The Utilization of Mouse Models to Study Gene Functions: The Role of Foxn3 and Chd2 in Murine Development and Cancer

Murine model organisms are an essential tool in the scientific community quest to decipher the molecular etiology of human diseases. Currently, several methods are used to induce or reproduce human diseases in mouse models using advanced genetic engineering techniques to mutate the wild-type genes. We utilized the Baygenomics gene-trap method to study the effects of two mammalian genes: FOXN3 and CHD2. The Forkhead Box (FOX) family of transcription factors shares a common DNA-binding domain and has been associated with organ development, differentiation, cell growth and proliferation, and cancer. Meanwhile, the CHD (Chromodomain helicase DNA binding protein) family of proteins is known to be involved in chromatin remodeling and regulation of gene expression. Phenotypic analysis of Foxn3 mutant animals revealed its indispensible role in craniofacial and embryonic development, embryonic lethality, expression of bone morphogenetic proteins, and spontaneous development of cancers in heterozygous and homozygous mutant mice. Preliminary evaluation of molecular mechanisms of FOXN3 signifies deregulation of cell-cycle checkpoint proteins Cyclin-B1 and CDK2 as the underlying etiology of tumors. Chd2 mutant mice exhibit spontaneous thymic and splenic lymphomas and reduced lifespan which can be restored through Chd2 re-expression in the thymus. At the molecular level, CHD2 deficiency reduces Puma (p53-upregulated modulator of apoptosis) induction after DNA damage in mouse thymocytes and HCT116 cells. Additionally, CHD2 is enriched at the Puma locus after DNA damage. CHD2-deficient cells also exhibit global reduction of active transcription markers H3K9-Acetylated and H4K8-Acetylated

Computational genomics of developmental gene regulation

The development of multicellular organisms requires the precise execution of complex transcriptional programs. The demands posed by development, coupled with the relatively late evolution of multicellularity, could have led to a separate mode of gene regulation for gene involved in, and regulated throughout development. I investigated the regulation of genes by enhancers using histone modifications coupled to gene expression, based on the observa- tion that developmental genes are surrounded by dense clusters of conserved enhancers which act in concert. Genes regulated by enhancers are much more likely to be developmentally regulated genes, and many enhancers at each loci co-ordinate to direct transcription across multiple tissues. CAGE-seq is a powerful tool for determining the structure of promot- ers. I analysed promoters in Amphioxus using CAGE-seq to determine if the diverse promoter architectures observed in vertebrates had ancestral ori- gins. Promoters in amphioxus can be divided into developmental and house- keeping promoters, which each have characteristic patterns of dinucleotide enrichment. Housekeeping promoters in Amphioxus have a novel promoter architecture, and a contain a high frequency of bidirectional promoters, which represents the ancestral vertebrate state. This set of genes highlight the mal- leability of promoter architecture during evolution. I developed a package in R/Bioconductor ‘heatmaps’ to enable effective visualisation of this, and other, data. Taken together, these results suggest a second mode of regulation in ver- tebrates governing the regulation of developmental genes.Open Acces

THE DYNAMIC CHROMATIN LANDSCAPE IN SACCHAROMYCES CEREVISIAE

The accurate and faithful segregation of chromosomes during mitosis is essential for cellular survival. Current paradigms of chromosome segregation focus on the mechanical contributions of the mitotic spindle without considering the biomechanical properties of the chromatin itself. In order to further our understanding of how the inherent physical properties of chromatin contribute to cellular processes like chromosome segregation, we have examined both histone and chromatin dynamics in the budding yeast Saccharomyces cerevisiae. During mitosis, the mitotic spindle exerts force on the pericentromeric chromatin, which adjusts structurally to accommodate this force. By measuring the fluorescence recovery after photobleaching (FRAP), we found that histones are more dynamic in the pericentromeric region as compared to the chromosome arm, and these increased recovery rates are dependent on spindle-based tension. The tension-dependent histone dynamics in the pericentromere are dependent on the chromatin remodeling activities of the Remodels the Structure of Chromatin (RSC) and Imitation Switch (ISWI) ATP-dependent chromatin remodeling complexes. Thus, balanced histone removal and reincorporation in the pericentromere provide a mechanism for accommodation of spindle-based tension and the maintenance of chromatin packaging. Having measured the dynamic nature of histone turnover within the chromatin polymer in response to spindle-based tension, we subsequently examined the spatio-temporal fluctuations of the chromatin polymer. We combined in vivo chromatin motion analysis and mathematical modeling to elucidate the physical properties of the chromatin polymer underlying dynamic fluctuations. The range of chromatin motion and its effective spring constants are found to vary along the length of the chromosome, in a manner dependent on tethering at the centromere. These polymer properties of the chromatin are dependent on both histone occupancy and cohesin packaging. As a whole, the work detailed in this dissertation contributes valuable insights into the importance of dynamic histone occupancy and chromatin motion in defining and maintaining the biomechanical polymer properties of chromosomes in vivo.Doctor of Philosoph

Novel functions for the BAF180 tumour suppressor: insights from mammalian and yeast systems

Genomic DNA is compacted into a protein-DNA complex known as chromatin, which regulates diverse cellular processes including transcription, DNA replication, recombination, DNA repair and the maintenance of genome integrity. The structure and activity of chromatin is regulated by DNA sequences, histone variants, posttranslational histone modifications and chromatin remodelling complexes. Chromatin remodelling complexes are multi-subunit entities that contain a single core catalytic ATPase subunit able to generate an array of nucleosome-related outputs. Importantly, recent studies have revealed that genes of the SWI/SNF family of chromatin remodeling complexes are frequently mutated in diverse cancers; however, their functional contributions in tumourigenesis are largely unclear. This work is comprised of four major results chapters, examining the roles of targeting subunits of the RSC SWI/SNF complex in budding yeast and the homologous BAF180 tumour suppressor protein in mammalian cells. We identify novel functions for these proteins that are directly relevant to tumourigenesis. In the first section we explored the contributions of the two isoforms of the RSC SWI/SNF complex in DNA repair. We found that the two isoforms provide both overlapping and distinct functions in this process. In the second section we identify a novel function for BAF180 in promoting centromeric sister chromatid cohesion. Importantly, this defect was transcription-independent and represents a paradigm shift in the field of chromatin remodeling and cancer. In the third section we show that PBRM1 missense mutations identified in cancer samples specifically impair a cohesion-related subset of functions when expressed in budding yeast. Moreover, these mutations completely ablated centromeric cohesion in human cells. In the final section we report the findings that novel HDAC inhibitors, which constitute a promising class of anticancer drugs, selectively sensitize cells lacking BAF180. These significant results suggest that HDAC inhibitors could be important tools for the treatment of BAF180-deficient tumours

Transcriptional Regulation During Adipocyte Differentiation: A Role for SWI/SNF Chromatin Remodeling Enzymes: A Dissertation

Chromatin has a compact organization in which most DNA sequences are structurally inaccessible and functionally inactive. Reconfiguration of thechromatir required to activate transcription. This reconfiguration is achieved by the action of enzymes that covalently modify nucleosomal core histones, and by enzymes that disrupt histone-DNA interactions via ATP hydrolysis. TheSWI/SNF family of ATP-dependent chromatin remodeling enzymes has been implicated not only in gene activation but also in numerous cellular processes including differentiation, gene repression, cell cycle control, recombination and DNA repair. PPARγ, C/EBPα and C/EBPβ are transcription factors with well established roles in adipogenesis. Ectopical expression of each of these factors in non-adipogenic cells is sufficient to convert them to adipocyte-like cells. To determine the requirements of SWI/SNF enzymes in adipocyte differentiation, we introduced PPARγ, C/EBPα or C/EBPβ into fibroblasts that inducibly express dominant-negative versions of the Brahma-Related Gene 1 (BRG1) or human Brahma (BRM), which are the ATPase subunits of the SWI/SNF enzymes. We found that adipogenesis and expression of adipocyte genes were inhibited in the presence of mutant SWI/SNF enzymes. Additionally, in cells expressing C/EBPα or C/EBPβ, PPARγ expression was SWI/SNF dependent. These data indicate the importance of these remodeling enzymes in both early and late gene activation events. Subsequently, we examined by chromatin immunoprecipitation (ChIP) assay the functional role of SWI/SNF enzymes in the activation of PPARγ2, the master regulator of adipogenesis. Temporal analysis of factors binding to the PPARγ2 promoter showed that SWI/SNF enzymes are required to promote preinitiation complex assembly and function. Additionally, our studies concentrated on the role of C/EBP family members in the activation of early and late genes during adipocyte differentiation. During adipogenesis, C/EBPβ and δ are rapidly and transiently expressed and are involved in the expression of PPARγ and C/EBPα, which together activate the majority of the adipocyte genes. Our studies determined the temporal recruitment of the C/EBP family at the promoters of early and late genes by ChIP assay during adipocyte differentiation. We found that all of the C/EBP members evaluated are present at the promoters of early and late genes, and the binding correlated with the kinetics of the C/EBPs expression. Binding of C/EBPβ and δ is transient, subsequently being replaced by C/EBPα. These studies demonstrated that C/EBPβ and δ are not only involved in the regulation of PPARγ and C/EBPα, but also in the activation of late expressed adipocyte genes

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin