It has become increasingly clear that transcription factors (TFs) play crucial roles in the development and day-to-day homeostasis that all biological systems experience. TFs target particular genes in a genome, at the appropriate place and time, to regulate their expression so as to elicit the most appropriate biological response from a cell or multicellular organism. TFs can often be grouped into families based on the presence of similar DNA binding domains, and these families are believed to have expanded and diverged throughout evolution by several rounds of gene duplication and mutation. The extent to which TFs within a family have functionally diverged, however, has remained unclear. We propose that systematic analysis of multiple aspects, or parameters, of TF functionality for entire families of TFs could provide clues as to how divergent paralogous TFs really are. We present here a multiparameter integrated network of the activity of the basic helix-loop-helix (bHLH) TFs from the nematode Caenorhabditis elegans. Our data, and the resulting network, indicate that several parameters of bHLH function contribute to their divergence and that many bHLH TFs and their associated parameters exhibit a wide range of connectivity in the network, some being uniquely associated to one another, whereas others are highly connected to multiple parameter associations. We find that 34 bHLH proteins dimerize to form 30 bHLH dimers, which are expressed in a wide range of tissues and cell types, particularly during the development of the nematode. These dimers bind to E-Box DNA sequences and E-Box-like sequences with specificity for nucleotides central to and flanking those E-Boxes and related sequences. Our integrated network is the first such network for a multicellular organism, describing the dimerization specificity, spatiotemporal expression patterns, and DNA binding specificities of an entire family of TFs. The network elucidates the state of bHLH TF divergence in C. elegans with respect to multiple functional parameters and suggests that each bHLH TF, despite many molecular similarities, is distinct from its family members. This functional distinction may indeed explain how TFs from a single family can acquire different biological functions despite descending from common genetic ancestry.

The immune system generates discrete lineages of cells that are designed to respond optimally to environmental cues and infectious agents. Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. Unlike conventional αβ T cells, γδ T cells express a preactivated or memory phenotype prior to pathogen encounter, and recent evidence indicates that effector functions may be programmed during thymic development. To better understand the development and function of γδ T cells, we analyzed the gene expression profiles of subsets of γδ T cells segregated by TCR repertoire and maturation state in the thymus. We also determined the impact of TCR signaling and trans-conditioning on γδ T cell subset-specific gene signatures by analysis of Itk-/- and Tcrb-/- γδ T cell subsets. Our analysis has defined three stages of γδ T cell subset-specific differentiation, and indicates that γδ T cells may consist of at least two separate lineages, distinguished by the expression of a Vγ2 or Vγ1.1 TCR, that arise from different precursors during thymic development. Key transcriptional networks are established in immature γδ T cells during the first phase of development, independent of TCR signaling and trans-conditioning, with Vγ2+ cells expressing modulators of WNT signaling, and Vγ1.1+ cells expressing high levels of inhibitor of DNA binding 3 (ID3), which regulates E2A/HEB proteins. The second stage involves the further specification of the Vγ2+ subset specific gene signature, which is dependent upon ITK-mediated signals. In the third stage, terminal maturation of γδ T cell subsets occurs, dependent on both TCR and trans-conditioning signals. The expression patterns of Vγ1.1+ subsets that differ in Vδ usage diverge, and all subsets further elaborate and reinforce their effector programming by the distinct expression of chemokine and cytokine receptors. Alteration of WNT signaling or E2A/HEB activity results in subset specific defects in effector programming, indicating that the transcriptional networks established at the immature stage are crucial for the functional maturation of γδ T cells. These data provide a new picture of γδ T cell development, regulated by multiple checkpoints that shape the acquisition of subset-specific molecular signatures and effector functions.

Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF. Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction.

There are two copies of each core histone in a nucleosome, however, it is unclear whether post-translational modifications on each molecule function redundantly or if symmetrical modifications are required to properly regulate gene expression. We tried to address this question by breaking nucleosomal symmetry and measuring its impact on gene expression. Our strategy includes re-engineering specific residues at the H3-H3 interface, generating pairs of mutant proteins, which were predicted by computational methods to form obligate heterodimers. Using S. cerevisiae as a model system, we tested the viability of strains with mutant histones, and analyzed the interaction between by co-immunoprecipitation from mononucleosome preparations. We also measured the changes of gene expression in the strains bearing single-tailed or tailless H3 heterodimers. The data suggested that the best computationally-derived H3 pair was frequently, but not exclusively heterodimeric in vivo. In order to obtain a more stringent H3 heterodimer, random mutagenesis was performed on four codons in the original computational design, and then genetic screening of the mutant libraries was performed.

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression. In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.

The proper workings of an organism rely on the accurate expression of genes throughout its lifetime. An important determinant for protein production is the availability of template mRNA molecules, the net effect of which is governed by their rates of synthesis vs. their rates of degradation. Normal mRNAs are proposed to be relatively stable in the cytoplasm while present in a protective, circularized conformation – the closed loop – through eIF4G-bridged interactions with 3’-bound poly(A) binding protein (Pab1p) and 5’-bound eIF4E. Introduction of a premature nonsense codon into an otherwise normal mRNA results in its rapid destabilization in cells, suggesting that not all stop codons behave the same, and events at premature termination events that lead to accelerated degradation of nonsense-containing mRNAs likely differ from those at normal termination, in which normal decay rates are maintained. The enhanced degradation observed for nonsense-containing mRNAs occurs through an evolutionarily conserved pathway involving the products of the UPF1, UPF2/NMD2, and UPF3 genes, the precise biochemical roles of which have remained elusive. We have developed a yeast cell-free translation system that allows us to assay biochemical events occurring at premature termination codons, compare them to those occurring at normal terminators, and study the role of Upf1p in these events. We find that premature termination is an inefficient process compared to normal termination and that one outcome of termination at a premature termination codon (PTC) is reinitiation at a nearby start codon. This in vitro post-termination reinitiation phenotype is dependent on the presence of Upf1p, a finding we have recapitulated in vivo. We also developed biochemical assays to define a role for Upf1p in translation following premature termination in vitro and find that Upf1p is involved in post-termination ribosome dissociation and reutilization. Supporting this idea are our findings that Upf1p predominantly cosediments with purified 40S ribosomal subunits. Finally, using our in vitro translation/toeprinting system, we have further characterized events leading to the formation of the mRNA closed loop structure and find that two states of the closed loop exist. The first requires the preinitiation 48S complex and includes Pab1p, eIF4G, eIF4E, and eIF3, whereas the second is formed after 60S joining and additionally requires the translation termination factors eRF1 and eRF3.

The utility of a model organism for studying biological processes is closely tied to its amenability to genome manipulation. Although tools for targeted genome engineering in mice have been available since 1987, most organisms including zebrafish have lacked efficient reverse genetic tools, which has stymied their broad implementation as a model system to study biological processes. The development of zinc finger nucleases (ZFNs) that can create double-strand breaks at desired sites in a genome has provided a universal platform for targeted genome modification. ZFNs are artificial restriction endonucleases that comprise of an array of 3- to 6-C2H2-zinc finger DNA-binding domains fused with the dimeric cleavage domain of the type IIs endonuclease FokI. C2H2-zinc fingers are the most common, naturally occurring DNA-binding domain, and their specificity can be engineered to recognize a variety of DNA sequences providing a strategy for targeting the appended nuclease domain to desired sites in a genome. The utility of ZFNs for gene editing relies on their activity and precision in vivo both of which depend on the generation of ZFPs that bind desired target sites high specificity and affinity. Although various methods are available that allow construction of ZFPs with novel specificities, ZFNs assembled using existing approaches often display negligible in vivo activity, presumably resulting from ZFPs with either low affinity or suboptimal specificity. A root cause of this deficiency is the presence of interfering interactions at the finger-finger interface upon assembly of multiple fingers. In this study we have employed bacterial-one-hybrid (B1H)-based selections to identify two-finger zinc finger units (2F-modules) containing optimized interface residues that can be combined with published finger archives to rapidly yield ZFNs that can target more than 95% of the zebrafish and human protein-coding genes while maintaining a success rate higher than that of ZFNs constructed using available methods. In addition to genome engineering in model organisms, this advancement in ZFN design will aid in the development of ZFN-based therapeutics. In the process of creating this archive, we have undertaken a broader study of zinc finger specificity to better understand fundamental aspects of DNA recognition. In the process we have created the largest protein-DNA interaction dataset for zinc fingers to be described that will facilitate the development of better predictive models of recognition. Ultimately, these predictive models would enable the rational design of synthetic zinc finger proteins for targeted gene regulation or genomic modification, and the prediction of genomic binding sites for naturally occurring zinc finger proteins for the construction of more accurate gene regulatory networks.

A key regulator of DNA damage-induced apoptosis is the tumor suppressor gene, p53. p53 is a transcription factor that upregulates genes involved in cell cycle arrest, apoptosis, and senescence. How p53 decides to activate one of these responses in response to DNA damage is largely unanswered. Many have hypothesized it is due to interaction with various signaling pathways and post-translational modification. The p53 tumor suppressor can be modified by SUMO-1 in mammalian cells, but the functional consequences of this modification are unclear. Conjugation to SUMO is a reversible post-translational modification that regulates several transcription factors involved in cell proliferation, differentiation, and disease. In Chapter II, we demonstrate that the Drosophila homolog of human p53 can be efficiently sumoylated in insect cells. We identify two lysine residues involved in SUMO attachment, one at the C-terminus, between the DNA binding and oligomerization domains, and one at the N-terminus of the protein. We find that sumoylation helps recruit Drosophila p53 to nuclear dot-like structures that can be marked by human PML and the Drosophila homologue of Daxx. We demonstrate that mutation of both sumoylation sites dramatically reduces the transcriptional activity of p53 and its ability to induce apoptosis in transgenic flies, providing in vivo evidence that sumoylation is critical for Drosophilap53 function. Many therapeutic cancer treatments rely on DNA-damaging agents to induce apoptosis in cancer cells. However, fifty percent of all human tumors lack functional p53 and p53 mutant cells are partially resistant to damage-induced apoptosis. Therefore, it is important to identify mechanisms to induce apoptosis independent of p53. Drosophila provides a good model system to study p53-independent apoptosis because it contains a single p53 homolog. In Chapter III, we describe a p53-independent mechanism that acts in parallel to the canonical DNA damage response pathway in Drosophila to activate apoptosis in response to inappropriately repaired chromosome breaks. Induction of chromosome aberrations by DNA damage followed by cell division results in segmental aneuploidy and reduced copy number of ribosomal protein genes. We find that activation of the pro-apoptotic gene hid by the JNK pathway acts in a p53-independent mechanism to induce apoptosis and limit the formation of aneuploid cells. Mutations in grp, the Drosophila Chk1 homolog, and puc, a negative regulator of the JNK pathway sensitize p53 mutant cells to IR-induced apoptosis. We propose a model in which the death of cells with reduced copy number of genes required for cell survival helps maintain genomic integrity following chromosome damage

In order to preserve its genomic integrity, an organism needs to detect and repair DNA double-strand breaks (DSBs) in a prompt and accurate fashion. This goal is accomplished by enabling an exquisitely sensitive DSB sensing apparatus as well as multiple and often overlapping pathways for repair. All of these processes are carried out on a highly organized and compacted chromatin substrate in the nucleus. An important question is whether chromatin plays an active role in the process and whether it helps in the signaling or repair of this damage. We have used Chromosome Conformation Capture (3C) to show that there are no large scale changes in chromosome structure at a single site-specific DNA double-strand break, although looping interactions between DSBs and donors can be detected. In a surprising result, we found that 3C detected a nucleus-wide decrease in interactions with the DSB. We have used a combination of 3C, fluorescence microscopy and chromatin immunoprecipitation to show that the decrease in interactions is a result of the relocalization of persistent DSB to the nuclear periphery. We also show that this is dependent on the recruitment of telomerase complex to the DSB, which then interacts with its natural partner in the Inner nuclear membrane, Mps3, and relocalizes the DSB to the periphery. Thus, a DSB that cannot be repaired is shunted into a pathway where the cell attempts to survive by putting a de novotelomere on the broken chromosome. Remarkably, this is not an irreversible phenomenon despite the recruitment of telomerase and the relocalization to the periphery. DSBs which are repaired slowly due to the presence of homology on a different chromosome, or merely usage of a kinetically slower form of repair, undergo this pathway switch, but can still recover and repair the DSB if homology is present. We also show that the role of the periphery is to ensure repair through de novotelomere formation or other non-canonical repair pathways. Indeed, loss of peripheral localization results in a dramatic suppression of the genomic instability of the Slx5/8 mutants, which have been implicated in the persistent DSB response at the Nuclear pores. Thus, the nuclear periphery is a special compartment where DSBs go after they cannot be repaired by canonical pathways. Specialized components such as telomerase, silencing proteins and components of the SUMO pathway, all seem to play roles in the healing of these chromosomes. Importantly, the SUN domain homologues of Mps3 have been shown to play roles similar to their yeast homologues in meiotic bouquet formation through their interactions with telomeres. Thus, they may represent a conserved mechanism for chromosome healing and telomere anchoring, despite the fact that mammalian telomeres are rarely found at the nuclear periphery. Such survival mechanisms may be expected to operate in cancer cells which may or may not have upregulated telomerase expression.

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a plethora of fundamental biological processes ranging from muscle contraction to the formation of memories. The studies described in this work focus on the transcriptional regulation of the CHRNB4 gene, which encodes the ß4 subunit of neuronal nAChRs. We previously identified a regulatory sequence (5´– CCACCCCT –3´), or “CA box”, critical for CHRNB4 promoter activity in vitro. Here I report transcription factor interaction at the CA box along with an in vivo analysis of CA box transcriptional activity. My data indicate that Sp1, Sp3, Sox10 and c-Jun interact with the CHRNB4 CA box in the context of native chromatin. Using an in vivo transgenic approach in mice, I demonstrated that a 2.3-kb fragment of the CHRNB4 promoter region, containing the CA box, is capable of directing cell-type specific expression of a reporter gene to many of the brain regions that endogenously express the CHRNB4 gene. Site-directed mutagenesis was used to test the hypothesis that the CA box is critical for CHRNB4 promoter activity in vivo. Transgenic animals were generated in which LacZ expression is driven by a mutant form of the CA box. Reporter gene expression was not detected in any tissue or cell type at ED18.5. Similarly, I observed dramatically reduced reporter gene expression at PD30 when compared to wild type transgenic animals, indicating that the CA box is an important regulatory feature of the CHRNB4 promoter. ChIP analysis of brain tissue from mutant transgenic animals demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the CHRNB4 promoter. I have also investigated transcription factor interaction at the CHRNB4 promoter CT box, (5´– ACCCTCCCCTCCCCTGTAA –3´) and demonstrated that hnRNP K interacts with the CHRNB4 promoter in an olfactory bulb derived cell line. Surprisingly, siRNA experiments demonstrated that hnRNP K knockdown has no impact on CHRNA5, CHRNA3 or CHRNB4 gene expression. Interestingly, knockdown of the transcription factor Purα results in significant decreases in CHRNA5, CHRNA3 and CHRNB4 mRNA levels. These data indicate that Purα can act to enhance expression of the clustered CHRNA5, CHRNA3 and CHRNB4 genes. Together, these results contribute to a more thorough understanding of the transcriptional regulatory mechanisms underlying expression of the CHRNB4 as well as the CHRNA5 and CHRNA3 genes, critical components of cholinergic signal transduction pathways in the nervous system.