Memoria para optar el título de Bioquímico / La transcripción del DNA, primera etapa de la expresión génica, corresponde a la conversión de un DNA molde a RNA por acción enzimática de la RNA polimerasa (RNAP). En las células de los organismos eucariontes existen tres clases de RNA polimerasas nucleares (RNAP I, II y III). Cada clase de RNAP reconoce promotores específicos mediante la formación de complejos de preiniciación (Pre-initiation Complex o PIC) entre la RNAP y el uso factores de transcripción generales (General Transcription Factors o GTFs), siendo estos últimos los que dan especificidad a cada PIC. Los promotores son secuencias de DNA, normalmente ubicadas río arriba del inicio de la transcripción (Transcription Start Site o TSS), relativamente conservadas en el genoma, que poseen todos los elementos en cis necesarios para el inicio y la regulación de la transcripción de un gen. Los promotores de la RNAP II son los más estudiados y se clasifican en los que poseen caja TATA y en los que no, llamados TATA-Less. Entre los factores de transcripción que se unen a estos promotores están TFIIB, TFIID, TFIIA, entre otros. La caja HomolD es un elemento del promotor mínimo que se encuentra en un promotor de tipo TATA-less de secuencia consenso CAGTCACA, descubierto en genes de proteínas ribosomales de Schizosaccharomyces pombe, y que es capaz de reclutar la RNAP II e iniciar la transcripción. Estudios recientes indican que para iniciar la transcripción desde estos promotores se necesitan los siguientes GTFs: TFIID, TFIIB, TFIIF, TFIIH y TFIIE. Además, se ha propuesto que el factor Rrn7 (antes conocido como miembro de la maquinaria de la RNAP I) podría interaccionar con algunos de estos GTFs, como por ejemplo TFIIB y/o TBP (miembro del complejo TFIID), lo que le conferiría a Rrn7 la facultad de unirse a la caja HomolD y dirigir la transcripción. Sin embargo, aún se desconoce cuáles de estos factores junto a Rrn7 conforman el PIC, que se ensambla sobre la caja HomolD. En este trabajo se estudió la participación de los factores TFIIB y TBP, como miembros candidatos del complejo de pre-iniciación formado sobre la caja HomolD. Mediante ensayos en geles de retardo o EMSA y ensayos de inmunoprecipitación de cromatina se analizaron las interacciones DNA-proteína y proteína-proteína. Para realizar los ensayos de EMSA fue necesario expresar y purificar la proteína Rrn7 recombinante utilizando una etiqueta de histidina, la que se utilizó junto a TBP y TFIIB. Además, se purificaron anticuerpos policlonales contra ambos factores, y fueron usados en ensayos de EMSA a modo de control. Los resultados de los EMSA muestran una intensificación de la banda representativa al complejo Rrn7-caja HomolD al agregar cada factor a la reacción (TFIIB o TBP). Los ensayos de ChIP se realizaron en una cepa silvestre de S. pombe utilizando los mismos anticuerpos purificados contra los factores TBP y TFIIB. El DNA obtenido, fue sometido a amplificación mediante PCR utilizando partidores específicos para la caja HomolD. Además se realizaron dos controles, uno utilizando partidores que amplificaron parte del promotor y del primer exón del gen nmt1 para la caja TATA como control positivo, y otros para una región del gen de actina (act1) como control negativo. Se observó amplificación del DNA representativo a la caja HomolD, al precipitar cada factor, reflejando la presencia de éstos en la región promotora. Los resultados obtenidos en este trabajo muestran in vitro (EMSA) un efecto potenciador de TBP y TFIIB sobre la formación del complejo Rrn7-caja HomolD, lo que es complementario a la presencia mostrada in vivo (ChIP) de estos factores en el promotor. De estos experimentos se concluye la participación de ambos factores en la formación del complejo de pre-iniciación sobre la caja HomolD. Además los resultados de EMSA indicaron que TFIIB posee un efecto potenciador mayor que TBP a una determinada concentración. Por otro lado, en ensayos EMSA ambos factores produjeron un desplazamiento o retardo de la banda del complejo Rrn7-HomoID, pero el desplazamiento provocado por TBP fue más intenso y estable, a diferencia del de TFIIB que fue solo temporal. Estos resultados sugieren que los roles que cumplen los factores TFIIB y TBP en el PIC en la caja HomolD, son el de un factor reclutador de Rrn7 al promotor y el de un factor potenciador de la interacción proteína-DNA, respectivamente / DNA transcription, the first stage of gene expression, is defined as the conversion of a template DNA to RNA by the enzymatic action of the RNA polymerase (RNAP). In the eukaryotic cells there are three classes of nuclear polymerases (RNAP I, II and III). Each class of RNAP recognizes specific promoters through the formation of pre initiation complexes (PIC) between the polymerase and general transcription factors (GTF). The latter provide the specificity to each PIC. These promoters are relatively conserved DNA sequences that usually are located upstream from the transcriptional start site (TSS), and have all the cis elements needed for the start and regulation of the transcription of a gene. RNAP II promoters are the most studied ones, and are categorized as those that have a TATA box or those that lack a TATA box, called TATA-less promoters. The factors that bind to these promoters are TFIIB, TFIIA and TFIID, among others. The HomolD box is a core promoter element (CPE) that is found in a TATAless type promoter, whose consensus sequence is CAGTCACA. This CPE was discovered in ribosomal protein genes of the fission yeast Schizosaccharomyces pombe, and recruits RNAP II and starts transcription. Recent studies have shown that to start transcription from these RNAP II promoters the GTFs: TFIID, TFIIB, TFIIF, TFIIH and TFIIE are needed. Furthermore, it has been proposed that the Rrn7 factor (formerly known as a member of the RNAP I machinery) interacts with some of these GTFs, for instance with TFIIB and/or TBP (member of the TFIID complex), which could confer Rrn7 the ability to bind the HomolD box and promote transcription. However, it is still unknown which of these factors in addition to Rrn7 conform the PIC that assembles over the HomolD box. In this work the participation of the factors TBP and TFIIB, as candidate members of the PIC formed over the HomolD box, was studied. Using Electrophoretic Mobility Shift Assays (EMSA) and Chromatin Immunoprecipitation assays (ChIP), DNA-protein and protein-protein interactions were analyzed. To carry out the EMSA studies, it was necessary to express and purify the recombinant Rrn7 protein using a 6xHis tag, which was then used with the factors TBP and TFIIB, also recombinant from S. pombe. Additionally, polyclonal antibodies for both factors were purified and used in EMSA experiments as a control. The EMSA results showed an intensification of the band representative of the complex when each factor was added to the reaction (TBP or TFIIB). ChIP assays were performed in a wild type S. pombe strain, using the same antibodies purified for TFIIB and TBP. The DNA obtained was amplified with PCR using specific primers for the HomolD box. Two controls were carried out, one using primers that amplified part of the promoter and the first exon of the nmt1 gene for the TATA box as a positive control. The other control used primers for a region of the actin gene (act1) as a negative control. When each factor was precipitated, an amplification of the HomolD box sequence, revealing the presence of both factors at the promoter region. Results obtained in this work show in vitro (EMSA) an enhancer effect of TBP and TFIIB on the formation of the Rrn7-HomolD box complex, which is complementary to the presence shown in vivo (ChIP) of these factors in the promoter. From these experiments it is concluded that both factors participate in the formation of the PIC over the HomolD box. Furthermore, the EMSA results indicate that TFIIB has a greater enhancer effect than TBP. On the other side, EMSA assays of both factors produced a shift of the complex Rrn7-HomolD band, but the one produced by TBP was more intense and stable, in comparison with the complex produced by TFIIB, which was temporal and less intense. These results suggest that TFIIB recruits Rrn7 to the PIC and that TBP enhances the interaction between protein and DNA of the PIC at the HomolD box

No description available.

The establishment of cell polarity is a vital requirement for cellular processes such as proper cell division, growth and movement. Cell polarization relies on different internal and external cues in order to reorient the cell growth machinery along the axis of polarity. The core mechanisms involved in establishment of polarized growth are highly conserved from yeast to humans. Cells of the fission yeast Schizosaccharomyces pombe grow in a highly polarized fashion, with cell growth restricted to the cell tips, making fission yeast an excellent model system to study polarized growth. Here I describe a system for long-term live-cell imaging of fission yeast polarized growth that is stress free, physiological and accessible to media change and drug addition. I use this improved imaging system along with yeast genetics and drug perturbations to address how cell polarity is established and maintained in fission yeast. I have shown that fission yeast growth polarity depends on competition and cooperation among three distinct internal polarity cues: 1) A microtubule-based cue involving Tea1/Tea4 polarity proteins positively regulates polarized growth, initially at the “old” cell end (i.e., the end that pre-existed in the mother cell) and later at the “new” cell end (i.e. the end that is generated by septation), in order to initiate the transition from monopolar to bipolar growth (also known as New End Take-Off, or “NETO”). 2) An actin cable-based cue “clears” polarity proteins from the new end immediately after cytokinesis thereby reinforcing old-end growth. As a result perturbation of actin cable-based transport by either deleting actin cable nucleator For3 or cable-based transporter Myo52 results in premature bipolar growth. 3) A novel “memory-based” growth polarity cue helps to establish polarized growth in the absence of the microtubule-based cue. This memory-based cue is dependent on the predicted transmembrane proteins Rax1/Rax2. In the absence of both Tea1/Tea4 cue and Rax1/Rax2 cue, cells depend on septation cue and grow exclusively from the cell ends generated by septation. Furthermore, both Tea1/Tea4 and Rax1/Rax2 cue are important to maintain polarized growth under various environmental stresses. In fission yeast, during interphase, nucleus is positioned at the centre of the cell and this precise positioning of nucleus, which is important for defining the position of cytokinetic ring is thought to be exclusively MT-dependent. Here I show that MT-independent nuclear movement exists in fission yeast and this nuclear movement is mediated by actin cables and type myosin myo52. Furthermore, I show that actin cable might be important for buffering the pushing forces generated by MTs on the nucleus. In this way both microtubules and actin cables are involved in nuclear movement in fission yeast.

Compact heterochromatin is essential for genome stability and hence cell survival. Studies in many organisms including humans underline the importance of pericentromeric heterochromatin in centromere function. Fission yeast centromeres share a common structural organisation with those of their metazoan counterparts. The fission yeast model has been pivotal in understanding many key events in the pathway leading to the assembly of pericentromeric heterochromatin. In particular, studies in this system have revealed that the RNA interference (RNAi) pathway connects with the chromatin modification machinery to impart proper heterochromatin formation. Transcription of the pericentromeres by RNA polymerase II (Pol II) produces double stranded RNA (ds RNA) which is processed by Dicer(Dcr1) into small interfering RNAs (siRNAs). These siRNAs are loaded onto the Argonaute protein Ago1, and target the Ago1- containing RITS (RNA-Induced Transcriptional Silencing) complex to the pericentromeres via complementary base-pairing of the siRNA to the nascent centromeric transcript. RITS then recruits the sole Histone H3-K9-methyl transferase, Clr4, as part of the Clr4-complex, CLRC. The resulting H3K9-methyl marks further result in the recruitment of downstream chromatin binding proteins including the HP1- homolgue Swi6 which plays a key role in cohesin retention. Additionally, the H3K9- methyl marks are required for stabilising the association of CLRC and RITS, thereby promoting a reinforcing loop within the RNAi-mediated heterochromatin pathway. Thus crosstalk between RITS and CLRC is important in establishing and maintaining silent chromatin at the pericentromeres. Stc1 has been proposed to act as a critical link that connects the RITS and CLRC complexes. Stc1 is required for heterochromatin establishment and maintenance at the pericentromere and association of RITS with CLRC is lost in the absence of Stc1. Moreover, Stc1 directly interacts with Ago1 and is essential for siRNA production. These and other previous observations (Bayne et al. 2010) highlight the key role played by Stc1 in the RNAi-mediated heterochromatin pathway. To understand how Stc1 mediates the specific cross-talk between RNAi and chromatin modification, I have investigated the nature of Stc1 interactions with the RNAi and chromatin modification machineries. Using in-vitro binding assays, I found that Stc1 directly interacts with the CLRC subunits Dos2 and Clr4. I also identified the RITS subunit Tas3 as a potential interactor of Stc1, in addition to Ago1. A collaborating research group elucidated the structure of Stc1 using NMR (He et al. 2013) and my study provides evidence for interactions via the distinct domains of Stc1. Stc1 utilises its disordered C-terminus to bind to Dos2 while the N-terminus, which contains a tandem zinc finger domain, acts as a multi-protein interaction interface binding the CLRC subunit Clr4 and RITS subunits Ago1 and Tas3, opening up possibilities for Stc1-containing distinct-complexes. My work provides new insights into the role of Stc1 and opens up future avenues of research key to understanding how heterochromatin domains are defined and maintained.

No description available.

No description available.

Cid1 is a cytoplasmic member of a novel class of regulatory poly(A) polymerases discovered recently in yeast, worms and vertebrates. Previous genetic studies in the fission yeast, Schizosaccharomyces pombe, suggested a role for Cid1 in the checkpoint response to replication stress, but it was not known how a poly(A) polymerase might contribute to this response. Further investigations into the mode of action of Cid1 were therefore undertaken in this study. Cid1 is likely to target specific RNAs for polyadenylation; potential RNA substrates were identified using the complementary methods of microarray hybridisation and whole proteome analysis using two-dimensional liquid chromatography. These experiments revealed that Cid1 does not affect RNAs during normal, unperturbed growth but instead alters the expression of specific subsets of genes during replication stress. Many RNAs affected by Cid1 in these circumstances were cell-cycle dependent and telomeric transcripts, including those encoding histones and a novel RecQ helicase, Rqh2. As Cid1 lacks an RNA recognition motif, it is unlikely to bind selectively to RNA targets on its own. Cid1-interacting proteins were identified using yeast two-hybrid and tandem affinity purification methods. From these studies, novel members of a Cid1 complex have been discovered including: a previously uncharacterised metallo-beta-lactamase, RNA-binding proteins, ribosomal proteins and a telomere-binding protein. Together, these approaches are leading to a model for the role of cytoplasmic polyadenylation by Cid1 in checkpoint control.

The Schizosaccharomyces pombe cdc21⁺ gene product is related to the Mcm2-3-5 family of replication proteins. By phylogeny analysis of their protein sequences and screening for cdc21⁺-related sequences using molecular probes I have suggested that at least six types of cdc21⁺-related genes may be present in the yeast genome. The isolation of interaction suppressors of the cdc21^ts mutant was attempted by overexpression of an S. pombe cDNA library. Two cDNAs were isolated, ts11⁺ and dom1⁺, whose overexpression specifically affected the viability of cdc21^ts cells under certain conditions. The predicted dom1 protein is 60% identical to the budding yeast HMG-like Nhp2 protein. I have studied the phenotype of S. pombe cells overexpressing the cdc21⁺ gene and amino-terminal truncations of it. Overexpression of the cdc21⁺ gene caused cell elongation but cells were not significantly affected in growth rate. Cells overexpressing the carboxyl-terminal part of cdc21⁺ arrested in S phase and also entered mitosis in the absence of nuclear division. The possibility that chromosomes in cdc21^ts arrested cells may be damaged was investigated by pulsed field gel electrophoresis. No differences could be found compared to wild-type chromosomes. I have also studied the arrest phenotype of cdc21 rad1 and cdc21 cdc2.3w double mutants. Both strains entered mitosis at the restrictive temperature indicating that cdc21^ts cells arrest in S phase and may contain DNA damage. I have generated two new mutant alleles of cdc21⁺. The first allele was made by deleting most of the cdc21⁺ open reading frame (ORF). The second allele was constructed by placing the cdc21⁺ ORF under control a regulatable promoter. The resulting construct was used to complement the cdc21 deletion. Both mutants were inviable under appropriate conditions arresting in S phase as elongated cells, although a proportion of them (15-20%) entered mitosis in the absence of nuclear division.

Thesis advisor: Charles Hoffman / Faithful chromosome segregation is necessary for the successful completion of mitosis and meiosis. The centromere is the site of kinetochore and microtubule attachment during chromosome segregation, and it is critical that the centromere is properly formed and maintained. Many proteins contribute to centromere formation, and this process has been extensively studied during the mitotic cell cycle. However, the roles of the centromere and its associated proteins during meiosis and their contribution to the fidelity of chromosome segregation process are not as well understood. Here, I aim to elucidate a mechanism that may contribute to aneuploidy in gametes, which is a major contributing factor in human infertility. In this study, I investigate the role of Abp1, the most prominent member of the transposase-derived protein family homologous to mammalian CENP-B in the assembly of centromeric chromatin during meiosis in the fission yeast Schizosaccharomyces pombe. I reveal that in contrast to its known role as a major regulator of LTR retrotransposons during the mitotic and meiotic cell cycles, Abp1 has a specialized role at the centromere during meiosis. My results indicate that Abp1 displays dynamic localization to the centromeres during meiosis compared to the vegetative cell cycle. I show that loss of abp1 impairs pericentromeric heterochromatin and the localization of Cnp1, a CENP-A ortholog, to the centromere central cores during meiosis. Moreover, Abp1 appears to suppress formation of meiotic neocentromeres by restricting deposition of Cnp1 at certain heterochromatin loci. Loss of abp1 has a drastic effect on chromosome segregation, resulting in dramatic frequency of aneuploidy. Furthermore, the genome surveillance role for retrotransposons by Abp1 appears to encompass centromeres as the mere insertion of an LTR sequence within the centromere central cores further exacerbates incidence of meiotic aneuploidy in abp1 null cells. This study provides intriguing insights into factors controlling the assembly of centromeric chromatin and its impact on the fidelity of chromosome segregation process during meiosis with important implications for advancing our understanding of the evolutionary forces driving the evolution of eukaryotic centromeres. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Hugh P. Cam / Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of strong recombination activity dubbed recombination hotspots, and regions of low recombination activity dubbed coldspots. Considerable attention has led to discoveries of a host of factors controlling the formation of hotspots. However, the determinants of coldspots are not as clearly defined. I have previously shown that CENP-B homologs of the fission yeast Schizosaccharomyces pombe have a genome surveillance role in regulating the nuclear organization and expression of Tf2 retrotransposons. Here, I reveal an additional role for CENP-Bs in suppressing meiotic recombination of Tf2s. I describe the development of a random sporulation assay to rapidly screen thousands of meiotic progeny for recombination across a locus in a variety of genetic backgrounds. Loss of any CENP-B family members (Abp1, Cbh1, Cbh2), results in increased HR at Tf2s. I show that Abp1, which acts as the primary determinant of HR suppression at Tf2s, is required to maintain proper recombination exchange of homologous alleles flanking a Tf2. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. I show that this suppression is likely mediated by Abp1 binding to specific motifs near the 3’end of flanking LTRs. I demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, I uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the non-homologous end joining pathway in ensuring the suppression of HR at Tf2s. Furthermore, I identify a role for the architectural protein condensin involved in 3D chromatin organization and chromosome condensation in restricting HR at Tf2s. My study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators, DNA repair pathway, and chromosome organizers to regulate meiotic recombination at interspersed repeats. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.