A methylation-phosphorylation switch determines Sox2 stability and function in ESC maintenance or differentiation.

Sox2 is a key factor for maintaining embryonic stem cell (ESS) pluripotency, but little is known about its posttranslational regulation. Here we present evidence that the precise level of Sox2 proteins in ESCs is regulated by a balanced methylation and phosphorylation switch. Set7 monomethylates Sox2 at K119, which inhibits Sox2 transcriptional activity and induces Sox2 ubiquitination and degradation. The E3 ligase WWP2 specifically interacts with K119-methylated Sox2 through its HECT domain to promote Sox2 ubiquitination. In contrast, AKT1 phosphorylates Sox2 at T118 and stabilizes Sox2 by antagonizing K119me by Set7 and vice versa. In mouse ESCs, AKT1 activity toward Sox2 is greater than that of Set7, leading to Sox2 stabilization and ESC maintenance. In early development, increased Set7 expression correlates with Sox2 downregulation and appropriate differentiation. Our study highlights the importance of a Sox2 methylation-phosphorylation switch in determining ESC fate.

The lysine methyltransferase SMYD2 methylates the kinase domain of type II receptor BMPR2 and stimulates bone morphogenetic protein signaling.

Lysine methylation of chromosomal and nuclear proteins is a well-known mechanism of epigenetic regulation, but relatively little is known about the role of this protein modification in signal transduction. Using an RNAi-based functional screening of the SMYD family of lysine methyltransferases (KMTs), we identified SMYD2 as a KMT essential for robust bone morphogenic protein (BMP)- but not TGFß-induced target gene expression in HaCaT keratinocyte cells. A role for SMYD2 in BMP-induced gene expression was confirmed by shRNA knockdown and CRISPR/Cas9-mediated knock-out of SMYD2 We further demonstrate that SMYD2 knockdown or knock-out impairs BMP-induced phosphorylation of the signal-transducing protein SMAD1/5 and SMAD1/5 nuclear localization and interaction with SMAD4. The SMYD2 KMT activity was required to facilitate BMP-mediated signal transduction, as treatment with the SMYD2 inhibitor AZ505 suppressed BMP2-induced SMAD1/5 phosphorylation. Furthermore, we present evidence that SMYD2 likely modulates the BMP response through its function in the cytosol. We show that, although SMYD2 interacted with multiple components in the BMP pathway, it specifically methylated the kinase domain of BMP type II receptor BMPR2. Taken together, our findings suggest that SMYD2 may promote BMP signaling by directly methylating BMPR2, which, in turn, stimulates BMPR2 kinase activity and activation of the BMP pathway.

Large-Scale Identification of Protein Crotonylation Reveals Its Role in Multiple Cellular Functions.

Lysine crotonylation on histones is a recently identified post-translational modification that has been demonstrated to associate with active promoters and to directly stimulate transcription. Given that crotonyl-CoA is essential for the acyl transfer reaction and it is a metabolic intermediate widely localized within the cell, we postulate that lysine crotonylation on nonhistone proteins could also widely exist. Using specific antibody enrichment followed by high-resolution mass spectrometry analysis, we identified hundreds of crotonylated proteins and lysine residues. Bioinformatics analysis reveals that crotonylated proteins are particularly enriched for nuclear proteins involved in RNA processing, nucleic acid metabolism, chromosome organization, and gene expression. Furthermore, we demonstrate that crotonylation regulates HDAC1 activity, expels HP1α from heterochromatin, and inhibits cell cycle progression through S-phase. Our data thus indicate that lysine crotonylation could occur in a large number of proteins and could have important regulatory roles in multiple nuclei-related cellular processes.

PHD finger protein 2 (PHF2) represses ribosomal RNA gene transcription by antagonizing PHF finger protein 8 (PHF8) and recruiting methyltransferase SUV39H1.

Regulation of rDNA transcription is central to cell growth and proliferation. PHF2 and PHF8 belong to a subfamily of histone demethylases that also possess a PHD domain-dependent di-/trimethylated histone 3 lysine 4 (H3K4me2/3) binding activity and are known to be enriched in the nucleolus. In this study, we show that, unlike PHF8 that activates rDNA transcription, PHF2 inhibits rDNA transcription. Depletion of PHF2 by RNA interference increases and overexpression of PHF2 decreases rDNA transcription, respectively, whereas simultaneous depletion of PHF8 and PHF2 restores the level of rDNA transcription. The inhibition of rDNA transcription by PHF2 depends on its H3K4me2/3 binding activity that is also required for PHF2 association with the promoter of rDNA genes but not its demethylase activity. We provide evidence that PHF2 is likely to repress rDNA transcription by competing with PHF8 for binding of rDNA promoter and by recruiting H3K9me2/3 methyltransferase SUV39H1. We also provide evidence that, whereas PHF8 promotes, PHF2 represses the transcriptional activity of RARα, Oct4, and KLF4 and a few PHF8 target genes tested. Taken together, our study demonstrates a repressive role for PHF2 in transcription by RNA polymerase I and II.

The E3 ubiquitin ligase SMAD ubiquitination regulatory factor 2 negatively regulates Krüppel-like factor 5 protein.

The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated posttranslationally. We identified SMAD ubiquitination regulatory factor 2 (SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced the protein stability of KLF5 as shown by cycloheximide chase assay, indicating that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1-165), a KLF5 amino-terminal construct that lacks the PY motif and DNA binding domain, was not degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and increased the expression of cyclin D1 and PDGF-A, two established KLF5 target genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, significantly inhibited the transcriptional activity of KLF5, as demonstrated by dual luciferase assay using the PDGF-A promoter, and suppressed the ability of KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation.

A small ubiquitin-related modifier-interacting motif functions as the transcriptional activation domain of Krüppel-like factor 4.

The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), regulates numerous biological processes, including proliferation, differentiation, and embryonic stem cell self-renewal. Although the DNA sequence to which KLF4 binds is established, the mechanism by which KLF4 controls transcription is not well defined. Small ubiquitin-related modifier (SUMO) is an important regulator of transcription. Here we show that KLF4 is both SUMOylated at a single lysine residue and physically interacts with SUMO-1 in a region that matches an acidic and hydrophobic residue-rich SUMO-interacting motif (SIM) consensus. The SIM in KLF4 is required for transactivation of target promoters in a SUMO-1-dependent manner. Mutation of either the acidic or hydrophobic residues in the SIM significantly impairs the ability of KLF4 to interact with SUMO-1, activate transcription, and inhibit cell proliferation. Our study provides direct evidence that SIM in KLF4 functions as a transcriptional activation domain. A survey of transcription factor sequences reveals that established transactivation domains of many transcription factors contain sequences highly related to SIM. These results, therefore, illustrate a novel mechanism by which SUMO interaction modulates the activity of transcription factors.

SUMO suppresses and MYC amplifies transcription globally by regulating CDK9 sumoylation.

Regulation of transcription is fundamental to the control of cellular gene expression and function. Although recent studies have revealed a role for the oncoprotein MYC in amplifying global transcription, little is known as to how the global transcription is suppressed. Here we report that SUMO and MYC mediate opposite effects upon global transcription by controlling the level of CDK9 sumoylation. On one hand, SUMO suppresses global transcription via sumoylation of CDK9, the catalytic subunit of P-TEFb kinase essential for productive transcriptional elongation. On the other hand, MYC amplifies global transcription by antagonizing CDK9 sumoylation. Sumoylation of CDK9 blocks its interaction with Cyclin T1 and thus the formation of active P-TEFb complex. Transcription profiling analyses reveal that SUMO represses global transcription, particularly of moderately to highly expressed genes and by generating a sumoylation-resistant CDK9 mutant, we confirm that sumoylation of CDK9 inhibits global transcription. Together, our data reveal that SUMO and MYC oppositely control global gene expression by regulating the dynamic sumoylation and desumoylation of CDK9.

Acidic domains differentially read histone H3 lysine 4 methylation status and are widely present in chromatin-associated proteins.

Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including nucleophosmin (NPM1), pp32, SET/TAF1ß, nucleolin (NCL) and upstream binding factor (UBF) are novel H3K4me2-binding proteins. These proteins exhibit a unique pattern of interaction with methylated H3K4, as their binding is stimulated by H3K4me2 and inhibited by H3K4me1 and H3K4me3. These proteins contain one or more acidic domains consisting mainly of aspartic and/or glutamic residues that are necessary for preferential binding of H3K4me2. Furthermore, we demonstrate that the acidic domain with sufficient length alone is capable of binding H3K4me2 in vitro and in vivo. NPM1, NCL and UBF require their acidic domains for association with and transcriptional activation of rDNA genes. Interestingly, by defining acidic domain as a sequence with at least 20 acidic residues in 50 continuous amino acids, we identified 655 acidic domain-containing protein coding genes in the human genome and Gene Ontology (GO) analysis showed that many of the acidic domain proteins have chromatin-related functions. Our data suggest that acidic domain is a novel histone binding motif that can differentially read the status of H3K4 methylation and is broadly present in chromatin-associated proteins.

MOF as an evolutionarily conserved histone crotonyltransferase and transcriptional activation by histone acetyltransferase-deficient and crotonyltransferase-competent CBP/p300.

Recent studies indicate that histones are subjected to various types of acylation including acetylation, propionylation and crotonylation. CBP and p300 have been shown to catalyze multiple types of acylation but are not conserved in evolution, raising the question as to the existence of other enzymes for histone acylation and the functional relationship between well-characterized acetylation and other types of acylation. In this study, we focus on enzymes catalyzing histone crotonylation and demonstrate that among the known histone acetyltransferases, MOF, in addition to CBP and p300, also possesses histone crotonyltransferase (HCT) activity and this activity is conserved in evolution. We provide evidence that CBP and p300 are the major HCTs in mammalian cells. Furthermore, we have generated novel CBP/p300 mutants with deficient histone acetyltransferase but competent HCT activity. These CBP/p300 mutants can substitute the endogenous CBP/p300 to enhance transcriptional activation in the cell, which correlates with enhanced promoter crotonylation and recruitment of DPF2, a selective reader for crotonylated histones. Taken together, we have identified MOF as an evolutionarily conserved HCT and provide first cellular evidence that CBP/p300 can facilitate transcriptional activation through histone acylation other than acetylation, thus supporting an emerging role for the non-acetylation type of histone acylation in transcription and possibly other chromatin-based processes.

Negative regulation of DNMT3A de novo DNA methylation by frequently overexpressed UHRF family proteins as a mechanism for widespread DNA hypomethylation in cancer.

Global DNA hypomethylation is a most common epigenetic alteration in cancer, but the mechanism remains elusive. Previous studies demonstrate that UHRF1 but not UHRF2 is required for mediating DNA maintenance methylation by DNMT1. Here we report unexpectedly a conserved function for UHRF1 and UHRF2: inhibiting de novo DNA methylation by functioning as E3 ligases promoting DNMT3A degradation. UHRF1/2 are frequently overexpressed in cancers and we present evidence that UHRF1/2 overexpression downregulates DNMT3A proteins and consequently leads to DNA hypomethylation. Abrogating this negative regulation on DNMT3A or overexpression of DNMT3A leads to increased DNA methylation and impaired tumor growth. We propose a working model that UHRF1/2 safeguards the fidelity of DNA methylation and suggests that UHRF1/2 overexpression is likely a causal factor for widespread DNA hypomethylation in cancer via suppressing DNMT3A.

SUMOylation regulates nuclear localization of Krüppel-like factor 5.

SUMOylation is a form of post-translational modification shown to control nuclear transport. Krüppel-like factor 5 (KLF5) is an important mediator of cell proliferation and is primarily localized to the nucleus. Here we show that mouse KLF5 is SUMOylated at lysine residues 151 and 202. Mutation of these two lysines or two conserved nearby glutamates results in the loss of SUMOylation and increased cytoplasmic distribution of KLF5, suggesting that SUMOylation enhances nuclear localization of KLF5. Lysine 151 is adjacent to a nuclear export signal (NES) that resembles a consensus NES. The NES in KLF5 directs a fused green fluorescence protein to the cytoplasm, binds the nuclear export receptor CRM1, and is inhibited by leptomycin and site-directed mutagenesis. SUMOylation facilitates nuclear localization of KLF5 by inhibiting this NES activity, and enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colon cancer cells. A survey of proteins whose nuclear localization is regulated by SUMOylation reveals that SUMOylation sites are frequently located in close proximity to NESs. A relatively common mechanism for SUMOylation to regulate nucleocytoplasmic transport may lie in the interplay between neighboring NES and SUMOylation motifs.

Protein inhibitor of activated STAT1 interacts with and up-regulates activities of the pro-proliferative transcription factor Krüppel-like factor 5.

Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that regulates proliferation of various cell types, including fibroblasts, smooth muscle cells, and intestinal epithelial cells. To identify proteins that interact with KLF5, we performed a yeast two-hybrid screen of a 17-day mouse embryo cDNA library with KLF5 as bait. The screen revealed 21 preys clustered in four groups as follows: proteins mediating gene expression, metabolism, trafficking, and signaling. Among them was protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) ligase that regulates transcription factors through SUMOylation or physical interaction. Association between PIAS1 and KLF5 was verified by co-immunoprecipitation. Structural determination showed that the acidic domain of PIAS1 bound to both the amino- and carboxyl-terminal regions of KLF5 and that this interaction was inhibited by the amino terminus of PIAS1. Indirect immunofluorescence demonstrated that PIAS1 and KLF5 co-localized to the nucleus. Furthermore, the PIAS1-KLF5 complex was co-localized with the TATA-binding protein and was enriched in RNA polymerase II foci. Transient transfection of COS-7 cells by PIAS1 and KLF5 significantly increased the steady-state protein levels of each other. Luciferase reporter and chromatin immunoprecipitation assays showed that PIAS1 significantly activated the promoters of KLF5 and PIAS1 and synergistically increased the transcriptional activity of KLF5 in activating the cyclin D1 and Cdc2 promoters. Importantly, PIAS1 increased the ability of KLF5 to enhance cell proliferation in transfected cells. These results indicate that PIAS1 is a functional partner of KLF5 and enhances the ability of KLF5 to promote proliferation.