The skeleton is a highly dynamic organ that undergoes constant remodeling to renew itself and maintain bone mass. It is subject to regulation from both hormones produced in peripheral tissues and neuronal control by the nervous system. Recent studies have shown that the skeleton is also an endocrine organ, releasing the hormone osteocalcin that increases insulin secretion and sensitivity in the pancreas and testosterone production in the Leydig cells of the testis. In my thesis study, I explore both the cell-nonautonomous regulation of osteoblast differentiation and the bone regulation of energy metabolism using cell-specific gene inactivation in the mouse. Early B-cell factor 1 (Ebf1) is a transcription factor whose inactivation in all cells results in high bone mass because of an increase in bone formation. To test if Ebf1 regulates bone formation cell-autonomously, I analyzed pattern of expression and its function in osteoblasts. I show here that in vivo deletion of in osteoblast progenitors does not affect osteoblast differentiation or bone formation accrual post-natally, indicating that the phenotype described in Ebf1 mice is not osteoblast-autonomous. Insulin signaling in osteoblasts contributes to whole body glucose homeostasis in the mouse and in humans by increasing the activity of osteocalcin. The osteoblast insulin signaling cascade is negatively regulated by ESP, a tyrosine phosphatase dephosphorylating the insulin receptor. is one of many tyrosine phosphatases expressed in osteoblasts, and this observation suggests that other protein tyrosine phosphatases may contribute to the attenuation of insulin receptor phosphorylation in this cell type. In this study, we sought to identify additional PTP(s) that like ESP, would function in the osteoblast to regulate insulin signaling and thus affect activity of the insulin-sensitizing hormone osteocalcin. For that purpose, we used as criteria, expression in osteoblasts, regulation by isoproterenol, and ability to trap the insulin receptor in a substrate-trapping assay. Here we show that the T-cell protein tyrosine phosphatase (TC-PTP) regulates insulin receptor phosphorylation in the osteoblast, thus compromising bone resorption and bioactivity of osteocalcin. Accordingly, osteoblast-specific deletion of TC-PTP (Ptpn2) promotes insulin sensitivity in an osteocalcin-dependent manner. This study increases the number of genes involved in the bone regulation of glucose homeostasis.

In eukaryotes, nuclear DNA is folded with histone proteins in the form of chromatin, and this structure plays a critical role in multiple biological processes, including development, DNA damage repair, and aging. Post-translational modifications of histones, such as acetylation and methylation, are essential regulators of chromatin structure and function. Consequently, misregulation of these post-translational modifications has causal roles in numerous diseases, including multiple types of cancer. However, the mechanisms that direct the localization of histone-modifying enzymes and regulate their activities are not fully understood. This thesis focuses on the characterization of a class of proteins containing the PWWP domain. This domain is often present in chromatin proteins, and it is predicted to recognize methylated histones based on structural analysis. Here, we have demonstrated that the PWWP domain proteins in fission yeast bind to methylated histones. Additionally, we have shown that proteins with this domain form complexes with diverse histone modifying activities to regulate multiple cellular processes. Methylation of histone H4 lysine 20 (H4K20me) is essential for the activation of a DNA damage checkpoint, which blocks the progression of cell cycle to allow sufficient time for DNA damage repair. In fission yeast, only the enzyme Set9 catalyzes H4K20me, and the mechanisms that underlie the regulation of this protein are poorly characterized. Here we showed that Set9 forms a stable complex with the PWWP domain containing protein Pdp1. The PWWP domain of Pdp1 binds to H4K20me, demonstrating that the PWWP domain constitutes a novel methyl-lysine recognition motif. Moreover, the binding of PWWP domain to methylated H4K20 plays a critical role in regulating Set9 activity, thus facilitating higher degrees of H4K20 methylation. Histone H3K9 methylation is critical for heterochromatin assembly in diverse organisms. The RNAi pathway is required for the formation of pericentric heterochromatin, although the exact role that RNAi plays in heterochromatin assembly remains a topic of significant debate. We discovered that a separate PWWP domain protein, Pdp3, forms a stable complex with the H3K14 histone acetyltransferase Mst2. Interestingly eliminating the enzymatic activity of the Pdp3-Mst2 complex obviates the requirement for the RNAi machinery in pericentric heterochromatin functions. Furthermore we demonstrated that one function of RNAi during heterochromatin assembly is to exclude the Pdp3-Mst2 complex, thus maintaining low levels of RNA polymerase II localization to pericentric regions in order to retain the parental histone modification patterns for its passage through generations. Altogether, my results have firmly demonstrated that the PWWP domain is a novel class of methyl-lysine binding motifs. Moreover, in fission yeast the PWWP domain proteins form stable complexes with other chromatin proteins to regulate diverse cellular processes.

Mutations within BRCA1 often contribute to breast cancer susceptibility. Many of these mutations cluster within two highly conserved regions that may be important for BRCA1's functions: the RING domain and the BRCT repeats. BRCA1's RING domain has E3 ubiquitin ligase activity, which is greatly enhanced when it forms a heterodimer with Bard1. This region is of particular interest because it displays the only known enzymatic activity of BRCA1. The BRCT repeats have phosphopeptide binding activity, which is necessary for BRCA1's interaction with DNA repair proteins BACH1, ABRAXAS, and CtIP. To test the importance of these two domains we generated cell lines and mouse models with point mutations that either eliminate the E3 ligase activity of Brca1 while maintaining its interaction with Bard1 (I26A) or eliminate its phosphopeptide binding activity (M1717R). We found that the E3 ubiquitin ligase activity of Brca1 is dispensable for its role in cell viability, embryonic development, double strand break repair, and tumor suppression. Interestingly, we were unsuccessful at generating homozygous BRCT mutant ES cells and homozygous M1717R Brca1 MEFs displayed a proliferation defect, spontaneous chromosomal aberrations, and centrosomal amplification. Our data shows that the BRCT repeats are crucial for BRCA1's role in DNA repair because BRCT mutant MEFs do not recruit Brca1 or Rad51 to IR induced DNA damage sites, they have a defect in homology directed repair, and M1717R Brca1 is not hyperphosphorylated in response to DNA damage in these cells. We were able to generate homozygous M1717R Brca1 mice, but with a very low frequency (<1%). All Brca1M17171R/M1717R mice produced thus far have been males and they are sterile. Our analysis indicates that the fertility defect is not due to a defect in meiosis. Introduction of the M1717R mutation in a conditional mammary or pancreatic tumor model also reduces the tumor latency to the same degree as the introduction of a Brca1 null mutation. Therefore our data shows that BRCA1 carries out the majority of its functions through its BRCT repeats.

During angiogenesis, nascent blood vessels sprout from pre-existing vasculature and recruit pericytes to induce maturation and vessel quiescence. Perictyes are associated with small vessels and capillaries where they share the basement membrane with the endothelium to provide vascular support. Pericytes are a critical component of the blood-brain barrier and regulate endothelial cell proliferation, vessel diameter, and vascular permeability. Endothelial cells express Notch1, whereas pericytes express both Notch1 and Notch3. Here we show that Notch signaling is essential for pericyte function. Through genetic manipulation and pharmacological tools we show that Notch regulates pericyte recruitment and pericyte/endothelial cell interactions. Notch1^+/-;Notch3^-/- mutant mice display decreased pericyte coverage and altered pericyte association with the retinal vascular plexus. Notch deficiency is associated with vascular anomalies where Notch1^+/-;Notch3-/- mice display retinal arteriovenous malformations (AVM) characterized by dilated vessels, vascular tangles and arteriovenous shunts that are similar to human brain AVMs. Disruption of pericyte/endothelial cell association is accompanied by an increase in vascular density, venule enlargement, and increased vascular permeability observed prior to AVM formation. In the ovary, we show that Jagged is essential for pericyte association with the endothelium where inhibition of Jagged-specific Notch activation results in luteal vessel dilation and hemorrhaging following ovarian hyperstimulation. By in vitro analysis of cultured pericytes we show that Notch1 and Notch3 induce plated derived growth factor receptor-β (PDGFR-β) expression to regulate cell migration. These findings expand the role for Notch in angiogenesis by demonstrating that Notch signaling in pericytes is essential for vascular development and function.

The dual specificity transcription factor Mga contains both a T-box binding domain and a basic helix-loop-helix zipper (bHLHZip) domain. Loss of Mga leads to embryonic lethality by E5.5. In vitro blastocyst culture and embryonic stem (ES) cell culture identify a lack of pluripotent inner cell mass (ICM) derived cells as the cause of embryonic lethality. Loss of Mga leads to increased apoptosis in E4.5 embryos, though there is no decrease in the amount of cell proliferation. Embryos with mutant Mga have fewer pluripotent ICM cells during delayed implantation, though the number of differentiated primitive endoderm cells remained initially stable. Despite the loss of pluripotent cells, there is no change in the pattern of expression of Nanog or Oct4, pluripotent cell markers, or Gata4, a primitive endoderm marker. Expression of Ornithine Decarboxylase (ODC), the rate-limiting enzyme in the synthesis of cellular polyamines, was identified as a possible cause of embryonic lethality based on a similar mutant phenotype as well as the presence of E-box sequences in genetic regulation loci. ODC is expressed at lower levels in the ICM of Mga mutants. Blastocyst and ES cell culture defects were rescued when cultured in the presence of exogenous putrescine, the metabolic product of ODC. These results suggest a mechanism for Mga to influence pluripotent cell survival through interactions with other bHLHZip domain proteins in the regulation of the polyamine pool in pluripotent cells of the embryo.

Binding proteins (both natural and man-made) have the ability to bind tightly and specifically with small molecules and other biopolymers. Binding proteins can function as human therapeutics, diagnostics, and as tools for scientific research. Given the wide range of potential applications, there is great interest in both academia and industry to develop methods for discovering novel binders. An important step in discovering new binders is called affinity maturation, when an initial hit that shows some ability to bind the target is further mutated in additional steps to improve binding affinity, specificity, solubility, pharmacokinetic profile. Ideally, the methods for affinity maturation would allow for cookbook protocols, be successful for arbitrary targets of interest, and be minimally resource intensive. Although traditional methods for affinity maturation have had some stunning successes over the past, the state of the field is still far from this ideal. In Chapter 1, I discuss the current state of the protein engineering field. In Chapter 2, I discuss the use of phenotypic selection for yeast-display protein binders, and test these systems on simple loop libraries. In Chapter 3, I construct a genetic system in yeast that can mutate a protein loop via homologous recombination, and test its recombination function. In Chapter 4, I mate libraries that target two different loops, and run FACS on the combinatorial libraries. In Chapter 5, I discuss future directions for the project.

Ribonucleoprotein complexes (RNPs) are involved in many essential cellular processes, of which the most prominent examples include the ribosome that functions in protein translation and the spliceosome which catalyzes pre-mRNA splicing. The biogenesis of RNPs often involves complex and elaborate pathways involving post-translational modifications, transit into specific cellular domains, and several auxiliary factors including assembly chaperones. One of the best-studied examples of such a chaperone is the survival motor neuron (SMN) protein, the disease gene in spinal muscular atrophy (SMA). SMN is part of a macromolecular protein complex and catalyzes the assembly of a heptameric core of Sm proteins onto small nuclear RNAs (snRNAs) to form spliceosomal snRNPs required for RNA splicing. The Sm and Sm-like (LSm) proteins are an evolutionarily conserved family of proteins that exhibit the propensity to form diverse heteromeric complexes with unique RNA-binding characteristics. The Sm/LSm proteins are thought to function as RNA chaperones whose association with their target RNAs plays a critical role in the maturation, transport, and stability of the resulting RNPs as well as modulation of RNA-RNA and RNA-protein interactions that are critical for RNP function. Sm/LSm containing RNPs have been shown to function in a variety of cellular pathways in addition to pre-mRNA splicing, including histone mRNA 3' end formation and mRNA decay. Interestingly, in addition to its direct binding to Sm proteins, SMN has been shown to associate in vitro with members of the LSm family as well as other RNA binding proteins, implicating the SMN complex in the biology of other cellular RNPs. The discovery of the full spectrum of RNPs that are dependent on SMN activity has important implications not only for our understanding of fundamental aspects of post-transcriptional gene regulation but also for SMA pathogenesis. To pursue this line of investigation, in this dissertation, I explore the hypothesis that SMN plays a general role in RNP assembly that extends to novel RNAs that function in diverse cellular pathways. First, I report the identification of a RNA polymerase III transcript of unknown function to be a novel cell type-specific RNA target of SMN function in ribonucleoprotein assembly. Second, I explore the role of SMN in the biology of the nuclear LSm2-8 complex active in splicing and the cytoplasmic LSm1-7 complex involved in mRNA decay. Finally, to facilitate the discovery of cellular pathways linked to SMN biology, I describe a novel cell-based model system for the phenotypic screening of genetic and pharmacological modifiers of SMN expression and function. Together, my studies significantly expand the repertoire of cellular RNAs that SMN is known to target and provide a unique platform for the identification of novel SMN-dependent cellular pathways, which have relevance for understanding RNA regulation and disease mechanisms and may help in the development of therapeutic approaches to SMA.

RNA polymerase II (RNAP II), transcribing messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), and non-coding RNAs (ncRNAs), is composed of 12 subunits. Rpb1, the largest subunit with catalytic polymerase activity, possesses a unique c-terminal domain (CTD) that consists of tandem heptad repeats with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). Somewhat reflecting the complexity of the organism, the number of repeats varies, from 26 in yeast to 52 in vertebrates. The CTD, intensively phosphorylated during transcription, serves a means to coordinate transcription and RNA processing- capping, splicing, and 3' end formation. For example, Ser 5, phosphorylated in the start of transcription, promotes the recruitment of capping enzyme, and Ser 2 phosphorylation facilitates RNA 3' end formation and transcription termination by acting as a landing pad for Pcf11. Detailed introduction is described in Chapter 1. Because of the importance of the CTD in these events, I created an Rpb1 conditional knock-out DT40 cell line (DT40-Rpb1) to further study the CTD with an initial focus on Thr 4, the function of which was unclear. Using DT40-Rpb1 system, we found that Thr 4 was phosphorylated in yeast, fly, chicken, and human cells, and cyclin-dependent kinase (CDK9) was likely the kinase to carry out this phosphorylation. We further provide evidence that Thr 4 functions in histone mRNA 3' end formation (presented mostly in chapter 2 of this thesis). Chapter 3 mainly describes the studies regarding Ser 2, Ser 5, and Ser 7. Based on the DT40-Rpb1 cell line, I created stable cell lines expressing an Rpb1 carrying a CTD with Ser 2, Ser 5, or Ser 7 mutated to alanine, and investigated the phenotypes of these cells. We found that cells expressing an Rpb1 with S2A or S5A mutation were defective in transcription and RNA processing. Contrary to previous findings, we found Ser 7 was not involved in snRNA expression and 3' end processing. In fact, no phenotypes associated with Ser 7 mutation were detected by our measurements. Extending previous Thr 4 studies, we showed in vitro and in vivo that Fcp1 dephosphorylated Thr 4. Finally, Chapter 4 describes what we have found the functions of CTD Tyr 1. Using the DT40-Rpb1 cells, I created stable cell lines expressing an Rpb1 with all Tyr residues mutated to phenylalanine (Phe). We found these cells were inviable, and the mutant Rpb1-Y1F was degraded to a CTD-less protein. Interestingly, the instability of Rpb1-Y1F was restored by reintroduction of one Tyr residue at the last heptad repeat. Further analysis provided evidence showing the involvement of Tyr phosphorylation in preventing Rpb1 from degradation by the 20S proteasome. Next, using ChIP assay, we showed Tyr phosphorylation was detected mostly at promoters, indicating a function of Tyr phosphorylation in transcription initiation. Indeed, transcription initiation defects were uncovered by assessing the recruitment of general transcription factors in cells with Y1F mutation. Extending this, we found an accumulation of upstream antisense RNAs in about one hundred reference genes by RNA-Seq analysis.

Immediate Early Genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP Responsive Element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.

Recent advances in the field of axon guidance have revealed complex transcription factor codes that regulate neuronal subtype identity and their corresponding axon projections. Retinal axon divergence at the optic chiasm midline is key to the establishment of binocular vision in higher vertebrates. In the visual system of binocular animals, the ipsilaterally and contralaterally projecting retinal ganglion cells are distinguished by the laterality of their axonal projections. Specific axon guidance receptors and their ligands are expressed in retinal ganglion cells (RGCs) and at the chiasm, tightly regulating the development of the ipsilateral (uncrossed) and contralateral (crossed) retinal projections. Though many factors are known, their dysfunction leads to only partial misrouting of RGC axons. Moreover, the complex transcription factor codes that regulate RGC subtype identity are only beginning to be uncovered. Numerous gaps remain in our understanding of how these guidance molecules are transcriptionally regulated and how they are induced by the patterning genes that set up the different domains in which these RGC subtypes reside. An even more elusive question within the field is how the ipsilateral and contralateral RGC subpopulations acquire their different cell fates. In this thesis, I present my work on dissecting out the molecular signatures of the ipsilateral and contralateral RGC populations during embryonic development through gene profiling followed by the functional characterization of one candidate from this screen. In Chapter 2, I developed a cell purification method based on retrograde labeling of these two cell populations from their divergent axonal projections followed by cell sorting. This method can be used in studies requiring purified populations of embryonic RGCs. In Chapter 3, I conducted a microarray screen of purified ipsilateral and contralateral RGCs using the above method. Through subsequent validation of the in vivo expression patterns of select candidates, I identified a number of genes that are differentially expressed in ipsilateral and contralateral RGCs. Subsequent functional characterization of these genes has the potential to uncover novel mechanisms for regulating axon guidance, cell differentiation, fate specification, and other regulatory pathways in ipsilateral and contralateral RGC development and function. The results of this screen also revealed that ipsilateral and contralateral RGC may have distinct developmental origins and utilize different strategies for differentiation. In Chapter 4, I demonstrate a novel role for cyclin D2, one of the above candidates, in the production of ipsilateral RGCs. The G1-active cyclin D2 is highly expressed in the ventral peripheral retina preceding and coincident with the developmental window of ipsilateral RGC genesis. I further found that ipsilateral RGC production is disrupted in the cyclin D2 null mouse. The expression of cyclin D2 in a distinct proliferative zone that has evolutionary significance in ipsilateral RGC production and its subtype-specific requirement during retinal development suggest that cyclin D2 may mark a distinct progenitor pool for ipsilateral RGCs. Thus, these studies offer an important advance in our understanding of neuronal subtype diversification within the retina.