The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.

Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes. Reversible acetylation on the ϵ‐amino group of α‐tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics. Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently. Here we report that β‐tubulin interacts with histone deacetylase‐6 (HDAC‐6) in a yeast two‐hybrid assay and in vitro. We find that HDAC‐6 is a micro tubule‐associated protein capable of deacetylating α‐tubulin in vivo and in vitro. HDAC‐6's microtubule binding and deacetylation functions both depend on the hdac domains. Overexpression of HDAC‐6 in mammalian cells leads to tubulin hypoacetylation. In contrast, inhibition of HDAC‐6 function by two independent mechanisms—pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC‐6 in embryonic stem cells)—leads to hyperacetylation of tubulin and microtubules. Taken together, our data provide evidence that HDAC‐6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non‐histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors.

Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the alphaC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered alphaC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2.

-methyladenosine (mA) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. mA is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of mA methyltransferase complex components in and mice. Like other components of this complex, Flacc controls mA levels and is involved in sex determination in We demonstrate that Flacc promotes mA deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the mA machinery.

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a ϳ10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.The signaling pathway centered on protein kinase B (PKB, 1 also called Akt) has emerged as a critical mediator of diverse cellular processes including metabolism, gene expression, migration, angiogenesis, proliferation, and cell survival (1, 2). PKB is tightly controlled and the consequences of its deregulation have been implicated in the development of cancers and diabetes (1, 2). The activity of PKB is markedly stimulated in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Upon stimulation, PKB is recruited to the plasma membrane through the binding of its N-terminal pleckstrin homology (PH) domain to phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), a lipid product of PI3K. PKB␣ is then activated by phosphorylation on two residues: Thr-308 in the activation loop and Ser-473 in the hydrophobic motif of the C-terminal tail (3). There is convincing evidence that Thr-308 is phosphorylated by 3-phosphoinositol-dependent kinase 1 (PDK1) (4, 5). In embryonic stem cells in which the PDK1 gene has been genetically disrupted, PKB is resistant to growth factor stimulation as consequence of loss of Thr-308 phosphorylation (5), but phosphorylation on Ser-473 still occurs. Like PKB, PDK1 also contains a PH domain that binds to PIP 3 (4, 6). Phosphorylation of Thr-308 in vivo is dependent on PI3K activity, but it is unclear if this requirement is necessary for the unfolding of PKB to allow access of PDK1 to Thr-308 site or direct activation of PDK1 through its PH domain (6, 7). Other results indicate that PI3K is important for PKB on Ser-473 because analysis of knock-in embryonic stem cells expressing PDK1 with a mutation in its PH domain revealed that PKB is not activated by insulin-like growth factor-1 (IGF-1), whereas ribosomal S6 kinase (RSK) is activated normally, indicating the importance of colocalization of PKB with PDK1 at the plasma membrane (8).Identification of the kinase responsible for phosphorylating Ser-473 has been a major challenge for a number of years but remains elusive. Several kinases have been r...