Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 161-184).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 137-154).

In vivo DNA is compacted tightly, via its association with histones and non-histone proteins, into higher-order chromatin structure. In this state, the DNA is refractory to the cellular factors that require access to DNA. The repressive nature of chromatin is alleviated in part by the action enzymes that modify chromatin structure. There are two major groups of chromatin modifying enzymes: those that post-translationally modify histones by the addition of small chemical moieties and those that utilize the energy derived from ATP hydrolysis to physically disrupt chromatin structure. The SWI/SNF enzyme belongs to this latter group. The SWI/SNF complex was identified originally in yeast. Several of its subunits are required for the expression of a subset of inducible genes. The ATPase activity is provided by the SWI2/SNF2 protein. In mammals, there are two biochemically separable SWI/SNF complexes that contain either BRG1 or BRM, both homologs of yeast SWI2/SNF2. The yeast and mammalian SWI/SNF complexes are able to disrupt the Dnase I digestion pattern of in vitro assembled mononucleosomes and arrays, as well as facilitate the accessibility of restriction nucleases and transcription factors. The mechanism by which SWI/SNF functions has yet to be elucidated. SNF5 is a component of the yeast SWI/SNF complex. It is required for sucrose fermentation and mating type switching. The mammalian homolog of Snf5 is SNF5/INI1. SNF5/INI1 was identified simultaneously by two groups as a protein that shares homology with Snf5 and via a yeast two hybrid assay as a protein that interacts with HIV integrase (INtegrase Interactor). INI1 is a component of all mammalian SWI/SNF complexes purified to date. In humans, mutations and/or deletions in INI1 are associated with a variety of cancers, including malignant rhabdoid tumors, choroid plexus carcinomas, medullablastomas, primitive neuralectodermal tumors, and some cases of leukemia. Furthermore, constitutional mutations within INI1in individuals presenting with these tumors support the role of INI1 as a tumor suppressor. In this thesis, we show that Ini1 also functions as a tumor suppressor in mice. Approximately 20% of mice heterozygous for Ini1 present with tumors. Most of these tumors are undifferentiated or poorly differentiated sarcomas with variable rhabdoid features. All tumors examined to date show loss of heterozygosity at the Ini1 locus. We also show that Ini1 is essential for embryonic development. Mice homozygous-null for Ini1die between days 4 and 5.5 post-fertilization due to an inability to adhere to their substratum, form trophectoderm, and expand their inner cell mass. We further characterize the function of Ini1 in tumor suppression by generating mice heterozygous for both Ini1 and either Rb or p53. While heterozygosity at the Ini1 locus appears to have no effect on the rate of tumorigenesis in Rb-heterozygous mice, many of the tumors arising in compound heterozygous mice present with an altered morphology. This finding suggests that Ini1 may contribute to tumor progression due to loss of Rb. In contrast, mice compound heterozygous for Ini1 and p53 show a marked reduction in the rate of tumorigenesis compared to p53-heterozygous mice. Furthermore, the tumor spectrum is altered in these compound heterozygous mice. These findings suggest that Ini1 may function normally to repress p53 activity. Lastly, we show that expression of the Ini1 tumor suppressor itself is regulated tightly. Tissues and cells heterozygous for Ini1 express roughly equivalent levels of Ini1 protein and mRNA as their wild-type counterparts. We further show that this compensation is mediated by an increase in the rate of transcription from the wild-type Ini1 allele. Moreover, when exogenous Ini1 is introduced into Ini1-heterozygous cells, expression from the Ini1 promoter is reduced. These data indicate that a compensatory mechanism exists to ensure that the steady-state levels of Ini1 are constant. In summary, research detailed in this thesis has contributed to our understanding of the regulation of Ini1 as well as the role this protein plays in mammalian development and tumor suppression.

Members of the Runx family of transcription factors play essential roles in the differentiation and development of several organ systems. Here we address the contribution of the osteoblast-related Runx gene, Runx2, to the osteogenic and chondrogenic differentiation of mesenchymal stem cells. Using a transgenic mouse model, we observe Runx2 transcription through one of its two known promoters (designated P1 in pre-cartilaginous mesenchymal condensations as early as E9.5. Runx2 gene activity is later repressed at the onset of cartilage formation, both in vivo and in vitro, necessitating examination of the regulation and function of Runx2 in mesenchymal stem cells. We demonstrate that Runx2 gene activity is repressed by the direct interaction of the homeodomain transcription factor Nkx3.2 with the proximal Runx2 P1 promoter. This repression was found to be required for the progression of BMP-induced chondrogenesis, thereby identifying Runx2 as a modulator of BMP activity in the chondrogenic as well as osteogenic differentiation program. To further understand the regulation of the Runx2 P1 promoter and to determine the contribution of P1-derived gene product, Runx2 Type II, to the formation of mineralized tissue, we have generated a Runx2 Type II-LacZ gene replacement mouse model in which the initial coding sequences and splice donor sites of the Type II isoform are replaced with the LacZ reporter gene. Activity of the endogenous P1 promoter can therefore be monitored by β-galactosidase production. Analysis of Runx2 Type II-LacZ mice demonstrates that the P1 promoter is transcriptionally most active in mature osteoblasts, but its product, Runx2 Type II is dispensable for embryonic skeletal formation. Lastly, we examine the link between growth control and osteogenic differentiation by tissue-specific deletion of the Mdm2 proto-oncogene in developing skeletal tissues of the mouse embryo. Loss of Mdm2 results in impaired bone formation, with skeletal elements exhibiting lower bone mineral content and higher porosity. Ex vivo cultures of calvarial osteoprogenitor cells exhibit severely decreased osteoblastogenesis and bone nodule formation accompanied by a failure to activate Runx2 gene activity. These findings suggest that Mdm2 is required for inhibition of p53 activity that ultimately allows for post-confluent proliferation and induction of Runx2 during maturation of the osteogenic phenotype. Taken together, our findings suggest that Runx2 modulates the commitment of progenitor cells to the osteogenic and chondrogenic lineages, and that Runx2 activity is inextricably linked to mechanisms that control cellular proliferation.

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

Several aspects of Drosophila telomere biology indicate that telomere protection can be regulated by an epigenetic mechanism. First, terminally deleted chromosomes can be stably inherited and do not induce damage responses such as apoptosis or cell cycle arrest. Second, the telomere protection proteins HP1 and HOAP localize normally to these chromosomes and protect them from fusions. Third, unprotected telomeres still contain HeT-A sequences at sites of fusions. Taken together these observations support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophilatelomeres from fusion. Work presented in this thesis demonstrates that the Drosophila proteins ATM and Nbs are required for the regulation of DNA damage responses similar to their yeast and mammalian counterparts. This work also establishes a role for the ATM and ATR DNA damage response pathways in the protection of both normal and terminally deleted chromosomes. Mutations that disrupt both pathways result in a severe telomere fusion phenotype, similar to HP1 and HOAP mutants. Consistent with this phenotype, HOAP localization at atm,atr double mutant telomeres is completely eliminated. Furthermore, telomeric sequences are still present, even at the sites of fusions. These results support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophila telomeres from fusion.

Bacteria lack nucleus and any other membrane-bound organelles. Hence all the cellular components, including proteins, DNA, RNA and other components are located within the cytoplasm. The region of the cell which encompasses the bacterial genomic DNA is termed ‘Nucleoid’. The nucleoid is composed largely of DNA and small amounts of proteins and RNA. The genomic DNA is organized in ways that are compatible with all the major DNA-related processes like replication, transcription and chromosome segregation. Proteins that play important role(s) in the structuring of DNA and having the potential to influence gene expression have been explored in all kingdoms of life. The organization of bacterial chromosome is influenced by several important factors. These factors include molecular crowding, negative supercoiling of DNA and NAPs (nucleoid-associated proteins) and transcription. Nucleoid-associated proteins are abundant and relatively low-molecular mass proteins which can bind DNA and function as architectural constituents in the nucleoid. Additionally, NAPs are involved in all the major cellular processes like replication, repair and gene transcription. At least a dozen distinct NAPs are known to be present in E. coli. HU, IHF (integration host factor), H-NS (histone-like nucleoid-structuring), Fis (Factor for inversion stimulation), Dps (DNA protection from starvation) are some of the abundant NAPs in E. coli. Most of these proteins bind DNA and show either DNA bending, bridging or wrapping which are directly relevant to their physiological role(s). As most of these proteins are involved in the regulation of transcription of many genes, they act as factors unifying gene regulation with nucleoid architecture and environment. Pathogenic bacteria have the ability to grow and colonize different environments and thus need to adapt to constantly changing conditions within the host. H-NS and IHF, being able to link environmental cues to the regulation of gene expression, play an important role in the bacterial pathogenesis. H-NS is one of the well studied NAPs in enterobacteria, and is known as a global gene silencer. It is also an important DNA structuring protein, involved in chromosome packaging. H-NS protein is a small (~15 kDa) protein, which is present at approximately 20000 copies/ cell. The most striking feature of H-NS is that although it binds DNA in a relatively sequence-independent fashion but is known to preferentially recognize and bind intrinsically curved DNA. It also constrains DNA supercoils in vitro, thereby affects DNA topology. H-NS also influences replication, recombination and genomic stability. In addition, it functions as a global regulator by regulating the expression of various genes which are linked to environmental adaptation. Various studies have shown the association of H-NS to AT-rich regions of the genome. About 5% of E. coli genes are regulated by H-NS, bulk of which are (~80%) negatively regulated. H-NS is involved in the silencing of horizontally-acquired genes, many of which are involved in pathogenesis, in a process known as xenogeneic silencing. H-NS is known to regulate the expression of various virulence factors like cytotoxins, fimbriae and siderophores in several pathogenic bacteria. Several studies have revealed that hns mutants show increased frequency of illegitimate recombination and reduction in intra-chromosomal recombination, indicating the involvement of H-NS in DNA repair/recombination. H-NS is known to act in several transposition systems, which it does so due to its ability to interact with other proteins involved and due to its DNA structure-specific binding activity. The prototypical IHF (Integration Host Factor) was originally discovered in E. coli as an essential co-factor for the site-specific recombination of phage λ. E. coli IHF belongs to DNABII structural family, along with HU and other proteins and consists of two subunits, IHFα and IHFβ. Thesubunits are ~10 kDa each and are essential for full IHF activity. Apart from its role in bacteriophage integration/excision, IHF also has roles in various processes such as DNA replication, transcription and also in several site-specific recombination systems. In most of these processes, IHF acts as an architectural component by facilitating the formation of nucleoprotein complexes by bending DNA at specific sites. IHF acts as a transcriptional regulator, influencing the global gene expression in E. coli and S. Typhimurium. Gene regulation by IHF requires its DNA architectural role, facilitating interactions between RNA polymerase and regulatory protein. The high intracellular concentration of IHF indicates that it might associate with DNA in a non-specific manner and contribute to chromatin organization. The binding of E. coli IHF causes the DNA to adopt U-turn and brings the non-adjacent sequences into close juxtaposition. IHF is also involved in gene regulation in several pathogenic organisms and is shown to regulate expression of many virulence factors. Despite extensive literature on NAPs, very little is known about NAPs and nucleoid architecture in M. tuberculosis. In the light of significant physiological roles played by NAPs in adaptation to environmental changes and in growth and virulence of bacteria, elucidation of their roles in M. tuberculosis is of paramount importance for a better understanding of its pathogen city. M. tuberculosis Rv3852 (hns) gene is predicted to encode a 134 amino acid protein with a molecular mass of 13.8 kDa. The amino acid sequence alignment revealed that M. tuberculosis H-NS and E. coli H-NS showed very low degree of sequence identity (6%). To explore the biochemical properties of M. tuberculosis H-NS, the sequence corresponding to Rv3852 was amplified via PCR, cloned and plasmid expressing M. tuberculosis hns was constructed. M. tuberculosis H-NS was over expressed and purified to homogeneity. E. coli H-NS was also over expressed and purified. Comparison of experimentally determined secondary structure showed considerable differences between M. tuberculosis and E. coli H-NS proteins. Chemical cross linking suggested that M. tuberculosis H-NS protein exists in both monomeric and dimeric forms in solution, consistent with the diametric nature of E. coli H-NS protein. Our studies have revealed that M. tuberculosis H-NS binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double stranded DNA with relatively higher GC content. It bound to the Holliday junction (HJ), the central recombination intermediate, with high affinity. Furthermore, similar to M. tuberculosis H-NS, E. coli H-NS was able to bind to replication and recombination intermediates, but at a lower affinity than M. tuberculosis H-NS. To gain insights into homologous recombination in the context of nucleoid, we investigated the ability of M. tuberculosis RecA to catalyze DNA strand exchange between single-strand DNA and linear duplex DNA in the presence of increasing amounts of H-NS. We found that M. tuberculosis H-NS inhibited strand exchange mediated by its cognate RecA in a concentration dependent manner. Similar effect was seen in the case of E. coli H-NS, where it was able to suppress DNA strand exchange promoted by E. coli RecA, but at relatively higher concentrations, suggesting that H-NS proteins act as ‘roadblocks’ to strand exchange promoted by their cognate RecA proteins. H-NS and members of H-NS-family of NAPs are known to form rigid nucleoprotein filament structures on binding to DNA, which results in gene-silencing and is also implicated in chromosomal organization. Studies have also shown that H-NS mutants defective in gene silencing also lack the ability to form rigid nucleoprotein filament structure and that nucleoprotein filament structure is responsive to environmental factors. Our studies employing ligase-mediated DNA circularization assays reveal that both E. coli and M. tuberculosis H-NS proteins abrogate the circularization of linear DNA substrate by rigidifying the DNA backbone. These results suggest that M. tuberculosis H-NS could form nucleoprotein filament-like structures upon binding to DNA and these structures might be involved in transcriptional repression, chromosomal organization and protection of genomic DNA. In summary, these findings provide insights into the role of M. tuberculosis H-NS in homologous and/or homeologous recombination as well as transcriptional regulation and nucleoid organization. The second part of the thesis concerns the characterization of M. tuberculosis integration host factor (mIHF). The annotation of whole-genome sequence of M. tuberculosis H37Rv showed the presence of Mtihf gene (Rv1388) which codes for a putative 20-kDa integration host factor (mIHF). Amino acid sequence alignment revealed very low degree of sequence identity between mIHF and E. coli IHFαβ subunits. Unlike E. coli IHF, mIHF is essential for the viability of M. tuberculosis. The three-dimensional molecular modeling of mIHF based upon co crystal structure of Streptomycin coelicolor IHF (sIHF) duplex DNA, showed the presence of conserved Arg170, Arg171, Arg173, which were predicted to be involved in DNA binding and a conserved Pro150, in the tight turn. The coding sequence corresponding to the M. tuberculosis H37Rv ihf gene (Rv1388) was amplified, cloned and plasmid over expressing M. tuberculosis ihf (pMtihf) was constructed. Using pMtihf as a template and using specific primers, mutant ihf encoding plasmids were constructed in which, the arginine at position 170, 171, or 173 was replaced with alanine or aspartate and proline at position 150 was substituted with alanine. To explore the role of mIHF in cell viability, we investigated the ability of M. tuberculosis ihf to complement E. coli ΔihfA or ΔihfB strains against genotoxic stress. Despite low sequence identity between Mtihf and E. coli ihfA and ihfB, wild type Mtihf was able to rescue the UV and MMS sensitive phenotypes of E. coli ΔihfAand ΔihfBstrains, whereas Mtihf alleles bearing mutations in the DNA-binding residues failed to confer resistance against DNA-damaging agents. To further characterize the functions of mIHF, wild type and mutant versions of mIHF proteins were over expressed and purified to near homogeneity. Circular dichroism spectroscopy of wild type mIHF and mIHF mutant proteins revealed that they have similar secondary structures. By employing size-exclusion chromatography and blue-native PAGE, we determined that mIHF exists as a dimmer in solution. To understand the mechanistic basis of mIHF functions, we carried out electrophoretic mobility shift assays. In these assays, we found that wild-type mIHF showed high affinity and stable binding to DNA containing attB and attP sites and also to curved DNA, but not those mIHF mutants bearing mutations in DNA-binding residues. Because wild type mIHF was able to rescue the UV and MMS sensitive phenotypes of E. coli ΔihfA and ΔihfB strains, we ascertained the effect of overexpression of mIHF proteins on the bacterial nucleoid. Our results revealed that wild type mIHF was also able to cause significant nucleoid compaction upon its overexpression, but mutant mIHF proteins were unable to cause compaction of E. coli nucleoid structure. M. smegmatis IHF is known to stimulate L5 phage integrase mediated site-specific recombination, we investigated the ability of mIHF to promote site-specific recombination. In vitro recombination assays showed that M. tuberculosis IHF effectively stimulated the L5 integrase mediated site-specific recombination. Since DNA-bending activity of E. coli IHF is necessary for its functions in various processes like initiation of replication, site-specific recombination, transcriptional regulation and chromosomal organization, we asked whether mIHF possesses DNA bending activity. We employed ligase mediated DNA circularization assays, which revealed that like E. coli IHF, mIHF was able to bend DNA resulting in the covalent closure of DNA to yield circular DNA molecules. Interestingly mIHF also resulted in the formation of slower migrating linear DNA multimers, albeit to a lesser extent, which suggest that both E. coli IHF and mIHF show DNA-bending, but the mechanism is distinct. Further studies using atomic force microscopy showed that depending upon the placement of preferred binding site (curved-DNA sequence) mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. Together, these findings provide insights into functions of mIHF in the organization of bacterial nucleoid and formation of higher-order nucleoprotein structures. Importantly, our studies revealed that the DNA-binding residues, the DNA bending mechanism and mechanism of action of mIHF during site-specific recombination were different from E. coli IHF protein. Together with extensive biochemical and in vitro data of bacterial growth, the findings presented in this thesis provide novel insights into the biological roles of H-NS and mIHF in M. tuberculosis.

The Runx family of transcription factors performs an essential role in animal development by controlling gene expression programs that mediate cell proliferation, growth and differentiation. The work described in this thesis is concerned with understanding mechanisms by which Runx proteins support this program of gene expression within the architectural context of the mammalian cell nucleus. Multiple aspects of nuclear architecture are influenced by Runx2 proteins including sequence-specific DNA binding at gene regulatory regions, organization of promoter chromatin structure, and higher-order compartmentalization of proteins in nuclear foci. This work provides evidence for several functional activities of Runx2 in relation to architectural parameters of gene. expression for the control of cell growth and differentiation. First, the coordination of SWI/SNF mediated chromatin alterations by Runx2 proteins is found to be a critical component of osteoblast differentiation for skeletal development. Several chromatin modifying enzymes and signaling factors interact with the developmentally essential Runx2 C-terminus. A patent-pending microscopic image analysis strategy invented as part of this thesis work - called intranuclear informatics - has contributed to defining the C-terminal portion of Runx2 as a molecular determinant for the nuclear organization of Runx2 foci and directly links Runx2 function with its organization in the nucleus. Intranuclear informatics also led to the discovery that nuclear organization of Runx2 foci is equivalently restored in progeny cells following mitotic division - a natural perturbation in nuclear structure and function. Additional microscopic studies revealed the sequential and selective reorganization of transcriptional regulators and RNA processing factors during progression of cell division to render progeny cells equivalently competent to support Runx2 mediated gene expression. Molecular studies provide evidence that the Runx proteins have an active role in retaining phenotype by interacting with target gene promoters through sequence-specific DNA binding during cell division to support lineage-specific control of transcriptional programs in progeny cells. Immunolocalization of Runx2 foci on mitotic chromosome spreads revealed several large foci with pairwise symmetry on sister chromatids; these foci co-localize with the RNA polymerase I transcription factor, Upstream Binding Factor (UBFl) at nucleolar organizing regions. A series of experiments were carried out to reveal that Runx2 interacts directly with ribosomal DNA loci in a cell cycle dependent manner; that Runx2 is localized to UBF foci within nucleoli during interphase; that Runx2 attenuates rRNA synthesis; and that this repression of ribosomal gene expression by Runx2 is associated with cell growth inhibition and induction of osteoblast-specific gene expression. This thesis has identified multiple novel mechanisms by which Runx2 proteins function within the hierarchy of nuclear architecture to control cell proliferation, growth and differentiation.

Haemophilus influenzae encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of H. influenzae, whose counterpart in E. coli has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in E. coli that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in H. influenzae, I targeted a gene predicted to be FNR-regulated, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The fnr, narP and nrfA mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrf promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the nrf promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the nrfpromoter is modulated by environmental redox signals. FNR of E. coli has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type H. influenzae after anaerobic pre-growth. Mutants of nrfA and narP have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, ytfE, which contributes to defense against nitrosative stress. The fnr and ytfE mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of ytfE might be important for in vivo infection.

Les cancers du sein ont la particularité de développer un microenvironnement tumoral important où les fibroblastes associés au cancer (CAF) jouent un rôle crucial dans la tumorigénèse via la sécrétion de différents facteurs de croissance, cytokines, protéases et composants de la matrice extracellulaire. Ces différents facteurs secrétés par les CAFs sont impliqués dans de nombreuses voies de signalisation suggérant que la reprogrammation des cellules cancéreuses par les CAFs peut affecter de nombreux gènes.Le séquençage des ARN messagers (RNAseq) de lignées cellulaires de cancer du sein cultivées en présence de milieux conditionnés de CAF, nous a permis d'identifier 372 gènes surexprimés in vitro par les facteurs sécrétés par les CAF et in vivo selon la teneur en cellules stromales des tumeurs mammaires. De façon inattendue, nous avons pu constater que les facteurs sécrétés par les CAF ainsi que le contenu en cellules stromales des tumeurs n'induisent pas de changements significatifs de méthylation de l'ADN mais activent de manière spécifique des gènes caractérisés par une signature méthylation. Différentes approches expérimentales, telles que l'inhibition de la méthylation de l'ADN, l'inhibition de l'expression de la protéine MBD2 (protéines de liaison à l'ADN méthylé) et des expériences d'immuno-précipitation de la chromatine (ChIP) ont permis de montrer l'implication de ces marques de méthylation et des protéines de liaison à l'ADN méthylé dans la réponse des cellules cancéreuses aux facteurs sécrétés par les CAFs. Ces résultats ont permis l'identification d'événements moléculaires impliqués dans la réponse des cellules tumorales aux signaux sécrétés par les cellules stromales dans les tumeurs mammaires mettant en lumière l'importance des marques épigénétiques dans la reprogrammation des cellules cancéreuses induites par les cellules stromales / Breast cancers develop in complex tissue environments where cancer associated fibroblasts (CAF) play a crucial role in tumorigenesis by secreting various growth factors, cytokines, proteases and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation, and epigenetic modulation suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation signature: hypermethylation at transcription start site (TSS) and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of MBD2, and ChIP assays) demonstrated the implication of DNA methylation and methyl DNA binding protein in the response of cancers cells to CAF-secreted factors. These data put in light the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicate that the interpreters of the DNA methylation signal play a major role in the response of the cancer cells to the microenvironment