Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1α : modulation by p38 MAPK

The transcriptional coactivator PPAR gamma coactivator 1 α (PGC-1α) is a key regulator of metabolic processes such as mitochondrial biogenesis and respiration in muscle and gluconeogenesis in liver. Reduced levels of PGC-1α in humans have been associated with type II diabetes. PGC-1α contains a negative regulatory domain that attenuates its transcriptional activity. This negative regulation is removed by phosphorylation of PGC-1α by p38 MAPK, an important kinase downstream of cytokine signaling in muscle and β-adrenergic signaling in brown fat. We describe here the identification of p160 myb binding protein (p160MBP) as a repressor of PGC-1α. The binding and repression of PGC-1α by p160MBP is disrupted by p38 MAPK phosphorylation of PGC-1α. Adenoviral expression of p160MBP in myoblasts strongly reduces PGC-1α's ability to stimulate mitochondrial respiration and the expression of the genes of the electron transport system. This repression does not require removal of PGC-1α from chromatin, suggesting that p160MBP is or recruits a direct transcriptional suppressor. Overall, these data indicate that p160MBP is a powerful negative regulator of PGC-1α function and provide a molecular mechanism for the activation of PGC-1α by p38 MAPK. The discovery of p160MBP as a PGC-1α regulator has important implications for the understanding of energy balance and diabetes

Ubiquitination regulates PTEN nuclear import and tumor suppression

The PTEN tumor suppressor is frequently affected in cancer cells, and inherited PTEN mutation causes cancer-susceptibility conditions such as Cowden syndrome. PTEN acts as a plasma-membrane lipid-phosphatase antagonizing the phosphoinositide 3-kinase/AKT cell survival pathway. However, PTEN is also found in cell nuclei, but mechanism, function, and relevance of nuclear localization remain unclear. We show that nuclear PTEN is essential for tumor suppression and that PTEN nuclear import is mediated by its monoubiquitination. A lysine mutant of PTEN, K289E associated with Cowden syndrome, retains catalytic activity but fails to accumulate in nuclei of patient tissue due to an import defect. We identify this and another lysine residue as major monoubiquitination sites essential for PTEN import. While nuclear PTEN is stable, polyubiquitination leads to its degradation in the cytoplasm. Thus, we identify cancer-associated mutations of PTEN that target its posttranslational modification and demonstrate how a discrete molecular mechanism dictates tumor progression by differentiating between degradation and protection of PTEN

A novel requirement for DROSHA in maintenance of mammalian CG methylation

In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem–loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation

SETDB1 Is Involved in Postembryonic DNA Methylation and Gene Silencing in Drosophila

DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3–9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian “Heterochromatin Protein 1”, to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development

Role of the Sin3-Histone Deacetylase Complex in Growth Regulation by the Candidate Tumor Suppressor p33(ING1)

Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain