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Investigating Mutant suppressor of synthetic lethality between htz1Δ and RPB2-2 SL in Saccharomyces cerevisiae Abstract Caleb Stubbs, Dr. Maria Santisteban.

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Presentation on theme: "Investigating Mutant suppressor of synthetic lethality between htz1Δ and RPB2-2 SL in Saccharomyces cerevisiae Abstract Caleb Stubbs, Dr. Maria Santisteban."— Presentation transcript:

1 Investigating Mutant suppressor of synthetic lethality between htz1Δ and RPB2-2 SL in Saccharomyces cerevisiae Abstract Caleb Stubbs, Dr. Maria Santisteban Department of Biology, UNC at Pembroke Histone H2A.Z is a H2A variant form of the highly conserved Histone H2A.Z/F family, which is found among most vertebrates. In yeast Saccharomyces cerevisiae H2A.Z (encoded by HTZ1) is not necessary for life, but htz1 mutants exhibit many different phenotypes in several cell processes including transcriptional regulation, gene silencing, and preventing the spread of heterochromatin, and mitotic chromosome transmission. Transcription regulation by Htz1 has been the focus of numerous studies. Htz1 containing nucleosomes have been shown to poise quiescent genes for activation and transcriptional initiation. We have also provided evidence for a role of Htz1 in transcription elongation. In order to elucidate the mechanism involved in regulating transcription elongation, we are using a dominant allele of the RNA Pol II gene, RPB2-2 SL identified in an unbiased screen for mutations synthetic lethal with deletion of the histone H2A.Z gene. Extragenic suppressors of the double mutant were isolated and efforts to identify them by complementing the suppressor phenotype have yielded the SET2 gene. SET2 encodes a histone methyltransferase, which has shown to play roles in transcription elongation. We are working to formally prove or disprove that a mutation in SET2 is indeed suppressing the synthetic lethality of htz1/ RPB2-2 SL. We are also investigating if htz1/ RPB2-2 SL may display a cryptic initiation phenotype, and if it does what effect the suppressors have on it. Introduction The role of chromatin structure in determining when and how genes are expressed has been proved by numerous studies. Histones in particular, which are wrapped by DNA to form nucleosomes, the basic unit of chromatin, play a big role in transcriptional regulation. Histone variants can be substituted in all core histones except H4 and are found in low amounts from yeast to humans. Early work demonstrated a direct role for Htz1 in transcription activation and showed that Htz1 function was partially redundant with histone remodeling/modifying complexes (1). More recently links to transcription elongation have been uncovered (3). Checking whether htz1 RPB2-2 SL is conferred by a set2 mutation: Strains MSY3621 (MATa) and MSY3622 (MATα) containing htz1 RPB2-2 SL sup* were mated to YMS030 (MATα) and YMS031 (MATa) containing htz1 RPB2-2 SL set2::KanMX. Both strains carry a plasmid with wild type HTZ1, ADE3, and URA3. Zygotes were isolated under a dissection microscope and grown on G418 plates at 28°C. After three days individual colonies were numbered and streaked onto GNA pre-sporulation media. Colonies were then moved to sporulation media (KAcetate 1%, Zn Acetate 0.005%) and incubated for three days. Signs of sporulation were observed in 4 of the 10 isolates. These were retained to do tetrad dissections. 1. Santisteban, M.S., Kalashnikova, T., and M. Mitchell Smith Histone H2A.Z regulates transcription and is partially redundant with nucleosome remodeling complexes. Cell 103: Vanessa Cheung, Gordon Chua, Nizar N Batada, Christian R Landry, Stephen W Michnick, Timothy R Hughes, Fred Winston Chromatin- and Transcription-Related Factors Repress Transcription from within Coding Regions throughout the Saccharomyces cerevisiae Genome /journal.pbio Santisteban, M.S., Hang, M.D., and Smith, M. Mitchell Histone Variant H2A.Z and RNA Polymerase II Transcription Elongation. Mol. Cell. Bio. 4. Nevan J. Krogan, Minkyu Kim, Amy Tong, Ashkan Golshani, Gerard Cagney, Veronica Canadien, Dawn P. Richards, Bryan K. Beattie, Andrew Emili, Charles Boone, Ali Shilatifard, Stephen Buratowski and Jack Greenblatt Methylation of Histone H3 by Set2 in Saccharomyces cerevisiae Is Linked to Transcriptional Elongation by RNA Polymerase II. Mol. Cell. Biol Swaminathan Venkatesh, Michaela Smolle, Hua Li, Madelaine M. Gogol, Malika Saint, Shambhu Kumar, Krishnamurthy Natarajan & Jerry L. Workman Set2 methylation of histone H3 lysine 36 suppresses histone exchange on transcribed genes /nature Michael J. Carrozza, Bing Li, Laurence Florens, Tamaki Suganuma, Selene K. Swanson, Kenneth K. Lee, Wei-Jong Shia, Scott Anderson, John Yates, Michael P. Washburn, and Jerry L. Workman Cell, Vol Picture from Wikipedia 8. Susan L. Forsburg. The art and design of genetic screens: yeast. September Nature Reviews Genetics. doi: / Li B, Gogol M, Carey, M, Patternden SG, Seidel C, et. Al. (2007) Infrequently transcribed long genes depend on the Set2/Rpd3S pathway for accurate transcription. Genes Dev 21: bp +2505bp -1147bp References Checking for Cryptic initiation: Because set2 mutation has been shown to have a cryptic initiation phenotype (9,2), we decided to test our suppressor mutant of the htz1 RPB2-2 SL synthetic lethality for this phenotype. We attempted to replace the FLO8 gene with KanMX-GAL1pr-FLO8-HIS3 cassette engineered by the Fred Winston lab (2). In this cassette, HIS3 coding sequence is inserted at the 3’ end of the FLO8 ORF, approximately two nucleotides away from the FLO8 cryptic initiation start site out-of-frame to the FLO8 coding region. The normal promoter was replaced with a GAL1 promoter to allow regulation of full length transcription on different types of medias. Acknowledgements: This work was supported by grant #5R25GM from the NIGMS (National Institute of General Medical Sciences) supporting the UNCP RISE Program. Also I would like to thank my mentor Dr. Maria Santisteban for allowing me to conduct research with her. Future of project If we find that a mutation in the SET2 gene is responsible for the suppression of the htz1 RPB2-2 SL synthetic lethality we will rescue and sequence the mutation. This would help us decide if the mutation could have an effect in the catalitic activity of the Set2 methyltransferase or other. We would then examine the global levels of histone H3 K36 methylation as well as the distribution of this mark throughout individual test genes. In parallel, we will be testing if the strains transformed with the KanMX-GAL1pr-FLO8-HIS3 cassette, and confirm to have integrated the cassette, are able to grow on SC-His +Gal media, which would indicate that they have a cryptic initiation defect. Mechanisms beyond histone H3 K36 methylation appear to control cryptic initiation. And different cryptic promoters in yeast maybe activated by different mechanisms. Even if we find that htz1 RPB2- 2 SL mutant has a cryptic initiation phenotype this may not be necessarily the result of an altered H3 K36 pattern. Changes in chromatin structure due to htz1Δ over the cryptic promoters could cause Rpb2-2 to initiate transcription at those locations. Our efforts have been focused on rpb2-2, a ts- allele of RPB2, the gene encoding the second largest subunit in RNA polymerase II, which is dominant synthetic lethality in combination with htz1Δ. Synthetic lethality results from combining two or more mutations that are individually tolerated (8). In order for htz1/ RPB2-2 SL to survive, a plasmid containing wild type HTZ1 gene is necessary. This plasmid also contains a URA3 gene as selectable marker and an ADE3 gene, which allows us to track it. Because the strain is ade2/ade3 double mutant, colonies are red when the ADE3 plasmid is present but they are sectoring/white as the plasmid is lost. In order to elucidate the defect of the htz1/ RPB2-2 SL double mutant, a suppressor screen of the synthetic lethality was carried out. Cloning attempts of one of the mutants by complementation of the suppression phenotype yield a plasmid containing SET2. SET2 gene encodes a histone methyltransferase, which methylates lys36 of histone H3 and is involved in transcription elongation (4,5,6). The role of SET2 during elongation appears to be linked to suppression of cryptic initiation (2,6,9). Evidence has shown that set2 allows cryptic initiation in a large set of genes (2). In this work we present the steps taken in validating set2 as the mutant suppressor gene and if, whether or not, it can suppress possible cryptic initiation of htz1/ RPB2-2 SL. Methods The KanMX-GALpr-FLO8-HIS3 cassette was amplified by PCR from FY2712 strain. Template DNA was from genomic prep. Forward primer was designed to anneal to positions relative to the start codon, and reverse primer annealed to the 2552 to 2534 positions. The picture above to the right shows the result of the PCR amplification; lanes 1-4 show the KanMX-GALpr-FLO8-HIS3 4.5kb cassette; lane 5 shows the amplification of the 3737bp FLO8 locus. DNA was purified with a Qiagen PCR purification kit and about 5 µg of DNA were used in the transformations. The following were transformed: MSY 1396 (wildtype), MSY 1499 (htz1  ), MSY1620 (htz1 RPB2-2 SL pHTZ1), and MSY3621 (htz1 RPB2-2 SL sup*). We have just recently ordered primers to verify the correct insertion of the cassette in the transformed strains. set2::KanMX sup* set2::KanMX sup* If set2 is the suppressor sup*/set2::KanMX SUP/SET2 sup*/SET2 sup*/set2::KanMX SUP/ SET2 SUP/set2::KanMX sup*/SET2 SUP/set2::KanMX If set2 isn’t the suppressor FY2712 FLO8-HIS 3 FY2719 wildtype FY2719/FY2712 Parental Ditype (PD) Tetratype (T)Nonparental ditype (NPD)


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