Karissa McClinic et al. (2006) Development & Evolution Meeting "Characterization of the Histone Variant H2A.Z in C. elegans"

Karissa McClinic, Brianne Ray, William Kelly

[Development & Evolution Meeting, 2006]

Eukaryotic genomes are packaged into complexes composed of both DNA and histone proteins which assemble to form chromatin. Chromatin is important in the regulation of genes, and can exist in an open state in which the DNA is actively transcribed or alternatively in a closed state where transcription is repressed. Three mechanisms that are known to change the structural state of chromatin include: chromatin remodeling, posttranslational modifications of histones, and incorporation of histone variants. A number of histone variants have been identified, and many are conserved from yeast to mammals. While it is known that histone variants can influence, or are correlated with gene expression, the mechanisms are poorly understood. H2A.Z is a variant of the core H2A histone, and is incorporated through an ATP-dependent chromatin-remodeling complex. In higher eukaryotes, H2A.Z is essential to development, and is thought to play a role in maintaining heterochromatic states. Our preliminary experiments in C. elegans have shown that RNAi targeted to H2A.Z leads to embryonic lethality with a few embryos escaping lethality and arresting as larvae. In addition, immunofluorescence staining with antibodies specific to H2A.Z have revealed that H2A.Z is excluded from the X chromosome in adult germ cells but present on the autosomes. These results indicate that H2A.Z is localized to actively transcribed chromatin and is essential for embryonic development. We are also investigating the chromatin remodeling machinery that is involved in the incorporation of H2A.Z into the nucleosome. Swr-1, an ATP-dependent chromatin-remodeling factor, is believed to remodel this variant into chromatin in budding yeast. We are testing a mutant containing a deletion in the Swr-1 C. elegans homolog. Using this mutation in combination with RNAi strategies, we hope to explore how Swr-1 works and interacts with H2A.Z. With C. elegans as a model system, we hope to better determine the role of this H2A.Z variant in metazoan genome regulation.

Karissa N. McClinic et al. (2007) International Worm Meeting "Characterization of htz-1 in C. elegans Development."

Karissa N. McClinic, Bill Kelly, Jason Lieb, Christina Whittle

[International Worm Meeting, 2007]

Eukaryotic genomes are packaged into nucleosomes that contain both DNA and histone proteins and self-assemble to form different hierarchies of chromatin. Chromatin is thus highly important in the regulation of eukaryotic genes. Three overlapping mechanisms are proposed to change and hence influence gene activity: energy-dependent chromatin remodeling, histone post-translational modifications, and active incorporation of histone variants. A number of histone variants have been identified, and many are conserved from yeast to mammals. H2A.Z is a variant of the core H2A variant, that in budding yeast is incorporated into the nucleosome through an ATP-dependent chromatin remodeling complex, SWR1. Using C. elegans as a model system, we are studying the role of the C. elegans H2A.Z, HTZ-1, throughout development and the machinery used to remodel H2A.Z into the nucleosome. Preliminary experiments in C. elegans show that RNAi targeted to H2A.Z leads to a high percentage of embryonic lethality with animals that escape embryonic lethality arresting in larval developmental stages. Immunofluorescence with antibodies specific to H2A.Z have revealed a genomic distribution that appears to be limited to actively transcribed chromatin. H2A.Z appears to be present throughout development, and in adult germ cells is not found on the X chromosome. We are also studying the effect of the SWR1 remodeling complex on the incorporation of H2A.Z into the nucleosome. Through RNAi experiments, we would like to identify how the components of the SWR1 complex interact with HTZ-1 to effect its genomic distribution. Furthermore, in collaboration with the Lieb lab at UNC, we have determined where H2A.Z is enriched in the C. elegans genome during embryogenesis by performing ChIP-chip assays.

Tom Ratliff et al. (2004) East Coast Worm Meeting "Identification of factors required for germline silencing"

David Han, Bill Kelly, Karissa McClinic, Tom Ratliff

[East Coast Worm Meeting, 2004]

Transcriptional repression (silencing) is a major theme in the establishment and maintenance of the C. elegans germline. Loss of silencing in the embryonic germline can cause the transformation of germ lineage precursors to somatic cells while in the post-embryonic germline desilencing can adversely affect the differentiation of germ cells in the gonad. A loss of silencing in the adult germline can be assayed by expression of repetitive extrachromosomal arrays, which are normally strongly silenced in this tissue. To genetically identify factors involved in germline silencing, we have isolated mutations that allow germline GFP expression from a repetitive extrachromosomal array. To date, all sig ( s ilencing i n the g ermline-defective) mutants recovered are variably sterile, suggesting that the genes identified by these mutations are required for fertility and that the germline silencing mechanism is an essential requirement in C. elegans . In addition to isolating new sig mutations, we are focusing on the characterization and cloning of sig-2 and sig-7 , each identified by a single mutation. The sig-2 mutant appears to have a germline-specific phenotype. In addition to the array desilencing defect, sig-2 animals are incompletely penetrant sterile. There is also a noticeable delay in the onset of oogenesis in the cohort of animals that eventually give rise to offspring. Mapping of sig-2 is in its very early stages. The sig-7 mutation causes a fully penetrant sterility and is notably the only sig mutation recovered thus far that has accompanying somatic defects. sig-7 animals are thin and have a protruding vulva, defects possibly related to our observation of increased transgene expression (let-858::gfp) in gut and vulval tissues. Using polymorphisms, we have physically mapped sig-7 to a small interval on LGI and are currently performing transformation rescue experiments.

Tom Ratliff et al. (2005) International Worm Meeting "The transgene desilencer sig-7 is an allele of the conserved cyclophilin cyn-14"

Tom Ratliff, Bill Kelly, David Han, Karissa McClinic

[International Worm Meeting, 2005]

Germline repression of the activity of transposons and other foreign elements benefits the species by ensuring the passage of a "pristine" genome between generations. A loss of silencing in the germline can be assayed by expression of repetitive extrachromosomal arrays that, like foreign elements, are normally strongly silenced in this tissue. To genetically identify factors involved in germline silencing, we have isolated mutations that allow germline GFP expression from a repetitive extrachromosomal array. We have identified a single recessive mutation in sig-7 (silencing in the germline-defective) that exhibits pleiotropic defects. sig-7 animals have a masculinized germline that makes excess sperm and no oocytes, resulting in fully penetrant sterility. The same transgene used in the sig screen (let-858::gfp) is expressed at markedly increased levels in gut and vulva cells of sig-7 larvae, suggesting the existence of an unanticipated mechanism of transgene repression in both germ and somatic cells. sig-7 vulvae protrude abnormally, indicating a morphogenesis and/or fate defect. Moreover, sig-7 synthetically interacts with lin-15A to produce a temperature-sensitive Multivulva phenotype. Thus, the sig-7 phenotype overlaps with several previously described genes that are implicated in repressive mechanisms (i.e. the mog and synMuv B class genes). To better understand sig-7 function, we cloned it and identified a splice-acceptor mutation in the curated gene, cyn-14 (cyclophilin). cyn-14 encodes a protein with two conserved regions, a peptidyl prolyl isomerase (PPI) domain and a RNA-recognition motif (RRM). PPIases have been shown to accelerate protein folding. The sig-7 mutation is predicted to disrupt the production of the RRM in CYN-14, suggesting that RNA-binding may be important for its function. The C-terminal region of CYN-14 is rich in basic residues that are predicted to serve as nuclear localization signals. CYN-14 is strongly conserved, with orthologs existing in S. pombe and up through humans. S. cerevisiae lacks a CYN-14 ortholog, which is intriguing given that its largely intron-less genome has been posited to be the result of massive retrotransposition. We are currently determining the CYN-14 expression pattern and assaying RNA-based mechanisms for defects in sig-7 mutants.