Doctor of Philosophy

dissertationThe Oct1/POU2F1 transcription factor was previously thought to constitutively occupy its cognate DNA binding sites, and to regulate the expression of housekeeping genes. This stereotype led to little attention being paid to Oct1 activity in dynamic cellular responses. In 2005, Oct1-/- mouse embryonic fibroblasts were shown to be highly sensitive to oxidative and genotoxic stresses, implicating Oct1 as a stress sensor. However, the mechanism connecting stress with Oct1 transcription regulation was unknown. To identify the mechanism by which Oct1 activity is regulated by posttranslational modifications in response to stress exposure, I used affinity purification and mass spectrometry to map specific Oct1 phosphorylation, O-GlcNAc modification and ubiquitination sites. Serine 385 (Chapter 2): I identified unique mechanisms of Oct1 regulation in response to stress and during mitosis, involving two different Oct1 phosphorylation events. Following genotoxic and oxidative stress, Oct1 binding specificity is mediated by phosphorylation of S385, switching from a monomeric into a dimeric conformation on different binding sequences. I confirmed this mechanism by genome wide ChIPseq. The homologous protein Oct4, a master regulator of embryonic stem cells, uses a similar mode of regulation. Serine 335 (Chapter 3): I identified an other phosphorylation event (pS335) as a negative regulator of Oct1 binding to DNA. I found that this phosphorylation is induced iv in mitotic cells as well as in stressed ones. The phosphorylated Oct1 is enriched in the mitotic spindle poles and midbody. Phospho-Oct1 is also K11-ubiquitinated. Using several different approaches, I showed that Oct1 directly regulates mitosis after being displaced from mitotic chromatin. Threonine 255 and Serine 728 (Chapter 4): In addition to phosphorylation and ubiquitination, I also identified O-GlcNAc modification of Oct1. Using an Oct1 glycosylation defective mutant, I found that the glycosylated residues of Oct1 regulate its DNA binding and transcriptional activity. Finally, I confirmed in embryonic stem cells that Oct1 and Oct4 share binding specificity for novel multimeric as well as conventional octamer motifs (Chapter 5). Further, multimeric binding motifs recruit Oct1 and Oct4 hetero-complexes suggesting extensive crosstalk between Oct1 and Oct4 in embryonic stem cells

Empirical Research on Herding Effects: Case of Real Estate Markets

This research investigates the existence of herding effects in the real estate market. Case-Shiller Index is used to demonstrate the relationship between herding and the markets. Following the approach in Christie and Huang (1995), we investigate the presence of herd behavior among individuals. Our research presents evidence that herding does not affect individual returns from the housing market. Instead, our findings support the prediction for individual return dispersion offered by the rational asset pricing model

Simultaneous deletion of Tet1 and Tet3 increases transcriptome variability in early embryogenesis

Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.J.K. was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. W.A.P. was supported by the National Science Foundation predoctoral graduate research fellowship while this work was being performed, and subsequently by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. L.C. was the recipient of a Feodor-Lynen fellowship from the Alexander von Humboldt foundation. M.L. is supported by the Max Planck Society within its International Max Planck Research School for Computational Biology and Scientific Computing program (IMPRS-CBSC). A.T. was the recipient of an Irvington postdoctoral fellowship from the Cancer Research Institute. This work was supported by NIH R01 Grants AI044432 and HD065812 (to A.R.) and a Director’s New Innovator Award (DP2-OD-008646-01) (to S.K.).This is the author accepted manuscript. The final version is available from The National Academy of Sciences via http://dx.doi.org/10.1073/pnas.151051011

Dynamic Regulation of Oct1 during Mitosis by Phosphorylation and Ubiquitination

Transcription factor Oct1 regulates multiple cellular processes. It is known to be phosphorylated during the cell cycle and by stress, however the upstream kinases and downstream consequences are not well understood. One of these modified forms, phosphorylated at S335, lacks the ability to bind DNA. Other modification states besides phosphorylation have not been described.We show that Oct1 is phosphorylated at S335 in the Oct1 DNA binding domain during M-phase by the NIMA-related kinase Nek6. Phospho-Oct1 is also ubiquitinated. Phosphorylation excludes Oct1 from mitotic chromatin. Instead, Oct1(pS335) concentrates at centrosomes, mitotic spindle poles, kinetochores and the midbody. Oct1 siRNA knockdown diminishes the signal at these locations. Both Oct1 ablation and overexpression result in abnormal mitoses. S335 is important for the overexpression phenotype, implicating this residue in mitotic regulation. Oct1 depletion causes defects in spindle morphogenesis in Xenopus egg extracts, establishing a mitosis-specific function of Oct1. Oct1 colocalizes with lamin B1 at the spindle poles and midbody. At the midbody, both proteins are mutually required to correctly localize the other. We show that phospho-Oct1 is modified late in mitosis by non-canonical K11-linked polyubiquitin chains. Ubiquitination requires the anaphase-promoting complex, and we further show that the anaphase-promoting complex large subunit APC1 and Oct1(pS335) interact.These findings reveal mechanistic coupling between Oct1 phosphorylation and ubquitination during mitotic progression, and a role for Oct1 in mitosis

Risk of the Cross-Sectional Returns in Foreign Exchange Markets

The cross-section of foreign exchange returns has substantial exposure to the risk captured by the marketwide moments. We investigate if the foreign exchange market risks are appropriately priced in exchange rates of individual countries. We use cross-sectional analysis to explore the correlation between the marketwide risks and risk premiums of foreign currencies. The results from analysis with the Fama and MacBeth regressions indicate that, while the market beta is negatively associated with the cross-sectional returns in foreign exchange markets, higher exposures to market-wide volatility, skewness, and kurtosis are positively related to individual countries’ exchange-rate risk premiums. These results are robust in the empirical setup

A general mechanism for transcription regulation by Oct1 and Oct4 in response to genotoxic and oxidative stress

Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. Here we show that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. We further show that stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, we show that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen we identify a large number of genes targeted by Oct1 specifically under conditions of stress, and show that several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure

Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells

Dioxygenases of the Ten-Eleven Translocation (TET) family are 5-methylcytosine oxidases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. We show that Tet1 and Tet2 have distinct roles in regulating 5hmC in mouse embryonic stem cells (mESC). Tet1 depletion diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is predominantly associated with decreased 5hmC in gene bodies. Enrichment of 5hmC is observed at the boundaries of exons that are highly expressed, and Tet2 depletion results in substantial loss of 5hmC at these boundaries. In contrast, at promoter/TSS regions, Tet2 depletion results in increased 5hmC, potentially because of the redundant activity of Tet1. Together, the data point to a complex interplay between Tet1 and Tet2 in mESC, and to distinct roles for these two proteins in regulating promoter, exon, and polyadenylation site usage in cells