Bok/Mtd (Bcl-2-related ovarian killer/Matador) is considered a pro-apoptotic member of the Bcl-2 family. Though identified in 1997, little is known about its biological role. We have previously demonstrated that Bok mRNA is upregulated following E2F1 over-expression. In the current work, we demonstrate that Bok RNA is low in quiescent cells and rises upon serum stimulation. To determine the mechanism underlying this regulation, we cloned and characterized the mouse Bok promoter. We find that the mouse promoter contains a conserved E2F binding site (-43 to -49) and that a Bok promoter-driven luciferase reporter is activated by serum stimulation dependent on this site. Chromatin immunoprecipitation assays demonstrate that endogenous E2F1 and E2F3 associate with the Bok promoter in vivo. Surprisingly, we find that H1299 cells can stably express high levels of exogenous Bok. However, these cells are highly sensitive to chemotherapeutic drug treatment. Taken together these results demonstrate that Bok represents a cell cycle-regulated pro-apoptotic member of the Bcl-2 family, which may predispose growing cells to chemotherapeutic treatment.

Chromatin is a multi-layered structure composed of DNA, nucleosomes, histone modifications, and associated proteins that critically affects genome function. Recently developed sequencing technologies enable genomewide characterization of certain aspects of chromatin structure, including nucleosome positioning and histone modifications. However, chromatin proteins present several challenges due to their dynamic nature and variable association with DNA. Chromatin proteins such as Polycomb regulators and heterochromatic factors play critical and global roles in epigenetic repression and hence new approaches are needed for their study. We first sought to identify sequences that recruit Polycomb repressive complex 2 (PRC2) in mammalian cells. We combined chromatin immunoprecipitation with sequencing (ChIP-seq) to map the candidate transcription factor YY1, and found that it does not correlate with PRC2 localization, suggesting that YY1 is not directly involved in PRC2 recruitment. We also identified GC-rich sequences that are necessary and sufficient for PRC2 recruitment. Yet attempts to map additional Polycomb proteins and other repressors using ChIP-seq proved difficult. Since chromatin proteins are often broadly, secondarily or transiently bound to DNA, they are difficult to crosslink. Antibody quality also varies, further hampering ChIP-seq technology. Here, we adapt DamID, a method for mapping chromatin regulators that uses a fusion enzyme and that does not rely on crosslinking or antibodies, for high-throughput sequencing. We show that DamID-seq can be used to globally characterize chromatin repressors in human cells. We used DamID-seq to map the binding of 12 chromodomain-containing and related proteins in K562 cells. We found that these proteins cluster into two modules: 1) Polycombrelated and 2) heterochromatin-related. Polycomb proteins bind developmental genes, while heterochromatin proteins bind broad olfactory receptor (OR) and zinc finger (ZNF) domains. Surprisingly, unlike other Polycomb proteins, CBX2 uniquely binds genes involved with modifying proteins. Our findings advance the model that the genome is compartmentalized into domains, and identify the distinct protein components that associate respectively with Polycomb and heterochromatin domains in human cells. We expect that DamID-seq, along with further advancements in characterizing the three-dimensional organization of chromatin, will bring us towards a better understanding of the role of chromatin in differentiation, development, and disease.

Eukaryotic centromeres are the region upon which kinetochores assemble, directing attachment of spindle microtubules and faithful segregation of chromosomes during mitosis and meiosis. Except for a transient disruption in mitosis when chromosomes are segregated, centromeres of fission yeast Schizosaccharomyces pombe remain closely associated with the nuclear periphery. Similar to multicellular eukaryotic centromeres, they also maintain unique chromatin architecture, with a central core defined by the presence of the conserved centromeric histone H3 variant CENP-A, designated Cnp1 in S. pombe, that is flanked by histone H3 containing heterochromatin. While much progress has been made in understanding chromatin-associated factors important for proper centromere function, many questions remain. In order to gain a better understanding of the factors involved in centromeric chromatin structure, we affinity purified and defined by mass spectrometry interactions among select proteins that had been implicated in proper Cnp1 localization and centromere function. These biochemical purifications revealed several proteins that may be involved in Cnp1 localization. Purification and analyses of Cnp1 also led us to the identification of the Mlp1/Tpr nucleoporin homolog Nup211. We have found that Nup211 interacts with components of the inner nuclear basket of the nuclear pore, and co-purifies with centromeric chromatin proteins. Cells lacking Nup211 have substantial chromosome segregation defects, as observed by synthetic growth assay, flow cytometric analysis, and fluorescent microscopy. A series of immunoprecipitation experiments have revealed that Nup211 associates with centromeric DNA, and that, surprisingly, cells lacking Nup211 have increased histone H3 lysine 9 methylation, a marker of heterochromatin, and a reduction in Cnp1 levels at the central core. Moreover, cells lacking Nup211 have decreased transcription at centromeric loci, disruption of the stereotypical nucleosome structure found at the central core of S. pombe, and show striking changes in the distribution of heterochromatic foci in the nucleus. By demonstrating that Nup211 is essential for the maintenance of normal central core chromatin state, these studies have shed light on a novel role for Nup211 in proper centromere structure and function in S. pombe, and suggest that Nup211 may play a role in preventing the invasion of flanking pericentric heterochromatin into the central core of centromeres.

DNA is packaged into the nucleus of a mammalian cell as a nucleoprotein complex called chromatin. Changes in chromatin structure occur during processes that are critical to an understanding of mammalian cell biology such as cell division. Existing fixed-cell techniques have provided insight into chromatin organization in the mammalian nucleus. In addition, fluorescence microscopy techniques have allowed us to study changes in chromatin structure in living cells. However, most of these fluorescence techniques cannot be used for tissue imaging or long-term imaging due to photobleaching. In this thesis, we demonstrate that a label-free technique called Stimulated Raman Scattering (SRS) microscopy can be used to solve these problems and study chromatin structure in living mammalian cells both in culture and in tissue. SRS is a vibrational microscopy technique that takes advantage of intrinsic contrast arising from specific chemical bonds in a molecule. Nucleic acids have specifc phosphate and CH vibrations that can be used to determine their cellular distributions. Imaging at specific phosphate peaks using fingerprint SRS microscopy allows the detection of polytene chromosomes in Drosophila salivary gland cells and condensed chromatin in metaphase mammalian cells. In addition, we develop a technique called multicolor SRS microscopy, in which we image at several wavelengths across the CH vibrational band, and then use linear combination to simultaneously determine the nucleic acid, lipid and protein distributions in living mammalian cells. This technique achieves greater contrast than imaging at the phosphate vibrational peak due to the stronger SRS signal in the high wavenumber CH band and so allows us to determine chromatin structure in interphase mammalian cells. This technique also allows long-term imaging of living mammalian cells and the imaging of tissue such as mouse skin. The technique is used to monitor mammalian cell division in culture and paves the way for similar studies in living tissue. This technique will provide insight into cell division, differentiation and apoptosis during development and in disease models such as cancer.

Regulation of gene expression is essential for many biological processes. Binding of transcription factors to DNA is a key regulatory step in the control of gene expression. It is commonly observed that DNA sequences with high affinity for transcription factors occur more frequently in the genome than the instances of genes bound or regulated by these factors. However, the mechanism by which transcription factors selectively identify and regulate these genes was unclear. I utilized the transcriptional control of the phosphate-responsive signaling pathway (PHO) in Saccharomyces cerevisiae as a model system to address this problem.

Lysine methylation is an important post-translational modification found on histones that is added and removed by histone lysine methyltransferases and demethylases, respectively. Lysine methylation occurs in a specific and well-regulated manner, and plays key roles in regulating important biological processes such as transcription, DNA damage and cell cycle. Regulation of the protein abundance of these methylation enzymes particularly by the ubiquitin-proteasome system has emerged as a key mechanism by which the histone methylation status of the cell can be regulated, allowing cells to respond rapidly to specific developmental and environmental cues. In my thesis, I focus on two histone lysine demethylases, KDM4A and PHF8, both of which appear to be regulated by E3 ligases; this regulation impacts their function in the cell. Chapter 2 shows that KDM4A is targeted for proteasomal degradation by the SCFFBXO22, and mis-regulation of KDM4A results in changes in global histone 3 lysine 9 and 36 (H3K9 and H3K36) methylation levels and impacts the transcription of a KDM4A target gene, ASCL2. Chapter 3 shows how PHF8 is targeted for proteasomal degradation by the APCCDC20 via a novel, previously unreported LxPKxLF motif on PHF8. I also found that similar to other APCCDC20 substrates like Cyclin B, PHF8 is an important G2-M regulator, loss of which results in cell cycle defects such as prolonged G2 and defective M phases. To further interrogate PHF8 biology, Chapter 4 describes the generation of a PHF8 conditional knockout mouse. PHF8 biology is interesting and relevant to human disease, as mutations are found in X-linked intellectual disability and autism. Complete loss of PHF8 by full body knockout in the mouse appears to be embryonically lethal, underscoring its key role in early development. This mouse model would allow us to extensively study the biochemistry and biology of PHF8 in the context of development and especially in brain function, where it is anticipated to play key roles. Overall, my dissertation work provides mechanistic and biological insights into how histone demethylases are dynamically regulated by the ubiquitin-proteasome system, providing an extra dimension to our understanding of how chromatin marks can be regulated.

EZH2 is a SET domain-containing methyltransferase and the catalytic component of the multimeric Polycomb- group (PcG) protein complex, PRC2. When in complex with other PRC2 members (EED, SUZ12, AEBP2, and RBBP4), EZH2 catalyzes methylation of H3K27, a histone modification associated with transcriptional repression and developmental regulation. As several PRC2 components are upregulated or mutated in a variety of human cancers, efforts to discover small-molecule modulators of PRC2 and understand its regulation may yield therapeutic insights. Identification of small-molecule probes with distinct chemotypes, MOAs, and selectivity profiles are not only of great value, but necessary in establishing comprehensive probe sets capable of illuminating the various roles of EZH2 in oncogenesis. Here we describe efforts to identify and characterize small-molecule modulators of PRC2 and further understand its regulation. Chapter II outlines the expression and purification of 5-component PRC2 (EZH2-EED-SUZ12-AEBP2-RBBP4) and the establishment of biochemical and cellular HTS assays. These assays were used to screen a diverse set of small molecules (>120,000), identifying biochemical PRC2 inhibitors and activators (described in Chapter III). One biochemical PRC2 inhibitor, BRD1835, appeared to inhibit PRC2 activity through a novel artifactual mechanism involving interaction with peptide substrate, leading to apparent peptide-competitive behavior and putative cellular activity (described in Chapter IV). The characterization of novel biochemical PRC2 activators, BRD3934 and BRD8284, is discussed in Chapter V. Chapter VI describes the use of an HCS assay to identify known bioactive compounds that alter intracellular levels of H3K27me3 through modulating H3K27me3-connected regulatory nodes or by targeting PRC2 directly. These efforts led to the discovery that an antifungal agent, miconazole, is capable of activating PRC2 activity in vitro, while a mucolytic agent, bromhexine, selectively ablates cellular H3K27me3 levels through targeting an activity distinct from PRC2. Finally, Chapter VII discusses novel PRC2-connected crosstalk mechanisms identified through screening libraries of uniquely modified histone peptides for their ability to bind or support methylation by PRC2. These studies enhance our understanding of PRC2 regulation by revealing the effects of H3R26 and H3K23me1 modifications on enzymatic activity, implicating their respective methyltransferases in PRC2 regulation.

The chromatin integrity in sperm cells is vital for successful pregnancy. In this study DNA-damage was evaluated in sperm cells from 50 men attending In Vitro Fertilization (IVF) or Intra Cytoplasmic Sperm Injection (ICSI) treatment. Male semen samples were purified with a two-shift gradient before the sperm cells were treated with the Halosperm® Test Kit and evaluated for DNA-damage. The samples were divided in two groups according to DNAFragmentation Index (DFI) of 30 % and the results correlated with fertilization, cleavage and pregnancy rate. Men with DFI ≥ 30 % had a higher fertilization and pregnancy rate and a lower cleavage rate compared to men with DFI ≤ 30 %. The conclusions were that fertilization in vitro may be independent of the degree of DNA-damage, the embryonic development could be seriously disrupted by damaged sperm cells, and the pregnancy rate showed no correlation to a DFI threshold of 30 %.

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.

In the nucleus of eukaryotic cells, DNA wraps around histone proteins to form nucleosomes, which in turn associate in a compact and dynamic fiber called chromatin. The physical properties of this fiber at different lengthscales, from the DNA double-helix to micrometer-sized chromosomes, are essential to the complex mechanisms of gene expression and its regulation. The present thesis is a contribution to the development of physical models, which are able to link different scales and to interpret and integrate data from a wide range of experimental and computational approaches. In the first part, we use Molecular Dynamics simulations of DNA oligomers to study doublehelical DNA at different temperatures. We estimate the sequence-dependent contribution of entropy to DNA elasticity, in relation with recent experiments on DNA persistence length. In the second part, we model the DNA-histone interactions within the nucleosome core particle,using DNA nanomechanics to extract a force field from a set of crystallographic nucleosome structures and Molecular Dynamics snapshots. In the third part, we consider the softer part of the nucleosome, the linker DNA between coreparticles which transiently associates with the histone H1 to form a "stem".We combine existing structural knowledge with experimental data at two different resolutions (DNA footprints and electro-micrographs) to develop a nanoscale model of the stem.