Dissertations / Theses on the topic 'Telomeric DNA; Genome'

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

This project was designed to isolate and characterise interstitial telomere repeat containing loci from the human and mouse genomes and to investigate the nature of the mouse telomere. Cloning of the internal telomere repeat loci proved to be extremely difficult and so alternative methods such as restriction enzyme analysis, hybridisation analysis, inheritance studies, and mapping within recombinant inbred and backcross mouse strains were employed to characterise these regions within the mouse genome. Similar methods were used to characterise mouse telomeres. From these experiments it was shown that, in the mouse, Trypanosoma-like (TTAGGG)_n telomere repeats are present at the telomeres and at interstitial sites. Within both of these regions, the (TTAGGG)_n repeats are present within distinct genetic loci that are stably inherited through subsequent generations. New variant generation is observed at both types of loci, takes place at a significantly higher frequency at the telomeric compared to interstitial loci and occurs during gametogenesis. It is possible that the higher rate of new variant generation at mouse telomers compared to internal sites may relate to their position within the mouse genome. Restriction enzyme and hybridisation sequence analysis demonstrated that both classes of loci are composed of telomere-related repeats and that an undefined simple repeat may also be present. Direct sequencing is required before the nature and organisation of simple repetitive DNA within these loci can be determined. Mapping of the interstitial, (TTAGGG)_n telomere repeat containing loci within the BxD Rl and Mus spretus/C57Bl/6 backcross mice demonstrated their presence within the protermini of chromosomes 9, 13 and X. It remains to be determined whether this distribution is functionally significant.

Upon fertilization, the early embryo sustains most of the cellular processes using the maternally deposited reserves in the egg itself until the zygotic gene expression takes charge. Among the plethora of essential components provided by the mother are small non-coding RNAs called PIWI-interacting RNAs (piRNAs), which provide immunity to the zygote against transposon challenge. In this thesis, I have presented three different functions of piRNAs in Drosophila melanogaster- in maintenance of genomic integrity, telomere protection and their role as an adaptive immune system against genomic parasites. In Chapter 2, I have described the phenotypic effects of the loss of piRNA function in early embryos. The mutations affecting the piRNA pathway are known to cause embryonic lethality. To describe this lethality in detail, I have shown that all the characterized piRNA mutants show compromised zygotic genomic integrity during early embryogenesis. In addition, two piRNA pathway components, Aubergine (Aub) and Armitage (Armi) are also required for telomere resolution during early embryogenesis. Aub and Armi recruit telomeric protection complex proteins, HOAP and HP1, to the telomeric ends and thus avoid activation of the Non-homologous end joining (NHEJ) DNA repair pathway at the telomeres. There are about 120 transposon families in Drosophila melanogaster and piRNA pathway mutations cause activation of many of the resident transposons in the genome. In Chapter 3, I have described the effects of infection by a single transposon, P-element, in naïve strains by introduction through the zygote. Activation of the P-element leads to desilencing of unrelated transposons, causing accumulation of germline DNA damage which is linked to severely reduced fertility in the hybrid females. However, there is partial restoration of fertility as the hybrid progeny age, which correlates with P-element piRNA production and thus P-element silencing. Additionally, a number of transposons mobilize into piRNA generating heterochromatic clusters in the genome, and these insertions are stably inherited in the progeny. Collectively our data shows that piRNA production can be triggered in the adults in an absence of maternal contribution and that piRNAs serve as an adaptive immune system which helps resolve an internal genetic conflict between the host and the parasite. In an effort to understand the phenotypic effects of piRNA dysfunction in Drosophila, we have uncovered new exciting roles for piRNAs in development and presented evidence how transposons can act as architects in restructuring the host genome.

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.

The integrity of the genome relies on the maintenance of chromosomes, the structural embodiment of the genetic material. Disruption of chromosome replication can lead to extensive genomic rearrangements, spanning kilobase (Kb) to megabase (Mb) regions. Some chromosome rearrangements are inherently dynamic, beginning as a single unstable rearrangement from which multiple rearrangements emerge. The rare formation and transient behavior of unstable chromosomes renders their study challenging. Here I characterize the genetic ontogeny of unstable chromosomes in a budding yeast model, from initial replication error to unstable chromosome formation to their resolution. I find that the initial error often arises in or near the telomere and frequently forms unstable chromosomes that later resolve to an internal "collection site" in the middle of the chromosome. The initial telomere-proximal unstable chromosome is increased in cells mutant for telomerase, the Tel1 checkpoint kinase and even the Rad9 checkpoint protein, with no known telomere-specific function. Defects in Tel1 and the Rrm3 DNA helicase, or the Tel1-MRX complex and 9-1-1 checkpoint clamp, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. I performed a candidate genetic screen of instability in telomere maintenance and DNA damage response (DDR) proteins to characterize the interplay of pathways regulating senescence and genomic instability. Collectively, my results suggest that unstable chromosomes form in or near damaged telomeres, independently of end degradation (Exo1-independent), by either nonhomologous end joining (partially Lig4-dependent) or by faulty template switch during replication (Lig4- and Rad52-independent). The telomere-proximal unstable chromosomes then rearrange further to the middle of the chromosome. These results implicate telomere replication errors as a common source of widespread genomic changes and make substantial progress to our understanding of the initiation and fate of unstable chromosomes in the eukaryotic genome.

A instabilidade genômica tem sido implicada como um dos principais fatores relacionados ao processo de envelhecimento. Esta é consequência do acumulo de danos no DNA em células somáticas continuamente expostas a fatores endógenos e exógenos. Um grupo de proteínas que desempenha diversos papéis na manutenção e estabilidade do genoma é formado pelas RecQ helicases, atuando em vários processos do metabolismo celular, tais como replicação do DNA, recombinação, reparo do DNA e manutenção dos telômeros. Algumas evidencias relacionam a expressão aberrante destas proteínas ao envelhecimento precoce. Com o objetivo de determinar os perfis de expressão transcricional de genes da família RecQ helicase e alguns genes envolvidos na via BER (Base excision repair), como PARP1, POL e APEX1 em células mononucleares do sangue periférico (PBMCs, do inglês Peripheral Blood Mononuclear Cells), comparamos grupos de indivíduos jovens (n = 20), idosos (n = 17) e centenários (n = 27). Além disso, foi também foi avaliado o comprimento telomérico em amostras de DNA desses indivíduos, buscando uma comparação entre os mesmos. Foi observada uma diminuição no nível de expressão transcricional do gene BLM nos grupos idoso e centenário quando comparados ao grupo jovem (p<0,05). Também foi observado uma diminuição na expressão do gene RECQL5 no grupo idoso comparado ao grupo jovem. Para os genes da via BER, foi observada uma repressão na expressão transcricional de PARP1 no grupo idoso em relação ao grupo jovem (p<0,05). Em relação ao comprimento telomérico, nossos resultados demonstraram associação entre a diminuição do comprimento telomérico e a idade. Obtivemos diferença significativa na comparação do comprimento telomérico de idosos e centenários comparados ao grupo jovem. Porém, não foi observada diferença entre os grupos idosos e centenários. Assim, nossos resultados mostram uma associação do processo de envelhecimento com a modulação de alguns genes da família RecQ helicase e participantes da via BER, e com o encurtamento telomérico. Os resultados gerados nesse trabalho são inéditos, sendo que relevantes para melhor compreensão do processo de envelhecimento.
Genomic instability plays a major role in the aging process due to the accumulation of DNA damage in somatic cells continuously exposed to endogenous and exogenous factors. A group of proteins essential in maintaining genome stability is composed by RecQ helicase, acting in several cell metabolism processes such as DNA replication, recombination, DNA repair and telomere maintenance. Some evidence related the aberrant expression of these proteins to premature aging. In order to determine the transcriptional expression profile of RecQ helicase gene family and some genes involved in the BER (Base excision repair) pathway, such as PARP1, POL and APEX1 in peripheral blood mononuclear cells (PBMCs), we compared groups of young (n = 20), elderly (n = 17) and centenarians (n = 27). Furthermore, it was also evaluated telomere length in DNA samples from these individuals. It was observed a decrease in the transcriptional expression of BLM gene in elderly and centenarians compared to the young group (p <0.05). It was also observed a decrease in expression of RECQL5 gene in the elderly compared to the younger group. For the BER genes, it was observed a transcriptional repression of PARP1 in the elderly group compared to the young group (p <0.05). Regarding the telomere length, our results demonstrated an association between reduction of telomere length and age. We obtained significant difference in comparing the telomere length of the elderly and centenarians compared to the younger group. However, no difference was observed between the elderly and centenarians groups. Thus, our results show an association of aging process with the modulation of certain genes from RecQ helicase family and participants of the BER pathway and the telomere shortening. The results generated in this study are promising, and relevant to better understanding the aging process.

Les extrémités des chromosomes comportent le télomère puis la région sous télomérique. Ces deux domaines se distinguent des autres régions chromosomiques car ils évoluent par des échanges entre les chromosomes hétérologues. Le télomère est une structure spécialisée constituant la fin des chromosomes et indispensable à leur stabilité. Il joue un rôle important dans l'organisation spatiale des chromosomes en particulier dans l'agglutination des extrémités chromosomiques en périphérie nucléaire. La région sous télomérique, adjacente au télomère est très redondante entre les chromosomes hétérologues et se termine avec les séquences uniques spécifiques à chaque chromosome. Sa fonction ainsi que sa structure ne sont pas bien connues. Plusieurs familles de séquences répétées y sont présentes. Certaines sont localisées uniquement à proximité du télomère, d'autres comme les minisatellites sont en majorité localisées dans les derniers mégabases des chromosomes. Nous avons étudié en détail une région sous télomérique présente sur une dizaine de chromosomes chez tous les individus. Nous montrons qu'elle s'est propagée par des translocations successives de domaines chromosomiques terminaux de 80 a 200 Kb, impliquant des processus de recombinaison divers. Ces translocations se sont produites après la séparation de l'homme et du chimpanzé. La stabilité de la région apparaît variable suivant les chromosomes ce qui se traduit par un polymorphisme des localisations de la région entre les individus. Cette région sous télomérique a évolué de façon très différente entre l'homme et le chimpanzé. Nous proposons que cette évolution pourrait être conditionnée par la présence de gènes adjacents à la région sous télomerique. Nous avons en effet montré que des gènes ubiquitaires se trouvent à quelques dizaines de Kb en aval de la région sous télomérique. Leur expression pourrait être influencée par la chromatine adjacente, c'est à dire par la nature de la région sous télomérique. Nous proposons enfin que l'évolution de la région sous télomérique constitue un modèle pour l'étude de l'évolution du génome humain.

Genregulation beeinflusst Phänotypen im Kontext von Gesundheit und Krankheit. In Krebszellen regulieren genetische und epigenetische Faktoren die Genexpression in cis. Das Neuroblastom ist eine Krebserkrankung, die häufig im Kindesalter auftritt. Es ist gekennzeichnet durch eine geringe Anzahl exonischer Mutationen und durch häufige Veränderungen der somatischen Kopienzahl, einschließlich Genamplifikationen auf extrachromosomaler zirkulärer DNA. Bisher ist wenig darüber bekannt, wie lokale genetische und epigenetische Faktoren Gene im Neuroblastom regulieren. In dieser Arbeit kombiniere ich die allelspezifische Analyse ganzer Genome (WGS), Transkriptome und zirkulärer DNA von Neuroblastom-Patienten, um genetische und cis-regulatorische Effekte zu charakterisieren. Ich zeige, dass somatische Dosis-Effekte der Kopienzahl andere lokale genetische Effekte dominieren und wichtige Signalwege regulieren. Genamplifikationen zeigen starke Dosis-Effekte und befinden sich häufig auf großen extrachromosomalen zirkulären DNAs. Die vorgestellte Analyse zeigt, dass der Verlust von 11q zu einer Hochregulation von Histonvarianten H3.3 und H2A in Tumoren mit alternativer Verlängerung der Telomere (ALT) führt, und dass erhöhte somatische Kopienzahl die Expression der TERT Gens verstärken können. Weitere Erkenntnisse sind, dass 17p-Ungleichgewichte und die damit verbundene Herunterregulierung neuronaler Gene sowie die Hochregulierung des genomisch geprägten Gens RTL1 durch Kopienzahl-unabhängige allelische Dosis-Effekte mit einer ungünstigen Prognose verbunden sind. Die cis-QTL-Analyse bestätigt eine zuvor beschriebene Regulation des LMO1 Gens durch einen Enhancer-Polymorphismus und charakterisiert das regulatorische Potenzial weiterer GWAS-Risiko-Loci. Die Arbeit unterstreicht die Bedeutung von Dosis-Effekten im Neuroblastom und liefert eine detaillierte Übersicht regulatorischer Varianten, die in dieser Krankheit aktiv sind.
Gene regulation controls phenotypes in health and disease. In cancer, the interplay between germline variation, genetic aberrations and epigenetic factors modulate gene expression in cis. The childhood cancer neuroblastoma originates from progenitor cells of the sympathetic nervous system. It is characterized by a sparsity of recurrent exonic mutations but frequent somatic copy-number alterations, including gene amplifications on extrachromosomal circular DNA. So far, little is known on how local genetic and epigenetic factors regulate genes in neuroblastoma to establish disease phenotypes. I here combine allele-specific analysis of whole genomes, transcriptomes and circular DNA from neuroblastoma patients to characterize genetic and cis-regulatory effects, and prioritize germline regulatory variants by cis-QTLs mapping and chromatin profiles. The results show that somatic copy-number dosage dominates local genetic effects and regulates pathways involved in telomere maintenance, genomic stability and neuronal processes. Gene amplifications show strong dosage effects and are frequently located on large but not small extrachromosomal circular DNAs. My analysis implicates 11q loss in the upregulation of histone variants H3.3 and H2A in tumors with alternative lengthening of telomeres and cooperative effects of somatic rearrangements and somatic copy-number gains in the upregulation of TERT. Both 17p copy-number imbalances and associated downregulation of neuronal genes as well as upregulation of the imprinted gene RTL1 by copy-number-independent allelic dosage effects is associated with an unfavorable prognosis. cis-QTL analysis confirms the previously reported regulation of the LMO1 gene by a super-enhancer risk polymorphism and characterizes the regulatory potential of additional GWAS risk loci. My work highlights the importance of dosage effects in neuroblastoma and provides a detailed map of regulatory variation active in this disease.

Preimplantation genetic diagnosis (PGD) refers to the testing of embryos produced through in vitro fertilization (IVF) in order to identify those unaffected by a specific genetic disorder or chromosomal abnormality. In this study, different methodologies were examined and developed for performance of PGD. Investigation of various whole genome amplification (WGA) methods identified multiple displacement amplification as a reliable method for genotyping single cells. Furthermore, this technology was shown to be compatible with subsequent analysis using single nucleotide polymorphism (SNP) microarrays. Compared to conventional methods used in this study to perform single cell diagnosis (e.g. multiplex PCR), WGA techniques were found to be advantageous since they streamline the development of PGD protocols for couples at high risk of transmitting an inherited disorder and simultaneously offer the possibility of comprehensive chromosome screening (CCS). This study also aimed to develop a widely applicable protocol for accurate typing of the human leukocyte antigen (HLA) region with the purpose of identifying embryos that will be HLA-identical to an existing sibling affected by a disorder that requires haematopoietic stem cell transplantation. Additionally, a novel microarray platform was developed that, apart from accurate CCS, was capable of reliably determining the relative quantity of mitochondrial DNA in polar bodies removed from oocytes and single cells biopsied from embryos. Mitochondria are known to play an important role in oogenesis and preimplantation embryogenesis and their measurement may therefore be of clinical relevance. Moreover, real-time PCR was used for development of protocols for CCS, DNA fingerprinting of sperm samples and embryos and the relative quantitation of telomere length in embryos (since shortened telomeres might be associated with reduced viability). As well as considering the role of genetics in terms of oocyte and embryo viability assessment and the diagnosis of inherited genetic disorders, attention was given to a specific gene (Phospholipase C zeta) of relevance to male infertility. A novel mutation affecting the function of the resulting protein was discovered highlighting the growing importance of DNA sequence variants in the diagnosis and treatment of infertility.

Le maintien de l'intégrité du génome est essentiel pour la survie cellulaire et la propagation de l'information génétique. Une mauvaise prise en charge des dommages de l’ADN et / ou une aberration de la maintenance de l’intégrité des télomères - les extrémités des chromosomes linéaires - provoquent chez l'homme des pathologies associées à une instabilité génétique. Ainsi, des dysfonctions télomériques sont à l’origine de la Dyskératose Congénitale (DC), et de sa forme rare et sévère, le Syndrome de Hoyeraal-Hreidarsson (HHS). Les DC et HHS se caractérisent principalement par une insuffisance médullaire progressive, des défauts développementaux et une prédisposition à développer des cancers. Par ailleurs, de nombreux syndromes associant déficits immunitaires et anomalies développementales sont causés par des défauts de réparation de l'ADN (cas de déficits immunitaires sévères, de l’Anémie de Fanconi (FA), de l’ataxie télangiectasie (AT), etc …). Au cours de ce travail, nous avons réalisé une étude phénotypique et génétique de patients atteints de deux pathologies aux caractéristiques cliniques distinctes. Ce travail de thèse a permis : 1) d'une part d'identifier des mutations de RTEL1 chez 3 patients atteints de HHS, décrivant ainsi une nouvelle cause moléculaire de cette pathologie. L'analyse des cellules de ces patients a révélé le rôle crucial que joue RTEL1 sur la stabilité du génome et le maintien des télomères dans des cellules humaines. 2) d'autre part, d'identifier un défaut en MYSM1, une histone déubiquitinase, dans un nouveau syndrome immuno-hématologique associé à des défauts de réparation de l’ADN présentant certaines similitudes avec l'anémie de Fanconi. Cette étude démontre pour la première fois, qu'outre son rôle dans la régulation transcriptionnelle, MYSM1 participe également aux mécanismes de réparation des lésions de l'ADN
Maintaining genome integrity is essential for cell survival and propagation of the genetic information. Improper management of DNA damages and / or aberrations in maintenance of telomere - the ends of linear chromosomes - causes humans disorders associated with genetic instability. Thus, in humans, telomere dysfunction causes Dyskeratosis Congenita (DC), and its rare and severe form, Hoyeraal-Hreidarsson Syndrome (HHS). DC and HHS are mainly characterized by progressive bone marrow failure, developmental defects and predisposition to cancer. In addition, many syndromes involving immunodeficiency and developmental abnormalities are caused by defects in DNA repair (e.g. severe immune deficiencies, Fanconi Anemia (FA), Ataxia Telangiectasia (AT),…). In this work, we performed a phenotypic and genetic study of patients with two syndromes presenting distinct clinical features. This work permitted : 1) on one hand, to identify RTEL1 mutations in patients with HHS and describe a new molecular cause of this disease. The analysis of patients’ cells revealed the crucial role for RTEL1 in genome stability and telomere maintenance in human cells. 2) on the other hand, to identify mutations in MYSM1, a histone deubiquitinase, in a new immuno-hematological syndrome associated with defects in DNA repair and sharing some similarities with Fanconi anemia. This study demonstrates for the first time that, in addition to its role in transcriptional regulation, MYSM1 is required to cope with DNA damages

Meiotic recombination is a prerequisite for exchange of genetic information in all Sexually reproducing organisms. This process is initiated by the formation of double stranded breaks (DSBs) in DNA followed by homology directed repair. The process is subjected to surveillance mechanisms that control DSB formation and allow for repair of DSBs by halting cell cycle progression. Interestingly, though generation of DSBs is an Essential event in meiosis they are nevertheless regarded as the most lethal forms of DNA damage. If left unrepaired a single DSB can lead to gene deletion, duplication, translocations and missegregation of large chromosome fragments leading to cell death. In Saccharomyces cerevisiae, genetic screens for mutants defective in meiotic recombination led to the identification of a group of genes called the RAD52 epistasis group which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2. A subset of these genes, namely MRE11, RAD50 and XRS2, have been shown by genetic studies to be essential for several nuclear events including sensing DSBs, double strand break repair (DSBR) by homologous recombination (HR) and non homologous end joining (NHEJ), telomere length maintenance, cell cycle activation in response to DSBs, mitotic and meiotic recombination. In vitro, Mre11 displays Mn2+-dependent endonuclease activity on ssDNA, 3'-5' Exonuclease on single- and double-stranded DNA, strand annealing and weak hairpin Opening activities. Mutational analyses have revealed two functional domains in Mre11- Then terminal nuclease domain involved in telomere length maintenance and DSB Processing and the C terminal DNA binding domain involved in DSB formation during Meiosis. Rad50, a 153 kDa protein shares homology with the SMC (Structural Maintenance of Chromosome) family of proteins which are involved in chromosome Condensation and cohesion. It consists of a bipartite N- and C terminal Walker A and Walker B motifs separated by a heptad repeat sequence which folds into an antiparallel Coiled-coil structure. The heptad repeats are separated by a metal binding globular region the Zn hook. Rad50 is an ATP-dependent DNA-binding protein. hRad50 regulates the exonuclease activity of hMre11. Unlike Mre11 and Rad50, which are evolutionarily conserved, Xrs2 is found only in S. cerevisiae and Nbs1 in mammals. Xrs2 appears to be sequence non-specific DNA- binding protein. Xrs2 in yeast or Nbs1 is its counterpart in mammals target Mre11 and Rad50 to the sites of DNA damage and mediate S-phase cell cycle checkpoint activation. Mutations in either one of the MRX subunits results in defects in repair of DSBs, activation of cell cycle checkpoint and shortened telomeres leading to genomic instability. Hypomorphic mutations in MRE11 and NBS1 lead to genetic disorders- A-TLD (ataxia-telangiectasia-like disorder) and NBS (Nijmegen breakage syndrome) respectively, that are phenotypic ally related to AT (ataxia-telangiectasia) caused by mutations in ATM. Patients with AT, A-TLD or NBS syndromes are hypersensitive to radiomimetic agents and are predisposed to cancer. Several lines of evidence suggest that S. cerevisiae strains bearing mre11Δ, rad50Δ or xrs2Δ display shortening of telomeres. Telomeres are the nucleoprotein ends of all linear eukaryotic chromosomes that are important in maintaining the integrity of the genome.Telomeres are comprised of repetitive G rich sequence most of which is double stranded but the extreme 3' end protrudes to form 3' single stranded overhang called the G tail. elopers are essential in preventing end-end fusion of chromosome, are important for chromosome replication, segregation and genome stability. Genetic studies have implicated the MRX complex in both telomerase-dependent and independent telomere length maintenance. Studies have indicated a direct role for S. cerevisiae MRE11 in the proper establishment of telomere end-structure. However, the molecular mechanism of MRX at telomeres is poorly understood. To understand the role(s) of MRX complex at telomeres, it is important to elucidate the biochemical activities of MRX complex as well as its individual subunits on the telomere DNA structures. Since, Mre11 complex is known to function in several processes related to DNA metabolism it becomes imperative to study the function of Mre11 complex on DNA substrates in the context of a given nuclear process. The 3' single trended telomeric sequence is capable of acquiring folded conformation(s) as a mechanism of end protection which is mediated by several telomere-specific and nonspecific ending proteins. In mammals, the 3' ssDNA has been demonstrated to fold into tloop configuration mediated by some of the components of sheltrin protein complex, wherein the ssDNA invades the duplex DNA resulting in the formation of a displacement loop (D loop). Evidence for the formation of t-loop has been shown in vitro with human telomeres. However, the formation of t-loops has not been demonstrated in S. cerevisiae. Nevertheless, there is growing body of evidence which suggests the formation of alternative DNA structures such as G4 DNA at the yeast telomeres. G quadruplexes (G quartets or G4 DNA) are thermodynamically stable structures formed by Hoogsteen base pairing between guanine residues. In a G quartet the four guanine residues are paired, where each guanine residue is an electron acceptor and a donor and stabilized by a metal cation. The presence of G rich motifs at the promoter regions, rDNA, telomeres and recombination hot spots indicate that G4 DNA has important functions in vivo. Although the existence of G4 DNA has been the subject of much debate, the identification of several proteins that promote (Rap1, Hop1, Topo I, TEBPβ), modify and resolve (POT1, TERT, KEM1, GQN1, BLM, WRN, Rte1) G4 DNA, together with the direct visualization of G4 DNA using G4 DNA specific antibodies and RNA interference have provided compelling for the existence of G4 DNA in vivo. To elucidate the function of MRX complex or its individual subunits at telomeres, the biochemical activities of purified MRX complex and its individual subunits on G4 DNA, D loop, duplex DNA and G rich ssDNA has been analyzed in this study. G4 DNA was assembled from S. cerevisiae telomeric sequence. G4 DNA was isolated and its identity was ascertained by chemical probing and circular dichroism. S. cerevisiae MRE11 and XRS2 was cloned and expressed in E. coli BL21 (DE3)plysS. S. cerevisiae RAD50 in pPM231 vector in S. cerevisiae BJ5464 strain was a gift from Dr. Patrick Sung (Yale University). Mre11, Rad50 and Xrs2 were overexpressed and purified to >98% homogeneity. The identity of the proteins was ascertained by Western bloting using polyclonal antibodies. Using purified proteins heterotrimeric MRX and heterodimeric MR and MX protein complexes were formed in the absence of ATP, DNA or Mn2+. The ability of M/R/X to bind to telomeric DNA substrates was studied by electrophoretic mobility shift assays. Mre11, Rad50, Xrs2 and MRX displayed higher binding affinity for G4 DNA over D loop, ss- or dsDNA. MRX bound G4 DNA more efficiently compared to its individual subunits as 10-fold lower concentration of MRX was able to shift the DNA into the protein-DNA complex. The protein-G4 DNA complexes were stable as >0.8 M NaCl as required to dissociate 50% of protein-G4 DNA complexes. Efficient competition by poly(dG), which is known to fold into G4 DNA, suggested that the protein-G4 DNA complex was specific. Competition experiments with tetra-[N-methyl- pyridyl]-porphyrin suggested that M/R/X recognizes distinct determinants and makes specific interactions with G4 DNA. G4 DNA is highly polymorphic and can exist as intramolecular or intermolecular (parallel and antiparallel) structures. High affinity binding of Mre11 to G4 DNA (parallel) over G2' DNA (antiparallel), ss- and dsDNA suggests the existence of parallel G4 DNA structures at the telomeres and that G4 DNA may be the natural substrate for MRX complex in vivo. Telomeres are elongated by telomerase that requires access to the 3' G-tail for its activity. Formation of G4 DNA structures renders the 3' G-tail inaccessible to telomerase thereby inhibiting telomere elongation. To elucidate the functional relevance of high affinity of M/R/X for G4 DNA, the ability of the complex to generate the appropriate DNA structure for telomere elongation has been analyzed. In this study, I considered the possibility that MRX could act as: (a) a helicase that opens up the G4 DNA structures making it accessible to telomerase or (b) as a nuclease that cleaves the G4 DNA generating substrates for telomerase. Helicase assay with Mre11, Xrs2, MX and MRX on G4 DNA and duplex DNA showed no detectable DNA unwinding activity. Interestingly, nuclease assays with Mre11 on G4 DNA showed that Mre11 cleaved G4 DNA in Mn2+-dependent manner and the cleavage was mapped to the G residues at the stacks of G quartets. Mre11 cleaved telomeric duplex DNA in the center of TGTG repeat sequence, G rich ssDNA at 5' G residue in an array of 3 G residues and D loop structure preferentially at the 5' ends at TG residues. Significantly, the endonuclease activity of Mre11 was abrogated by Rad50. Xrs2 had no effect on the endonuclease activity of Mre11. Structural studies on Rad50 and Mre11 showed that binding of ATP by Rad50 positions the Rad50 catalytic domain in close proximity to the nuclease active site of Mre11. In yeast, disruption of ATP binding Walker motifs results in a null phenotype, suggesting that ATP is required for Rad50 functions in vivo. hRad50 is known to regulate the exonuclease activity of hMre11 in the presence of ATP. Therefore, can ATP modulate the effect of S. cerevisiae (Sc) Rad50 on ScMre11? To address this question, I monitored the ATPase activity of Rad50 in the absence or presence of DNA. Rad50 hydrolyzed ATP in a DNA-independent manner; however, ATPase activity was enhanced in the presence of Mre11 and Xrs2. However, Rad50 exhibited a low turnover indicating that ATP could function as a switch molecule. Based on these observations, the effect of ATP on the nuclease activity was examined. The binding of ATP and its hydrolysis by Rad50 attenuated the inhibition exerted by Rad50 on the Mre11 endonuclease activity. Cleavage of G4 DNA, D loop, duplex DNA and ssDNA required ATP hydrolysis, since no cleavage product was observed when ADP or ATPγS was substituted for ATP. This observation was corroborated using a hairpin DNA substrate that mimics a intermediate in VDJ recombination, thereby confirming the generality of regulation of Rad50 on the endonuclease activity of Mre11. Does Rad50 regulate the exonuclease activity of Mre11 as well? To address this question, exonuclease activity of Mre11, MR and MRX on 3' labeled duplex DNA and G4 DNA was assayed. Rad50 had no measurable effect on the exonuclease activity of Mre11. Based on previous studies and my observations, I propose a model for the role of MRX in telomere length maintenance and its regulation by the ATP-binding pocket of Rad50. MRX binds telomeric DNA substrates in a non-productive complex, which is converted to a catalytically active complex upon binding of ATP by Rad50. ATP induces conformational changes, repositioning the complex such that the catalytic site of Mre11 now has access to the substrate. Following cleavage of DNA by Mre11, the release of ADP and inorganic phosphate, generate the cleaved product. The cleaved DNA is now accessible to telomerase or telomere binding proteins. In summary, the data presented in my PhD thesis demonstrates that Mre11 is a structure- and sequence-specific endonuclease. The natural substrate for telomerase is the 3' ssDNA. G quartets at telomeres not only protect the ends from degradation but also make the ends inaccessible for telomerase activity. Genetic studies have shown that cells proficient for telomerase activity but lacking any one of the components of the MRX complex display shortening in telomere length. The ability of Mre11 to cleave G4 DNA at the stacks of G quartets therefore, suggests a mechanism by which the 3' ssDNA is rendered accessible to telomerase or other telomere binding proteins. Yeast telomeres are characterized by the presence of subtelomeric Y' elements proximal to the terminal TG1- 3 repeat sequences. The Y' element has been shown to be amplified by telomerase in a fraction of mutants with short telomeres. The mechanism by which Y' DNA is amplified is unclear. The ability of Mre11 to cleave telomere duplex DNA at the center of TGTG repeats could contribute to the generation of appropriate substrate for elongation by telomerase, thereby contributing to Y' DNA amplification. Telomere length is maintained by homeostasis between processes that contribute to telomere elongation and those that cause attrition in telomeric ends. Overelongated telomeres are brought to wild type telomere size by a unique recombinational single step deletion process termed telomere rapid deletion (TRD). TRD involves invasion of the elongated 3' G tail into the proximal telomeric tract resulting in the formation of the D loop structure. Following branch migration the D-loop is nicked and resolved into a deleted telomere and a circular liner product. Cells deleted for MRE11, RAD50 or XRS2 are deficient in TRD process. It has been hypothesized that Mre11 could be a candidate for cleaving the D-loop structure. The endonuclease activity of Mre11 on D-loop structure, preferentially at the 5' ends at TG residues demonstrated in this study, show that Mre11 could function as the nuclease required to generate the deleted telomere in TRD. MRX complex is involved in several processes involving DNA metabolism. It is important that the activities of the complex are regulated in the in vivo context. Complex formation and the interaction of the individual subunits with nucleotide cofactors and metal ions constitute a mode of regulation. This study shows that Rad50 regulates the endonuclease, but not exonuclease activity of Mre11. The binding of ATP and its hydrolysis by Rad50 brings in the regulatory factor necessary to keep the uncontrolled nuclease activity of MRX in check, thus preventing any deleterious effects on telomere length. Telomere maintenance by telomerase is activated in 80% of cancer cells. Inhibition of telomerase by G quartets provides a new drug targets for potential anti-cancer drugs. It is, therefore, likely that understanding the biological consequences of G quadruplex interactions would provide a better insight in development of therapeutics for cancer.

In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

Le matériel génétique (l’ADN) d’un organisme contient l’information nécessaire à sa survie, sa croissance et sa reproduction. La perte de cette information affecte grandement la santé de l’organisme et cette altération est l’un des facteurs les plus courants dans le vieillissement ou le cancer. Quasiment toutes les cellules d’un organisme contiennent une copie de ce matériel génétique, communément appelé le génome, et font usage de plusieurs mécanismes pour en réparer les sections endommagées ainsi que pour le copier avec précision lors de la division cellulaire. Nous avons cherché à étudier les processus cellulaires qui maintiennent la stabilité génomique en inactivant systématiquement chacun des gènes avec la technique de criblage par CRISPR afin d’en étudier les rôles. Nous avons effectué ces criblages à l’échelle du génome dans des lignées cellulaires humaines en combinaison avec des perturbations chimiques dans le but d’identifier l’effet du traitement chimique ou le rôle de gènes qui exacerbent ou atténuent la perturbation. Nous nous sommes d’abord concentrés sur le resvératrol, une molécule initialement extraite de plantes qui a démontré des propriétés antivieillissement dans certains organismes modèles ainsi que la capacité d’inhiber la prolifération cellulaire. Notre criblage génétique a révélé que le resvératrol inhibait la réplication de l’ADN. En comparant les effets cellulaires du resvératrol à l’hydroxyurée, un agent connu pour causer du stress réplicatif, nous avons montré que ces deux traitements menaient à une diminution similaire de la progression de la fourche de réplication ainsi qu’à une activation de la signalisation en réponse au stress réplicatif. Nous avons également démontré que l’inhibition de la réplication de l’ADN dans les cellules humaines par le resvératrol est l’un des effets principaux de la molécule sur la prolifération cellulaire et ne requiert pas la présence de la déacétylase d’histone Sirtuin-1, protéine qui a été suggérée comme étant la cible principale du resvératrol pour son effet antivieillissement. Nous avons également étudié la perturbation d’un second processus cellulaire, soit le maintien des télomères. Ces séquences spéciales aux extrémités des chromosomes sont indispensables à la protection du génome et leur érosion graduelle est contrebalancée par l’activité enzymatique de la télomérase. Nous avons effectué un crible génétique par CRISPR à l’échelle du génome dans une lignée cellulaire dont nous avons inhibé la télomérase en utilisant BIBR1532, un inhibiteur spécifique de la télomérase. Nous avons découvert une forte interaction génétique entre la télomérase et C16orf72, un gène non-annoté que nous avons nommé TAPR1. Nous avons montré que les cellules déficientes en TAPR1 possèdent des niveaux élevés de la protéine p53, un facteur de transcription central à la réponse cellulaire aux dommages télomériques et aux dommages à l’ADN. Nous suggérons que TAPR1 agit comme un inhibiteur de la stabilité protéique de p53. En somme, ces travaux mettent en évidence la capacité des cribles génétiques CRISPR à approfondir nos connaissances sur le fonctionnement des processus de maintien de la stabilité génomique chez l’humain.
The genetic material (DNA) of an organism contains the necessary information for survival, growth and reproduction. Loss of this information strongly impacts the health of the organism and is the leading factor in aging and cancer. Almost all cells in an organism contain a copy of said genetic material (genome) and employ several mechanisms to repair any damaged section of the genome and to accurately copy it during cell division. We sought to understand the cellular processes by which cells maintain genome stability by systematically inactivating individual genes to uncover their role using pooled CRISPR-Cas9 screening. We employed genome-wide CRISPR screening in human cell lines in combination with specific chemical perturbations to identify gene deletions that enhance or suppress the phenotype of the chemical treatment, thereby shedding light on the effect of the treatment or the role of said enhancer/suppressor genes. We first focused on resveratrol; a small molecule first discovered in plants that has been suggested to extend lifespan in model organisms while also inhibiting cell proliferation ex vivo. Chemical-genetic screening pinpointed a role of resveratrol in inhibition of DNA replication. When we compared the cellular effects of resveratrol to hydroxyurea, a known inducer of replicative stress, we found that both treatments led to slower replication fork progression and activation of signaling in response to replicative stress. Importantly, we showed that the inhibition of DNA replication by resveratrol in human cells is a primary effect on cell proliferation and independent of the histone deacetylase Sirtuin-1, which has been implicated as the primary target in lifespan extension by resveratrol. We then studied the perturbation of a second cellular process, namely telomere maintenance. These specialized sequences at the termini of chromosomes are critical for the protection of chromosome ends and their erosion is counteracted by the enzymatic activity of telomerase. We performed a genome-wide CRISPR screen in cells that were concomitantly treated with a specific telomerase inhibitor, BIBR1532. We uncovered a strong genetic interaction between telomerase and a previously unannotated gene, C16orf72, which we named TAPR1. We found that TAPR1-depleted cells led to elevated p53 levels, a transcription factor central for the cellular response to telomeric and global DNA damage. We propose that TAPR1 is a negative regulator of p53 protein levels by promoting its turnover. Altogether, these studies highlight the power of CRISPR-Cas9 in genetic screening to uncover novel insight into the human genome stability maintenance network.

La sénescence réplicative est le phénomène associé à un arrêt de croissance permanent causé par le raccourcissement progressif des télomères à chaque division. Lorsqu’ils atteignent une longueur critique, les télomères perdent leur structure terminale protectrice en t-loop, ce qui révèle l’extrémité du chromosome et déclenche une Réponse aux dommages à l’ADN (RDA) p53-dépendante. Le nombre de télomères ouverts nécessaire à la mise en place de la sénescence n’est pas connu, mais plusieurs évidences suggèrent que la cellule pourrait en tolérer un certain nombre avant de s’arrêter définitivement. Dans ce projet, nous utilisons un dominant négatif de Tin2 (Tin2DN), un membre du complexe nucléo-protéique nommé le télosome qui stabilise la t-loop, pour démontrer que la dysfonction chromatinienne télomérique seule ne suffit pas à déclencher un arrêt de croissance permanent. Lorsqu’il est exprimé, Tin2DN induit la formation de foyers de dommages de 53BP1, la RDA ainsi qu’un arrêt de croissance transitoire. De façon surprenante, nous observons que les cellules qui ont subi ce premier arrêt de croissance ré-entrent dans le cycle cellulaire et se divisent, et ce malgré la présence de foci télomériques. Cette réentrée cause l’apparition de cassures secondaires ainsi qu’une accumulation d’instabilités génomiques, telles que des ponts chromosomiques ou des micro-noyaux. Cet échappement des points de blocages du cycle cellulaire pourrait être expliqué par notre observation que la dysfonction télomérique induite par Tin2DN n’active que très faiblement p53 et p21, et pratiquement pas la kinase chkChk2. Néanmoins, en inhibant directement l’activité de p53, nous n’observons plus aucun arrêt de croissance mais une accumulation de foci et d’instabilités génomiques, avec une forte occurrence de catastrophes mitotiques. L’ensemble de ces résultats propose un nouveau modèle d’entrée en sénescence réplicative : l’ouverture des télomères induits une faible RDA menant à un premier arrêt de prolifération transitoire p53-dépendant. Les cellules échappent à cet arrêt et se divisent, mais l’ouverture des télomères ayant causé des fusions chromosomiques, la division crée alors de nouvelles cassures doubles brins dans le génome qui déclencheront une forte RDA et un nouvel arrêt de croissance permanent, la sénescence réplicative.
Replicative senescence is the physiological permanent growth arrest caused by telomeres shortening, at each round of replication. Once they have reach a critical length, the telomeres lose their t-loop structure, revealing the chromosome extremity that triggers a p53-dependant DNA damage response (DDR) and leads to proliferation arrest. The number of shortened telomeres that are necessary to onset senescence is not known, but accumulating evidences suggest that the cell is able to tolerate a certain level of telomere uncapping before stopping its divisions. Here, we used an inducible dominant negative form of Tin2 (Tin2DN), a member of the shelterin complex that stabilizes the t-loop, to show that telomeres uncapping alone is not sufficient to induce a stable growth arrest. When expressed, Tin2DN leads to the openingverture of the t-loop, creating a DDR with the formation of 53BP1 DNA damage foci (DDF) and a transient growth arrest. Indeed, we observed that the cells were re-entering the cell cycle and dividing, despite their uncapped DDF harbouring telomeres. As telomere uncapping creates chromosome fusions, such division leads to the apparition of secondary DNA breaks, with an accumulation of genomic instabilities, such as chromosomes bridges or micronuclei. We observed that Tin2 DN-induced telomere uncapping leads to a very weak activation of p53 and p21, with almost no phosphorylation of chkChk2. Nevertheless, when we infected our cells with a shp53, the primary growth arrest did not occur, leading to an amplification of the damages, with strong signs of instability and mitotic catastrophe. Altogether, these results propose a new model for replicative senescence: telomere uncapping induces a weak DDR that leads to a transitory growth arrest. The cells divide with fused chromosomes, creating new randomly distributed double strand breaks that trigger a stronger DDR and a permanent growth arrest. In that model, replicative senescence is not directly induced by telomere uncapping, but by an amplification of DNA damages through mitotic catastrophe.