RNA polümeraas II sõltuva transkriptsiooni elongatsiooni mehhanismide uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Kõigis tuumaga rakkudes on geneetilise informatsiooni kandjad - DNA molekulid - kõvasti kokku pakitud, et tagada nende mahtumine rakutuuma. DNA lahtipakkimine on oluline mitmete rakuliste protsesside läbiviimiseks, sealhulgas transkriptsiooniks. Transkriptsiooni käigus kopeeritakse informatsioon DNA-lt mRNA-ks ning seejärel valkudeks. Seda protsessi viib läbi RNA polümeraas II, mille tööprotsess jagatakse kolmeks etapiks – initsiatsioon, elongatsioon ja terminatsioon. Käesolevas töös uuriti põhiliselt elongatsiooni käigus toimuvaid muutusi DNA-l. Esmane DNA kokkupakkimise tase on nn nukleosoomne struktuur. Sellisel juhul on DNA seotud histoonivalkudega. On teada, et kui geeni transkriptsioonitase on kõrge, siis eemaldatakse nukleosoomid DNA-lt. Uurisime, mis määrab selle ala ulatuse, kust nukleosoomid transkriptsiooni käigus kaovad. Selgus, et DNA struktuur muutub ainult siis, kui ta on seotud elongeeriva RNA polümeraasiga ning nuklesoome ei eemaldata sealt, kuhu polümeraas ei jõua. Eelpool mainitud nukleosoomne DNA struktuur on RNA polümeraasile suureks takistuseks. Veel suuremaks takistuseks peetakse aga DNA ja valkude kompleksi, mida nimetatakse heterokromatiiniks ehk vaigistatud kromatiiniks. Heterokromatiini puhul on DNA seotud lisaks histoonidele veel teistegi valgukompleksidega. Vaatasime, kas juba transkribeeriv polümeraas suudab heterokromatiini läbida. Saime teada, et suudab küll ning vaigistavad kompleksid eemaldatakse DNA-lt elongeeriva polümeraasi möödumisel. Nii varasemates töödes kui ka meie katsetes ilmnes, et transkriptsiooni indutseerimisel on mudelgeenil näha rohkem RNA polümeraasi geeni alguses kui geeni lõpus. Kuna katsed olid tehtud kasutades rakkude kogu populatsiooni, tahtsime teada, kas ka ühe raku tasemel on RNA polümeraas transkribeeritaval geenil samamoodi jaotunud. Leidsime, et üksikul transkribeeritaval DNA fragmendil on polümeraasi molekulid jaotunud ühtlaselt. Antud tööst saadud tulemused on oluliseks täienduseks baasteadmistele transkriptsiooni elongatsioonist ning rikastavad arusaama RNA polümeraas II sõltuva transkriptsiooni elongatsiooniprotsessist.In all eukaryotic cells the carriers of genetic information - DNA molecules - are tightly packed to fit in the nucleus. Unpacking DNA is crucial for several cellular processes, including transcription. During RNA polymerase II (RNAPII)-dependent transcription one strand of DNA is used to synthesise complementary mRNA. RNA synthesis is divided into three main phases – initiation, elongation and termination. The main aim of the current thesis was to elucidate the mechanisms of elongation and concurrent changes in the higher structure of DNA. In the nucleus DNA is assembled into chromatin by forming complexes with histone proteins. The structural elements consisting of DNA and histone proteins are called nucleosomes. When a gene is highly transcribed, nucleosomes are fully evicted from the coding region of the gene. Firstly we showed that this eviction is tightly connected to elongating RNAPII and nucleosomes are not removed from the area where RNAPII does not reach. In addition to nucleosomes, RNAPII encounters even bigger obstacles on its way called heterochromatic complexes or silenced chromatin. These complexes have been considered impenetrable for RNAPII. We managed to show that elongating RNAPII is able to remove the heterochromatic complexes and transcribe silenced chromatin. The transcription levels and levels of elongating RNAPII on different genes vary significantly as the requirements for the gene products are not identical. Several genome-wide studies have addressed the question of the distribution of the components of transcriptional machinery. But as these studies draw conclusions on the average signal from the whole cell population they fail to describe processes occurring in a single cell. Thus we aimed to determine the distribution of elongation RNAPII molecules on single chromatin fragment. We found that RNAPII molecules are distributed along the gene evenly. The results obtained from our studies add new information to the basic knowledge of gene transcription and enhance the understanding about the mechanisms of RNAPII-dependent elongation

Distribution and Maintenance of Histone H3 Lysine 36 Trimethylation in Transcribed Locus

<div><p>Post-translational modifications of core histones play an important role in the epigenetic regulation of chromatin dynamics and gene expression. In <i>Saccharomyces cerevisiae</i> methylation marks at K4, K36, and K79 of histone H3 are associated with gene transcription. Although Set2-mediated H3K36 methylation is enriched throughout the coding region of active genes and prevents aberrant transcriptional initiation within coding sequences, it is not known if transcription of one locus impacts the methylation pattern of neighbouring areas and for how long H3K36 methylation is maintained after transcription termination. Our results demonstrate that H3K36 methylation is restricted to the transcribed sequence only and the modification does not spread to adjacent loci downstream from transcription termination site. We also show that H3K36 trimethylation mark persists in the locus for at least 60 minutes after transcription inhibition, suggesting a short epigenetic memory for recently occurred transcriptional activity. Our results indicate that both replication-dependent exchange of nucleosomes and the activity of histone demethylases Rph1, Jhd1 and Gis1 contribute to the turnover of H3K36 methylation upon shut-down of transcription.</p></div

Dynamics of H3K36me3 in demethylase deletion strains.

<p>The relative amount of histone H3 (<b>A, C, E, G, I</b>) and H3K36me3 (<b>B, D, F, H, J</b>) was determined at 2.6 kb of the <i>GAL-VPS13</i> upon transcription repression in wild type (<b>A</b> and <b>B</b>), <i>rph1Δ</i> (<b>C</b> and <b>D</b>), <i>jhd1Δ</i> (<b>E</b> and <b>F</b>), <i>gis1Δ</i> (<b>G</b> and <b>H</b>) and <i>rph1Δjhd1Δgis1Δ</i> triple deletion mutant (ΔΔΔ <b>I</b> and <b>J</b>) strains. All samples were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120200#pone.0120200.g001" target="_blank">Fig. 1</a>.</p

Replication-dependent loss of H3K36me3 after transcriptional repression in wt and <i>rph1Δjhd1Δgis1Δ</i> strains.

<p>The relative amount of histone H3 (<b>A</b> and <b>C</b>) and H3K36me3 (<b>B</b> and <b>D</b>) was determined upon glucose-mediated transcriptional repression at 2.6 kb in the coding region of <i>GAL-VPS13</i> in G1-arrested wt (<b>A</b> and <b>B</b>), and in <i>rph1Δjhd1Δgis1Δ</i> cells (ΔΔΔ; <b>C</b> and <b>D</b>). (<b>E</b>) Comparison of H3K36me3 turnover in wt and <i>rph1Δjhd1Δgis1Δ</i> strains grown asynchronously, or kept G1-arrested throughout the experiment. The Student <i>t</i> test was used to evaluate the statistical significance of differences between indicated samples. * indicates p<0.05; ** indicates p<0.005; *** indicates p<0.0001. All samples were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120200#pone.0120200.g001" target="_blank">Fig. 1</a>. Cell cycle profiles of G1-arrested wt and <i>rph1Δjhd1Δgis1Δ</i> strains are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120200#pone.0120200.s002" target="_blank">S2 Fig.</a></p

Yeast strains.

<p>Yeast strains.</p

Distribution of H3K36me3 in <i>GAL-VPS13</i> locus.

<p>(<b>A</b>) Schematic representation of the 9435 bp long <i>GAL-VPS13</i> locus. The <i>GAL-VPS13-3kb–term</i> strain contains the <i>FBA1</i> transcription termination region inserted at 3 kb from the <i>VPS13</i> promoter (3 kb-terminator, black rectangle). Vertical lines beneath the gene indicate the positions of PCR probes 2.6 kb (a), 3 kb (b), 3.6 kb (c) and 4 kb (d) downstream from the start-codon of <i>GAL-VPS13</i>. (<b>B</b>) ChIP assay followed by qPCR was used to determine the relative amount of histone H3 in the coding region of <i>GAL-VPS13</i> upon transcriptional activation in galactose (black bars, Gal) and repression in glucose (grey bars, Glu). (<b>C</b>) The relative amount of H3K36me3 was determined in the coding region of <i>GAL-VPS13</i> upon transcriptional activation in galactose (black bars, Gal) and repression in glucose (grey bars, Glu). (<b>D</b>) The occupancy of Set2 upon transcriptional activation in galactose (black bars, Gal) and inactivation in glucose (grey bars, Glu) in the coding region of <i>GAL-VPS13</i>. In all assays, the ChIP signal obtained from nontranscribed region of the right arm telomere of chromosome VI (Tel6) was set as 1 and all samples are presented as relative to that. In addition, the H3K36 trimethylation signal in C was normalised to total H3 occupancy. Error bars show standard error of at least 3 independent experiments.</p

Rpb9-deficient cells are defective in DNA damage response and require histone H3 acetylation for survival

Rpb9 is a non-essential subunit of RNA polymerase II that is involved in DNA transcription and repair. In budding yeast, deletion of RPB9 causes several phenotypes such as slow growth and temperature sensitivity. We found that simultaneous mutation of multiple N-terminal lysines within histone H3 was lethal in rpb9Δ cells. Our results indicate that hypoacetylation of H3 leads to inefficient repair of DNA double-strand breaks, while activation of the DNA damage checkpoint regulators γH2A and Rad53 is suppressed in Rpb9-deficient cells. Combination of H3 hypoacetylation with the loss of Rpb9 leads to genomic instability, aberrant segregation of chromosomes in mitosis, and eventually to cell death. These results indicate that H3 acetylation becomes essential for efficient DNA repair and cell survival if a DNA damage checkpoint is defective

Acetylation of H3 K56 Is Required for RNA Polymerase II Transcript Elongation through Heterochromatin in Yeast▿

In Saccharomyces cerevisiae SIR proteins mediate transcriptional silencing, forming heterochromatin structures at repressed loci. Although recruitment of transcription initiation factors can occur even to promoters packed in heterochromatin, it is unclear whether heterochromatin inhibits RNA polymerase II (RNAPII) transcript elongation. To clarify this issue, we recruited SIR proteins to the coding region of an inducible gene and characterized the effects of the heterochromatic structure on transcription. Surprisingly, RNAPII is fully competent for transcription initiation and elongation at the locus, leading to significant loss of heterochromatin proteins from the region. A search for auxiliary factors required for transcript elongation through the heterochromatic locus revealed that two proteins involved in histone H3 lysine 56 acetylation, Rtt109 and Asf1, are needed for efficient transcript elongation by RNAPII. The efficiency of transcription through heterochromatin is also impaired in a strain carrying the K56R mutation in histone H3. Our results show that H3 K56 modification is required for efficient transcription of heterochromatic locus by RNAPII, and we propose that transcription-coupled incorporation of H3 acetylated K56 (acK56) into chromatin is needed for efficient opening of heterochromatic loci for transcription