INO80, a member of the multi-subunit SWI2/SNF2 superfamily, is involved in transcription regulation, DNA repair and replication. Not much is known about its substrate specificity and remodeling mechanism or how it differs in comparison to SWI/SNF or ISWI. Site-directed mapping of histone-DNA contacts showed that INO80 generally remodels mononucleosomes by moving them to the center of DNA. The length of extranucleosomal DNA was found to play an important role in nucleosome binding as well as remodeling by INO80 much like ISW2 and ISW1a. INO80 preferentially binds to nucleosomes containing >20bp of extranucleosomal DNA. Similarly, INO80 remodeling of mononucleosomes with different lengths of extranucleosomal DNA showed that at least 33bp of extranucleosomal DNA on one side of the nucleosome was required for initiation of remodeling. These data suggest that INO80 behaves much like ISW2 and ISW1a complexes based on their requirement for extranucleosomal DNA. INO80 does not unravel or displace nucleosomes like SWI/SNF. There are several key aspects of how INO80 interacts with and remodels nucleosomes that are quite distinct from SWI/SNF, ISW2, and ISW1a. Previously SWI/SNF and ISW2 were shown to initiate nucleosome movement by translocating along nucleosomal DNA two helical turns from the dyad axis. Nucleosome movement by INO80 instead requires translocation by the complex along nucleosomal DNA near the entry/exit site at the dimer-tetramer interface. Sliding interference of INO80 by the presence of nicks indicated that torsional strain at the site of translocation is required for nucleosome mobilization by INO80. Hydroxyl radical footprinting of the INO80-nucleosome complex shows found that INO80 interactioninteracts with extranucleosomal DNA at, the entry-exit site and to lesser extent at the dyad axis, but it lacks the protection found indoes not contact 2 helical turns from the dyad like ISW2 and SWI/SNF at two helical turns from the dyad axis as determined by photoaffinity cross-linking studies. The catalytic subunit (Ino80) rather than being found associated 2 helical turns from the dyad, was bound to extranucleosomal DNA and nucleosomal DNA near the entry-exit site. Other subunits (Arp8p, Arp5p and Nhp10) were also found to be contacting both nucleosomal and extranucleosomal DNA. Site-specific histone cross-linking studies revealed that Ino80, Arp5 and Arp4 interact extensively with the histone dimer of the nucleosome in comparison to H3-H4 tetramer. Although N-terminal histone tails are often important for chromatin remodeling, INO80 shows no requirement of histone tails for its nucleosome binding and mobilizing activities. The deviation of INO80 from the canonical model of how ATP-dependent remodelers interact and mobilize nucleosome is apparently due to its unique role as a member of the remodeling complexes that promote the exchange of H2A/H2B dimer from core nucleosome particle.

The dynamic nature of eukaryotic chromatin enables the packaging of large amounts of genetic material in a small space. At the same time, it provides controlled access to genomic DNA for a variety of nuclear processes for example, transcription and DNA repair. The transition between open and closed chromatin states is largely governed by ATP-dependent chromatin remodeling complexes, which operate on nucleosomes in concert, to modulate chromatin structure and composition. Exchange of the canonical and variant forms of histones in nucleosomes, and altering the spacing between consecutive nucleosomes, are two major ways which regulate chromatin-based processes and chromatin higher-order organization. The evolutionarily conserved INO80 and ISW1a complexes mediate these two aspects of nucleosome remodeling, respectively. Despite sharing conserved domain architecture of the core remodeling machinery, chromatin remodelers differ significantly in their modes of interaction with nucleosomes, and how they alter histone-DNA contacts. In this study, we have used a site-specific photocrosslinking approach coupled with peptide mapping to determine the interactions of subunits and domains of the S. cerevisiae INO80 and ISW1a complexes with nucleosomes. We find that specific interactions of remodelers with different regions of the nucleosome largely dictate their specialized functions and mechanisms. The ATP-dependent helicase-like (ATPase) domains of remodelers belonging to the ISWI and SWI/SNF families translocate along DNA close to the center of nucleosomes in order to mobilize, space or disassemble nucleosomes. In contrast, we observed that INO80 has a strikingly distinct mechanism, which is different even from its paralog SWR1. INO80 mobilizes nucleosomes as well as catalyzes the exchange of histone variant H2A.Z for the canonical histone H2A, while SWR1 mediates the reverse exchange of H2A for H2A.Z, without being able to mobilize nucleosomes. We have found that INO80, in order to promote H2A-H2B dimer exchange, translocates along DNA at the H2A-H2B interface close to the edge of nucleosomes and persistently displace DNA from H2A-H2B. Blocking either DNA translocation or the accumulation of DNA torsions close to the edge of the nucleosome interferes with this dimer exchange by INO80. SWR1 and other SWI/SNF and ISWI remodeling complexes translocate along DNA at the H3-H4 interface and do not persistently displace DNA from the histone octamer as does INO80. This study shows for the first time an ATP-dependent chromatin remodeler that invades nucleosomes at the DNA entry site instead of the center − a more logical approach for the displacement of H2A-H2B. We also investigated nucleosomal DNA interactions of other INO80 subunits and domains to understand the architecture of INO80 bound to nucleosomes. We found that the HSA (helicase-SANT-associated) domain of Ino80 along with actin-related protein (Arp) subunits Arp8 and Arp4 bind to the extranucleosomal DNA and is potentially involved in a coupling mechanism with the ATPase domain to regulate its activity. We also mapped the DNA binding regions of Arp8 and Arp4, which might be involved in recruiting INO80 to genomic sites. The ISWI remodeler ISW1a regulates the distance (spacing) between nucleosomes in an array by simultaneously interacting with two nucleosomes and directionally remodels one of them. We mapped DNA interactions of ISW1a subunits in mono- and di-nucleosomes. Our results show that the catalytic Isw1 subunit specifically interacts with the region of DNA translocation and DNA entry site of the asymmetrically positioned nucleosome in a di-nucleosome, which is preferentially mobilized. In contrast, the Ioc3 subunit interacts extensively with the linker DNA as well as the extranucleosomal DNA of the un-remodeled nucleosome. This bias in nucleosomal DNA interactions of ISW1a enables directional remodeling, which reveals the molecular basis of nucleosome spacing. We have identified a novel domain within the non-catalytic Ioc3 subunit of ISW1a that regulates nucleosome spacing. We found that when this domain is deleted, the catalytic Isw1 subunit loses its specificity and interacts with both the nucleosomes of a di-nucleosome substrate. This is consistent with the domain-deleted ISW1a mobilizing both nucleosomes efficiently, leading to the loss of its nucleosome spacing activity. In summary, this dissertation explores how different remodeling complexes have customized and regulated modes of nucleosome interaction in order to accomplish specialized remodeling outcomes. INO80 places its ATPase domain for translocation at the H2A-H2B dimer interface and persistently displaces DNA from its surface to promote H2A.Z exchange. Nucleosome spacing by ISW1a requires the catalytic Isw1 subunit to engage with and reposition one out of two consecutive nucleosomes in an array, while the Ioc3 subunit likely monitors the distance between them.

The ATP-dependent chromatin remodeling complex SWR1 exchanges a variant histone H2A.Z-H2B dimer for a canonical H2A-H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 Mega-Dalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single hetero-hexameric Rvb1/2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multi-subunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvb1/2 ring and a distinct substrate-handling mode by SWR1, thereby providing the first structural framework for understanding the complex dimer-exchange reaction.

Les extrémités des chromosomes contiennent des répétitions de séquences d’ADN appelées télomères qui empêchent l’activation inopportune de la réponse aux dommages de l'ADN afin de préserver l'intégrité génomique. Les télomères raccourcissent à chaque cycle de réplication d’ADN et la télomérase a pour fonction de contrebalancer cette érosion en allongeant les télomères. Les cellules somatiques n’expriment pas la télomérase, donc leur durée de vie est normalement limitée par ce raccourcissement progressif des télomères qui conduit à l'activation de la voie p53 entraînant un arrêt de la croissance cellulaire. En revanche, les cellules cancéreuses acquièrent l'immortalité cellulaire principalement en réactivant la télomérase ou en utilisant des méthodes alternatives d'allongement des télomères basées sur la recombinaison d’ADN. Auparavant, dans notre laboratoire, un criblage CRISPR à l'échelle du génome a été réalisé dans la lignée cellulaire pré-B NALM-6 traitée avec la molécule BIBR1532, un inhibiteur de la télomérase. Ces résultats suggéraient que cinq sous-unités du complexe de remodelage de la chromatine INO80, lorsque supprimées indépendamment, réduisaient la prolifération des cellules ayant un raccourcissement des télomères induit par le BIBR1532. Mon objectif était d'étudier cette interaction génétique afin de comprendre les processus biologiques impliqués dans cette létalité synthétique. Après l'élimination des gènes codant à la fois pour la sous-unité enzymatique de la télomérase humaine (hTERT) ainsi que les sous-unités spécifiques du complexe INO80 humain, nous avons constaté que les cellules double-négatives avaient une capacité proliférative réduite, ce qui démontre que l’interaction génétique mesurée par criblage CRISPR est bel et bien spécifique. Étant donné le rôle du facteur de transcription p53 dans la réponse cellulaire au raccourcissement télomérique, nous avons exploré l’importance de cette voie de signalisation pour l’interaction entre le complexe INO80 humain et la télomérase. Après l’activation de p53 avec un traitement avec la molécule nutlin-3a, les niveaux d'expression de plusieurs cibles de p53 tels que MDM2 et CDKN1A ont augmenté dans les cellules ayant une délétion du gène NFRKB, codant pour une sous-unité du complexe INO80 humain. Les cellules ayant une délétion du gène UCHL5, codant pour le partenaire d’interaction de NFRKB, ont également montré une augmentation de l’expression de MDM2 lorsque traitées avec nutlin-3a. Enfin, la perte de télomérase (hTERT) modifie les niveaux d'expression des composants de la 2 voie p53 CDKN1A, BAX et MDM2. En conclusion, la suppression des gènes codant pour des sous-unités du complexe INO80 telles que NFRKB ou UCHL5 est nuisible aux cellules ayant une délétion de la télomérase. Le complexe INO80 humain peut être impliqué dans l'inhibition de la voie p53, en réponse à l'activation de p53 soit par des télomères courts ou avec un traitement avec nutlin-3a. Des recherches plus approfondies sur cette interaction génétique pourraient mener au développement de nouvelles thérapies combinatoires afin d’inhiber la croissance des cellules cancéreuses. / The ends of chromosomes contain telomeric repeats that prevent the DNA damage response from being activated in order to preserve genomic integrity. Telomerase functions to alleviate incomplete DNA replication at telomeres, and to repair those telomeres damaged by various means including oxidative damage. The lifespan of telomerase negative somatic cells is normally restricted by gradual telomere shortening which can lead to the activation of the p53 pathway resulting in cellular growth arrest. Cancer cells often elongate their telomeres in order to acquire cellular immortality predominantly by reactivating telomerase or by using recombination-based, alternative telomere lengthening methods. Previously in our lab, a genome-wide CRISPR screen was conducted in the pre-B cell line NALM-6 treated with a small molecule inhibitor of telomerase, BIBR1532. These previous results suggested that five subunits of the INO80 chromatin-remodeling complex, when independently deleted, reduced cellular proliferation in cells with BIBR1532 induced telomere shortening. My goal was to investigate this genetic interaction in order to understand the biological processes implicated in this synthetic lethal relationship. After the knockout of the genes encoding both the enzymatic subunit of human telomerase (hTERT) and specific subunits of the human INO80 complex, I found that the proliferative capacity of NALM-6 cells was reduced. This result indicates the genetic interaction identified by CRISPR screening is in fact specific. In addition, after p53 stimulation with nutlin-3a treatment, expression levels of the p53 pathway component MDM2 were altered after the knockout of the genes encoding specific subunits of the human INO80 complex, NFRKB and UCHL5, individually. CDKN1A expression was also altered after nutlin-3a treatment and NFRKB knockout. Finally, the loss of telomerase (hTERT) alters the expression levels of the p53 pathway components CDKN1A, BAX and MDM2. In conclusion, the deletion of the genes encoding specific subunits of the INO80 complex, including NFRKB and UCHL5, is harmful to cells after hTERT knockout. The human INO80 complex may be involved in inhibiting the p53 pathway, in response to p53 activation by short telomeres or nutlin-3a treatment. Further investigation into this synthetic lethal relationship may shed light on new combinatorial therapeutics in cancer.