiconservatively, through a mechanism that involves splitting of the nucleosome into two H3/H4 dimers. Histone inheritance has been intensely investigated and numerous studies have shown that during replication, tetramers of H3/H4 are not split, but instead are segregated to one daughter strand or the other (Annunziato, 2005). Consistent with this, Xu et al. observe that tetramers containing H3.1, which is deposited in a DNA replication-dependent manner, are passed to daughter strand intact. Their data suggest that distinct mechanisms are used to distribute H3.1 and H3.3 (and their associated modifications) [...]