Hong-Duk Youn

O-GlcNAc Regulates Pluripotency and Reprogramming by Directly Acting on Core Components of the Pluripotency Network

O-linked-N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of diverse cellular processes, but its role in embryonic stem cells (ESCs) and pluripotency has not been investigated. Here we show that O-GlcNAcylation directly regulates core components of the pluripotency network. Blocking O-GlcNAcylation disrupts ESC self-renewal and reprogramming of somatic cells to induced pluripotent stem cells. The core reprogramming factors Oct4 and Sox2 are O-GlcNAcylated in ESCs, but the O-GlcNAc modification is rapidly removed upon differentiation. O-GlcNAc modification of threonine 228 in Oct4 regulates Oct4 transcriptional activity and is important for inducing many pluripotency-related genes, including Klf2, Klf5, Nr5a2, Tbx3, and Tcl1. A T228A point mutation that eliminates this O-GlcNAc modification reduces the capacity of Oct4 to maintain ESC self-renewal and reprogram somatic cells. Overall, our study makes a direct connection between O-GlcNAcylation of key regulatory transcription factors and the activity of the pluripotency network.

CtBP represses p300-mediated transcriptional activation by direct association with its bromodomain.

Histone acetyltransferase coactivators bind to acetylated histones through their bromodomains and catalyze the acetylation of histone H3 and H4 tails for transcriptional activation. C-terminal binding protein (CtBP) serves as a transcriptional corepressor by recruiting histone deacetylases. However, the precise mechanism by which CtBP represses transcription has not been determined. In this study we found that CtBP1 directly associates with p300 by binding to the PXDLS motif in the bromodomain of p300. Moreover, CtBP1 blocks the accessibility of p300 to histones in an NADH-sensitive manner and thus represses p300-mediated histone acetylation and transcriptional activation. In addition, an NADH-nonresponsive, monomeric mutant, CtBP1 (G183V), was found to strongly repress p300-mediated transcriptional activation. Thus, the dissociation of NADH from CtBP1 leads to the repression of p300-driven general transcriptional activity by CtBP1. These results suggest a novel mechanism whereby CtBP1 serves as an energy-sensing repressor of histone acetyltransferase(s) and thus affects general transcription.

Histone demethylase LSD1 is required to induce skeletal muscle differentiation by regulating myogenic factors

During myogenesis, transcriptional activities of two major myogenic factors, MyoD and myocyte enhancer factor 2 (Mef2) are regulated by histone modifications that switch on and off the target genes. However, the transition mechanism from repression to activation modes of histones has not been defined. Here we identify that lysine specific demethylase 1, (LSD1) is responsible for removing the repressive histone codes during C2C12 mouse myoblast differentiation. The potent role of LSD1 is suggested by the increment of its expression level during myogenic differentiation. Moreover, by performing co-immunoprecipitation and ChIP assay, physically interaction of LSD1 with MyoD and Mef2 on the target promoters was identified. Their interactions were resulted in upregulation of the transcription activities shown with increased luciferase activity. Interruption of demethylase activity of LSD1 using shRNA or chemical inhibitor, pargyline, treatment led to aberrant histone codes on myogenic promoters during skeletal muscle differentiation. We also demonstrate that inhibition of LSD1 impairs C2C12 mouse myoblast differentiation. Our results show for the first time the regulatory mechanism of myogenesis involving histone demethylase. Altogether, the present study suggests a de-repression model and expands the understanding on the dynamic regulation of chromatin during myogenesis.

Histone chaperones regulate histone exchange during transcription

Transcription by RNA polymerase II is accompanied by dynamic changes in chromatin, including the eviction/deposition of nucleosomes or the covalent modification of histone subunits. This study examined the role of the histone H3/H4 chaperones, Asf1 and HIR, in histone mobility during transcription, with particular focus on the histone exchange pathway, using a dual histone expression system. The results showed that the exchange of H3/H4 normally occurs during transcription by the histone chaperones. Both Asf1 and HIR are important for histone deposition but have a different effect on histone exchange. While Asf1 mediated incorporation of external H3/H4 and renewal of pre‐existing histones, HIR opposed it. The balance of two opposing activities might be an important mechanism for determining current chromatin states.

p53 regulates glucose metabolism by miR-34a.

Cancer cells rely mainly on glycolysis rather than mitochondrial respiration for energy production, which is called the Warburg effect. p53 mutations are observed in about half of cancer cases, and p53 controls the cell cycle and cell death in response to cellular stressors. p53 has been emphasized as a metabolic regulator involved in glucose, glutamine, and purine metabolism. Here, we demonstrated metabolic changes in cancer that occurred through p53. We found that p53-inducible microRNA-34a (miR-34a) repressed glycolytic enzymes (hexokinase 1, hexokinase 2, glucose-6-phosphate isomerase), and pyruvate dehydrogenase kinase 1. Treatment with an anti-miR-34a inhibitor relieved the decreased expression in these enzymes following DNA damage. miR-34a-mediated inhibition of these enzymes resulted in repressed glycolysis and enhanced mitochondrial respiration. The results suggest that p53 has a miR-34a-dependent integrated mechanism to regulate glucose metabolism.

Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells

This study examined the role of calcineurin, a major calcium‐dependent protein phosphatase, in dephosphorylating Ser‐9 and activating glycogen synthase kinase‐3β (GSK‐3β). Treatment with calcineurin inhibitors increased phosphorylation of GSK‐3β at Ser‐9 in SH‐SY5Y human neuroblastoma cells. The over‐expression of a constitutively active calcineurin mutant, calcineurin A beta (1–401), led to a significant decrease in phosphorylation at Ser‐9, an increase in the activity of GSK‐3β, and an increase in the phosphorylation of tau. Km of calcineurin for a GSK‐3β phosphopeptide was 469.3 μM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK‐3β were co‐immunoprecipitated in neuron‐derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK‐3β. We conclude that in vitro, calcineurin can dephosphorylate GSK‐3β at Ser‐9 and form a stable complex with GSK‐3β, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK‐3βin vivo.

Menin represses JunD transcriptional activity in protein kinase Cθ-mediated Nur77 expression

TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors. It has been shown that the Nur77 promoter is activated by at least two signaling pathways, one mediated by calcium and the other by protein kinase C (PKC). MEF2D has been known to regulate Nur77 expression in a calcium- dependent manner. The mechanism by which calcium regulates MEF2D is through dissociation of calcium-sensitive MEF2 corepressors (Cabin1/ HDACs, HDAC4/5) and the association with calcineurin-activated transcription factor NF-AT and the coactivator p300. However, little is known about how PKC activates the Nur77 promoter. Herein, we report that PKCθ targets AP-1 like response element in the Nur77 promoter where JunD constitutively binds. PKCθ triggers mitogen-activated protein kinase- inediated phosphorylation of JunD, and increases transcriptional activity of JunD, cooperatively with p300. Menin is identified as the transcriptional corepressor for JunD via recruitment of mSin3-istone deacetylases. In fact, Menin represses PKCθ/ p300-mediated transcriptional activity of JunD in T cell. Its dynamic regulation of histone modifiers with JunD is responsible for PKCθ-synergistic effect on Nur77 expression in T cell.

The tumor suppressor, parafibromin, mediates histone H3 K9 methylation for cyclin D1 repression

Parafibromin, a component of the RNA polymerase II-associated PAF1 complex, is a tumor suppressor linked to hyperparathyroidism-jaw tumor syndrome and sporadic parathyroid carcinoma. Parafibromin induces cell cycle arrest by repressing cyclin D1 via an unknown mechanism. Here, we show that parafibromin interacts with the histone methyltransferase, SUV39H1, and functions as a transcriptional repressor. The central region (128–227 amino acids) of parafibromin is important for both the interaction with SUV39H1 and transcriptional repression. Parafibromin associated with the promoter and coding regions of cyclin D1 and was required for the recruitment of SUV39H1 and the induction of H3 K9 methylation but not H3 K4 methylation. RNA interference analysis showed that SUV39H1 was critical for cyclin D1 repression. These data suggest that parafibromin plays an unexpected role as a repressor in addition to its widely known activity associated with transcriptional activation. Parafibromin as a part of the PAF1 complex might downregulate cyclin D1 expression by integrating repressive H3 K9 methylation during transcription.

Global protein expression profiling of budding yeast in response to DNA damage.

Exposure to DNA‐damaging agents can activate cell cycle checkpoint and DNA repair processes to ensure genetic integrity. Such exposures also can affect the transcription of many genes required for these processes. In the budding yeast Saccharomyces cerevisiae, changes of global gene expression as a result of a DNA‐damaging agent were previously identified by using DNA chip technology. DNA microarray analysis is a powerful tool for identifying genes whose expressions are changed in response to environmental changes. Transcriptional levels, however, do not necessarily reflect cellular protein levels. Green fluorescent protein (GFP) has been widely used as a reporter of gene expression and subcellular protein localization. We have used 4156 yeast strains expressing full‐length, chromosome‐tagged GFP fusion proteins to monitor changes of protein levels in response to the DNA‐damaging agent, methyl methanesulphonate (MMS). Through flow cytometry, we identified 157 proteins whose levels were increased at least three‐fold following treatment with MMS. Of 157 responsible genes, transcriptions of 57 were previously not known to be induced by MMS. Immunoblot experiments with tandem affinity‐tagged yeast strains under the same experimental conditions confirmed these newly found proteins as inducible. These results suggest, therefore, that the 57 protein expressions are regulated by different mechanisms, such as post‐translational modifications, and not by transcriptional regulation. Copyright

Cabin1 restrains p53 activity on chromatin

The tumor suppressor p53 has been proposed to bind target promoters upon genotoxic stress. However, recent evidence shows that p53 occupies some target promoters without such stress, suggesting that a negative regulator might render p53 transcriptionally inactive on these promoters. Here we show that calcineurin binding protein 1 (Cabin1) is a negative regulator of p53. Downregulation of Cabin1 induces activation of a subset of p53 target genes. Cabin1 physically interacts with p53 on these target promoters and represses p53 transcriptional activity in the absence of genotoxic stress, by regulating histone modification and p53 acetylation marks. Knockdown of Cabin1 retards cell growth and promotes cell death after DNA damage in a p53-dependent manner. Thus, Cabin1 inhibits p53 function on chromatin in the quiescent state; the presence of inactive p53 on some promoters might allow a prompt response upon DNA damage.