6,315 research outputs found
The role of chromatin on RNA polymerase II transcription termination
Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012Tem-se tornado cada vez mais evidente que a transcrição no ambiente da cromatina é um processo extremamente complexo e finamente regulado. É agora claro que a maquinaria transcricional eucariótica está adaptada para explorar a presença de nucleossomas de variadíssimas e sofisticadas maneiras. Após activação, os genes sofrem drásticas mudanças na estrutura da cromatina. Isto é possível devido a um sistema de montagem da cromatina dependente da transcrição que integra a acção coordenada de numerosos factores proteicos capazes de modificar as propriedades da cromatina. Estes factores vão cooperar ou competir de forma a alterar o estado da cromatina entre permissivo e não permissivo, levando à activação ou repressão da transcrição. O trabalho apresentado nesta tese focou-se na terminação da transcrição. O objectivo foi investigar de que forma está a cromatina implicada na fase final da transcrição. Assim, levantou-se a hipótese que características específicas da cromatina são observadas nas regiões do DNA onde a RNA polimerase II se dissocia do DNA molde. Para testar esta hipótese, foram utilizadas técnicas bioquímicas, como por exemplo, imunoprecipitação de cromatina e digestão com nucleases. Os resultados revelaram um comportamento dinâmico da RNA polimerase II dependente da transcrição em regiões downstream do limite 3´ dos genes. Além disto, os resultados sugerem que os nucleossomas localizados nesta região são desmontados ineficientemente pelos complexos transcricionais da RNA polimerase II. De facto, experiências adicionais demonstraram que regiões a jusante do local de poliadenilação, onde a terminação ocorre, exibem reduzido recrutamento de chaperones de histonas. Finalmente, foi possível confirmar um enriquecimento de modificações específicas de histonas nas regiões 3’ que flanqueiam os genes. Em conjunto, os resultados aqui apresentados permitem sugerir um modelo pelo qual o estado da cromatina a jusante do local de poliadenilação facilita os eventos moleculares que levam à terminação da transcrição.It has become increasingly evident that transcription in a chromatin environment is an extremely complex and finely tuned process. It is now clear that the eukaryotic transcriptional machinery is adapted to exploit the presence of nucleosomes in a variety of sophisticated ways. Nucleosomes are now seen as a crucial component of this process, rather than a simple passive barrier. Upon activation, genes undergo severe changes in chromatin structure. This is achieved through a transcription-dependent chromatin assembly system that involves the orchestrated action of numerous protein factors capable of modifying chromatin properties. These proteins will cooperate or compete to change the chromatin state between permissive and non-permissive, leading to activation or repression of transcription. Herein we focused on transcription termination. We aimed at investigating whether chromatin is a determinant of the final stage of transcription. In this sense, we hypothesized that specific chromatin features are observed at the DNA regions where RNA Polymerase II dissociates from the transcribed template. To test this hypothesis, we used a biochemical approach, which includes techniques such as chromatin immunoprecipitation and nuclease digestion assays. Our results reveal a transcription-dependent dynamic behavior of RNA Polymerase II molecules in regions downstream the 3’ boundary of genes. Our data further suggest that the nucleosomes occupying this region are inefficiently disassembled by the RNA polymerase II transcription complexes. Notably, the regions downstream the poly(A) site, where termination takes place, exhibited reduced recruitment of histone chaperones. Finally, we were able to confirm the presence of histone modifications enriched at the 3’ flanking region of genes. Altogether, these data allow us to envisage a model by which the chromatin landscape downstream the poly(A) site facilitates the molecular events that drive transcription termination
Nucleases and histone acetyltransferases in DNA repair and immune diversity
DNA repair mechanisms are essential for genome maintenance and adaptive immunity. A careful balance must be achieved whereby highly accurate and efficient canonical repair protects the genome from accumulating mutations that lead to aging and cancer, and yet mutation and error-prone non-canonical repair is required for generating immune diversity. Immune diversity is achieved within a tightly regulated environment in which mutator proteins are directed to the antibody locus to introduce a swathe of DNA damage. This produces high affinity antibodies that recognise an infinite number of invading pathogens. This process of secondary antibody diversification is dependent on both active transcription and DNA repair. Downstream of histone signalling, DNA repair nucleases are recruited to remove the damaged bases. The structure of damaged regions in the DNA can have very different conformations depending on whether the source of the damage is endogenous or exogenous. Specific DNA nucleases recognise particular DNA substrates and generate DNA intermediates that are repaired in conjunction with polymerases and ligases. Despite their multitude and importance to DNA repair, very few nucleases have been characterised, while the activities of some studied nucleases remain controversial. Conventional techniques for studying DNA nucleases have several disadvantages; they are hazardous, laborious, time-consuming, and capture nuclease activity in a discontinuous manner. Recognising a need for a safer, faster alternative, a fluorescence-based method has been developed for the study of DNA nucleases, nickases and polymerases. Key histone modifications that are known to orchestrate canonical DNA repair have since been discovered to regulate non-canonical repair at the antibody locus. The Kat5 histone lysine acetyltransferase functions highly upstream of DNA repair and promotes active transcription, yet a role for Kat5 in secondary antibody diversification has not yet been established. Using chemical inhibitors to prevent the catalytic activities of Kat5, and the genetic method of an inducible degron system for rapid and reversible downregulation of Kat5, a role for Kat5 in secondary antibody diversification is recognised, and the research contributes to our current understanding of the DNA repair signal transduction pathway
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Implementation of an extrachromosomal system for the detection of novel DNA double strand break repair genes in S. cerevisiae
The outcome of DNA damage is diverse and generally adverse. Acute effects arise from disturbed DNA metabolism, triggering cell-cycle arrest or cell death. Long-term effects result from irreversible mutation contributing to oncogenesis and genome instability. In view of the many types of lesions, several pathways were developed to repair these lesions: nucleotide-excision repair (NER), base-excision repair (BER), mismatch repair (MMR), homologous recombination (HR) and non-homologous end joining (NHEJ). The double-strand break (DSB) is the most dangerous type of DNA lesion, it can be repaired mainly by HR (by crossover (CO), gene conversion (GC), and singlestrand annealing (SSA)) or by non-homologous end-joining (NHEJ). When, after replication a second identical DNA copy is available, HR seems to be preferred, other wise cells rely on NHEJ, which is more error prone. Up to now, all DSB repair processes have been studied separately, although it is clear that all of them can occur simultaneously in different proportions. We have created a new molecular plasmid system to simultaneously detect all four types of recombinational DBS repair. To this end, we constructed an in vivo/in vitro HNS plasmid system (HNS: HR, NHEJ, SSA), based upon two topologically different DNA molecules, which allows us to follow all four recombination processes at once. The plasmids, named pURRA8A and pRURA8A, contain two truncated non-functional URA3 genes in direct or inverted orientation respectively, sharing a central homologous region where a l-Scel site was introduced artificially. The plasmids also carry a centromere sequence and two phenotypic markers TRP1 and ADE8. DSB can be induced in vitro at the homologous (l-SceI) or non-homologous (BamHI) region. Yeast transformation with linearized plasmids was performed in strain YPH 250 and isogenic knockout strains lacking either the RAD52 gene involved in HR and SSA, HDF1 (yKU70) and NEJ1 involved in NHEJ. In addition MRE11 complex and MSH2 null mutants were studied. Distribution of DSB repair events among the various pathways was monitored by phenotypic and PCR analysis. The rad52 knockout mutant showed lower levels of CO and SSA, while the hdf1 mutant showed a decrease in conservative and non conservative NHEJ, as expected. These results confirm the validity of the HNS system for monitoring all 4 repair pathways simultaneously. The HNS system has been used to identify new genes involved in DNA damage response. DBS were induced in vivo by the expression of the l-Sce I endonuclease under Gal1-promoter control. After transformation, we performed transposon mutagenesis using the mTn-lacZ/LEU2 library system and selected cells that lost the ability to recombine. From the initial 7000 mutants tested, we selected initially 150 that were rescreening to obtain finally 33 mutants. The identification of the locus of the transposon insertion of all of them was performed. Some known genes (RAD50, SWR1, MCK1, SIN4, RSC2, SWE1 and DBP1) as well as unknown ones (YLR238W, YLR089C, YMR278W) were selected. Null mutants of all them were constructed, DSBR events profile as well as MMS sensitivity were determined. Relative rates of DSB repaired by HR, NHEJ and SSA were examined in all the selected null mutants strains. By comparing the distribution of DSBR by different mechanisms, we were able to obtain a strain-specific profile in which the relative proportions of repair events occurring in the cell were characteristic of that mutation. RSC2 (RSC complex component) and YLR235w (FHA containing protein) genes were selected to further characterisation. Epistatic null mutants analysis with key recombination genes (yku70 and rad52) was performed. MMS sensitivity and survival was analysed, as well as DSB induction in-vivo. This enables us to verify the involvement of a particular gene product in DNA damage response. In particular, we showed that Ylr238w is involved in DNA damage transcription regulation
Linker Histone Functions of HMO1- Implications for DNA repair
The DNA of eukaryotic cells does not exist in free linear strands; it is tightly packaged and wrapped around nuclear proteins in order to be accommodated it inside the nucleus. The basal repeating unit of chromatin, termed the nucleosome, provides the first level of compaction of DNA into the nucleus. Nucleosomes are interconnected by linker DNA and associated linker histones to form 30 nm fibers. The highly diverse linker histones are critical for compaction and stabilization of higher order chromatin structure by binding DNA entering and exiting the nucleosome. The lysine-rich C-terminal domain (CTD) of metazoan H1 is crucial for such stabilization. This study concerns the functions of Saccharomyces cerevisiae Hmo1p, an high mobility group (HMGB) family protein unique in containing a terminal lysine-rich domain and functions in stabilizing genomic DNA. My study suggests that Hmo1p shares with mammalian linker histone H1 the ability to stabilize chromatin, as evidenced by the absence of Hmo1p or deletion of the Hmo1p CTD creating a more dynamic chromatin environment that is more sensitive to nuclease digestion and in which chromatin remodeling events associated with DNA double strand break repair occur faster; such chromatin stabilization requires the lysine-rich extension of Hmo1p. Further, my data indicates that Hmo1p functions in the DNA damage response by directing lesions towards the error-free pathway. My results suggest that Hmo1p controls DNA end resection and favors the classical non- homologous end joining (NHEJ) over alternate end Joining (A-EJ) that is error-prone process. In all, my study identifies a novel linker histone function of Hmo1p in Saccharomyces cerevisiae with the ability to stabilize genomic DNA, and appears to go beyond conventional linker histone function
Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways
Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.European Research Council ERC2014-ADG669898 TARLOOPMinisterio de Economía y Competitividad BFU2016-75058-PJunta de Andalucía BIO123
Mechanism of chromatin reassembly at the yeast PHO5 promoter upon repression
The goal of this study has been to elucidate the mechanisms responsible for rebuilding nucleosomes at the PHO5 promoter upon rerepression.
In this work, I could unambiguously show that histones are incorporated at the PHO5 promoter upon repression. Regarding the source of these histones, I provide evidence that a significant fraction of the deposited histones originate from a soluble histone pool, i.e. a histone source in trans. Promoter closure occurs with strikingly rapid kinetics and is independent of replication. In agreement with the finding that PHO5 repression does not require cell division, I found that histone chaperones which are associated with replication-independent nucleosome assembly are important for rapid PHO5 promoter closure. Strains deleted for histone chaperones involved in replication-dependent nucleosome assembly did not exhibit any defect in promoter closure. Other factors contributing to rapid PHO5 repression turned out to be nucleosome remodelers, whose characteristic mode of action is chromatin assembly in trans. Nucleosome remodeling mutants typically catalyzing nucleosome movements in cis are not implicated in PHO5 promoter reassembly. The phenomenon of trans-deposition of histones upon repression is not restricted to the PHO5 promoter but is also found at two other phosphate regulated promoters, PHO8 and PHO84. By its rapid mode of action, this mechanism contributes to efficiently shutting off transcription. This might also hold true for other yeast genes.
In the second part of this work I present results that indicate a role for the histone chaperone Asf1p in the activation of the PHO5 gene. Interestingly, the induction of PHO5 in an asf1 mutant is dependent on the phosphate concentration of the growth medium. Full induction occurs only when the medium is completely free of phosphate. The abundance of even trace amounts of phosphate precludes PHO5 activation altogether
Chromatin binding factor Spn1 contributes to genome instability in Saccharomyces cerevisiae, The
2018 Spring.Includes bibliographical references.Maintaining the genetic information is the most important role of a cell. Alteration to the DNA sequence is generally thought of as harmful, as it is linked with many forms of cancer and hereditary diseases. Contrarily, some level of genome instability (mutations, deletions, amplifications) is beneficial to an organism by allowing for adaptation to stress and survival. Thus, the maintenance of a "healthy level" of genome stability/instability is a highly regulated process. In addition to directly processing the DNA, the cell can regulate genome stability through chromatin architecture. The accessibility of DNA for cellular machinery, damaging agents and spontaneous recombination events is limited by level of chromatin compaction. Remodeling of the chromatin for transcription, repair and replication occurs through the actions of ATP remodelers, histone chaperones, and histone modifiers. These complexes work together to create access for DNA processing and to restore the chromatin to its pre-processed state. As such, many of the chromatin architecture factors have been implicated in genome stability. In this study, we have examined the role of the yeast protein Spn1 in maintaining the genome. Spn1 is an essential and conserved transcription elongation factor and chromatin binding factor. As anticipated, we observed that Spn1 contributes to the maintenance of the genome. Unexpectedly, our data revealed that Spn1 contributes to promoting genome instability. Investigation into a unique growth phenotype in which cells expressing a mutant form of Spn1 displayed resistance to the damaging agent, methyl methanesulfonate revealed Spn1 influences pathway selection during DNA damage tolerance. DNA damage tolerance is utilized during replication and G2 to bypass lesions, which could permanently stall replication machinery. This pathway congruently promotes and prevents genome instability. We theorize that these outcomes are due to the ability of Spn1 to influence chromatin structure throughout the cell cycle
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