Browsing by Subject "Embryonic Stem Cells"
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Item Analysis of Interrelationships Among NAD+, PARP1, ADP-Ribosylation, and Splicing in Murine Embryonic Stem Cells(2022-05) Jones, Aarin; Banaszynski, Laura; DeBerardinis, Ralph J.; Wang, Yingfei; Kraus, W. LeeThe differentiation of embryonic stem cells (ESC) into a lineage-committed state is a dynamic process involving changes in epigenetic modifications, gene expression, RNA processing, and cellular metabolism. Previous studies have implicated poly(ADP-ribose) polymerase 1 (PARP1), an abundant nuclear enzyme that plays key roles in a variety of nuclear processes, in ESC self-renewal and lineage commitment. Given the diverse molecular functions of PARP1, I sought to determine the potential regulatory role of PARP1 in determining ESC state. PARP1 functions both as an enzyme, through its NAD+-dependent ADP-ribosyltransferase catalytic activity, and as a structural protein, through its NAD+-independent nucleic acid binding activity. I observed a dramatic induction of PARP1 catalytic activity during the early stages of mESC differentiation (e.g., within 12 hours of LIF removal) leading me to query the regulation and outcome of PARP1-mediated ADP-ribosylation in mESCs. NAD+ is synthesized through three main pathways - De novo, Salvage, and Preiss-Handler - and is constrained within cellular compartments. I found that both pathway usage and subcellular localization were dynamic during differentiation in a PARP1-dependent manner, with transition from De novo to Salvage pathway usage and increases in nuclear NAD+ levels upon differentiation feeding PARP1 catalytic activity. Using an NAD+ analog-sensitive PARP (asPARP) chemical biology approach, I characterized the PARP1-mediated ADP-ribosylated proteome during mESC differentiation. PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. In addition, I observed a genome-wide dysregulation of splicing events upon loss of PARP1 in transcriptomic analysis. These results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC cell state, specifically in the splicing program during differentiation.Item Identification and Characterization of the Multifunctional Epigenetic Regulator CFP1 as an ERK1/2 Substrate(2014-11-21) Klein, Aileen Melanie; Sternweis, Paul C.; Cobb, Melanie H.; Goodman, Joel M.; Conrad, NicholasEpigenetic regulation of gene transcription occurs as an integration of multiple layers of signals at a genetic locus. These signals can include local chromatin structure, covalent modifications to both histone proteins and DNA, the presence of transcription factors, and modification directly to the transcriptional machinery. Our lab is interested in the control of cellular processes by the mitogen activated protein kinases ERK1/2. In a yeast two-hybrid screen with activated ERK2 (extracellular signal-regulated kinase 2) to find novel interacting partners, our lab identified CFP1 (CxxC finger protein 1), a DNA-binding protein that is a vital component of the H3K4 trimethylating Set1A/B complexes to promote gene transcription. CFP1 has also been shown to interact physically and functionally with the major maintenance DNA methyltransferase DNMT1. We are interested in defining how substrate targeting of CFP1 by ERK1/2 regulates downstream transcriptional outcomes. Interaction between ERK2 and CFP1 in cells was validated by co-immunoprecipitation from isolated mononucleosomes. Active ERK2 can phosphorylate CFP1 on multiple sites in vitro, an observation supported by studies in cells. Some of the most likely in vivo ERK1/2 phosphorylation sites include serine 224 and threonine 227. CFP1 is essential for focusing trimethylation of H3K4 at promoters, a histone modification that supports transcription from these loci. We hypothesized that phosphorylation of CFP1 by ERK1/2 during mitogenic signaling may support trimethylation of H3K4 and transcription of ERK1/2-regulated target genes. Introduction of CFP1 containing the mutation T227V into HeLa cells blocked global H3K4 trimethylation to a similar extent as CFP1 depletion. On the other hand, CFP1 S224A shows diminished transactivation capacity against a model transcriptional substrate. Neither of these mutants fail to interact with Set1B in a pulldown, suggesting that these sites may be important for Set1 complex targeting or activity towards chromatin. Consistently, CFP1 knockdown hinders induction of several ERK1/2-regulated immediate early gene targets in response to serum treatment. It will be of interest to test whether this is dependent on stable or inducible H3K4 trimethylation and what impact overexpression of point mutants will play in their transcription. Regulation of H3K4 trimethylation through CFP1 phosphorylation might represent a novel regulatory input to support transcription of ERK1/2-regulated genes.Item Modulation of Transcription Factor Chromatin Association and Gene Transcription Program in Embryonic Stem Cell and Triple Negative Breast Cancer by Poly (ADP-Ribose) Polymerase 1(2016-03-30) Liu, Ziying; Zhang, Chun-Li; Kraus, W. Lee; Kim, Tae-Kyung; Morrison, Sean J.; Li, BingPoly(ADP-ribose) polymerase-1 (PARP-1), also referred to as ADP-ribosyltransferase Diphtheria toxin-like 1 (ARTD1), is an abundant nuclear protein that plays key roles in a variety of nuclear processes, including the regulation of transcription. PARP-1 possesses an intrinsic enzymatic activity that catalyzes the transfer of ADP-ribose (ADPR) units from nicotinamide adenine dinucleotide (NAD+) onto target gene regulatory proteins, thereby modulating their activities. Although great strides have been made in the past decade in deciphering the seemingly opposing and varied roles of PARP-1 in gene regulation, many puzzles remain in this field. Using a combination of cell biology, molecular biology, genomics and biochemistry methods, I investigated the functions of PARP-1 in regulating gene transcription program in mouse embryonic stem cells and human triple negative breast cancer cells. I found that in mouse embryonic stem cells, PARP-1 functions as a pre-pioneering factor, stabilizing transcription factor Sox2 interaction with nucleosomes. This function is required for maintaining gene transcription program in embryonic stem cells. Depletion of PARP-1 causes disrupted embryonic stem cell gene expression profile, including decreased expression of Nanog, as well as increased expression of differentiation genes. Furthermore, using human triple negative breast cancer cells, I showed that this gene transcriptional regulation mechanism through PARP-1-Sox2 interplay is conserved in different physiological models. Interestingly, inhibiting PARylation activity causes gain of Sox2 binding to a set of genomic locations in TNBC cells, indicating that PARylation activity plays an antagonizing role in PARP-1-regulated Sox2 chromatin interaction. In summary, our results illustrate how PARP-1 can act at the level of the nucleosome to produce global effects on transcription factor binding and biologically important gene expression outcomes.Item Nuclear Receptors in Lung Cancer(2007-05-22) Jeong, Yangsik; Mangelsdorf, David J.; Minna, John D.Lung Cancer is a fatal disease with new diagnoses of more than 150,000 Americans every year. Although it has a relatively well-known etiology (e.g. smoking) and has been widely researched, clinical tools and markers for early diagnosis, prognostic prediction, and therapeutic interventions remain limited. Here, for the first time, I propose a novel translational approach for providing diagnostic, prognostic, mechanistic, and therapeutic information by studying of the expression of the nuclear receptor (NR) superfamily in lung cancer. Using quantitative real-time PCR, mRNA expression levels for the 48 members of the NR superfamily were profiled in 56 lung cell lines. Based on the resulting dataset, further analysis was performed to show the diagnostic and therapeutic potential of the NR profile using both an in vitro cell response assay and an in vivo mouse xenograft model with cognate ligand treatment for selected nuclear receptors. In addition, the NR profiles of 30 microdissected and pair-matched patient tissue samples provided a subset of NRs showing dramatic differences in expression and subgroupings that demonstrate individual variations between the normal and corresponding tumor. Furthermore, I identified several individual NRs as well as a subgroup of NRs with prognostic power. The relevance of NRs to disease pathogenesis was then studied in genetically manipulated human bronchial epithelial cells (HBEC3) and in transgenic K-rasV12 mice, a well-known genetic model for lung adenocarcinoma. In the HBEC3 panel, the induced expression of peroxisome proliferator activating receptor gamma (PPARγ) in the parental HBEC3 introduced by oncogenic K-rasV12 is decreased in a subset of tumorigenic clones derived from the parental cells. It appears to be strongly correlated to the expression of cylooxygenase 2 (COX2), which is shown to be decreased with PPARγ ligand treatment. In the transgenic model, I demonstrated that expression of a subgroup of NRs in wild type mice becomes altered in histologically normal tissues that harbor the K-ras mutation, and become further altered in tumor tissues of the mutant. This observation suggests that NR profiling also provides a valuable tool for understanding disease pathogenesis in lung cancer.Item Requirement of a High-Flux Metabolic State for Mouse Embryonic Stem Cell Self-Renewal(2010-11-02) Alexander, Peter Barton; McKnight, Steven L.Unbiased profiling of global metabolite levels has revealed that cultured mouse embryonic stem (ES) cells exist in a unique metabolic state. Metabolites fluctuating dramatically in response to ES cell differentiation include purine nucleotides, acetyl-CoA, the amino acid threonine, and folic acid derivatives. These altered metabolic pathways, collectively known as the high-flux backbone (HFB) of metabolism, are surmised to be responsible for the rapid proliferation of this cell type. In particular, the amino acid threonine is shown here to be critical for mouse ES cell self-renewal. Gene and protein expression analysis has revealed that the enzyme threonine dehydrogenase (TDH) has the potential to play a major role in the establishment of HFB metabolism. TDH breaks down threonine into glycine and acetyl-CoA, molecules which are used to drive purine biosynthesis and ATP production, respectively. Using multiple approaches, we show here that TDH is strongly expressed both in ES cells and in the inner cell mass of the mouse blastocyst. Identification of potent and specific small molecule inhibitors has made possible the targeted elimination of the TDH enzyme in mouse ES cells. Using these compounds, we have determined that metabolic flux through this pathway is essential for ES cell selfrenewal. TDH inhibition is shown to cause an alteration in the cell’s metabolic state that results in increased autophagic activity and cell death. This study also reports on the generation of TDH conditional knockout mice, which will enable further elucidation of the role of HFB metabolism in adult and developing animals.Item [Southwestern News](2005-11-15) Siegfried, AmandaItem [UT Southwestern Medical Center News](2009-07-09) Siegfried, AmandaItem [UT Southwestern Medical Center News](2006-12-05) Siegfried, Amanda