The Sonic hedgehog (Shh) signalling pathway plays important roles during development and in cancer. Here we report a Shh-induced epigenetic switch that cooperates with Gli to control transcription outcomes. Before induction, poised Shh target genes are marked by a bivalent chromatin domain containing a repressive histone H3K27me3 mark and an active H3K4me3 mark. Shh activation induces a local switch of epigenetic cofactors from the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) to an H3K27me3 demethylase Jmjd3/Kdm6b-centred coactivator complex. We also find that non-enzymatic activities of Jmjd3 are important and that Jmjd3 recruits the Set1/MLL H3K4 methyltransferase complexes in a Shh-dependent manner to resolve the bivalent domain. In vivo, changes of the bivalent domain accompanied Shh-activated cerebellar progenitor proliferation. Overall, our results reveal a regulatory mechanism that underlies the activation of Shh target genes and provides insight into the causes of various diseases and cancers exhibiting altered Shh signalling.
Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. The protein kinase MST1 (mammalian Ste20-like kinase 1) plays a major role in oxidative stress-induced cell death in primary mammalian neurons. However, the mechanisms that regulate MST1 in oxidative stress responses remain largely unknown. In the present study, we demonstrate that the protein kinase c-Abl phosphorylates MST1 at Y433, which triggers the stabilization and activation of MST1. Inhibition of c-Abl promotes the degradation of MST1 through C terminus of Hsc70-interacting protein (CHIP)-mediated ubiquitination, and thereby attenuates cell death. Oxidative stress induces the c-Abl-dependent tyrosine phosphorylation of MST1 and increases the interaction between MST1 and FOXO3 (Forkhead box O3), thereby activating the MST1-FOXO signaling pathway, leading to cell death in both primary culture neurons and rat hippocampal neurons. The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST1 suggests that the c-Abl-MST1 signaling cascade plays an important role in cellular responses to oxidative stress.
A method for studying steady-state kinetics of nucleotide analogues incorporation into DNA strand by non-gel CE (NGCE) with LIF was developed. Nucleoside analogue is a kind of antiviral agent used to inhibit viral replication in infected cells, especially HIV. Steady-state parameter K(m) for nucleotide analogues is determined to imply the relationship between nucleoside analogues and the enzyme in the DNA chain elongation and predict the antiviral efficacy in vivo. Samples were prepared by single nucleotide incorporation assays catalyzed by Taq DNA polymerase at 58 degrees C and HIV reverse transcriptase (RT) at 37 degrees C, and then were separated using NGCE under optimized conditions: 25 mmol/L Tris-boric-EDTA buffer (pH 8.0) with 7 mmol/L urea in the presence of 20% w/v PEG 35000 at 30 degrees C and -20 kV. K(m(dTTP)), K(m(d4TTP)) and K(m(AZTTP)) were measured by NGCE for the first time and their values for Taq DNA polymerase were 0.29+/-0.04, 32.1+/-3.3 and 74.5+/-6.6 micromol/L, respectively. For HIV RT, the values were 0.15+/-0.05, 0.31+/-0.03 and 0.17+/-0.03 micromol/L, respectively. The trend of data for HIV RT measured by NGCE was consistent with that measured by PAGE. The reported method by NGCE for the K(m) determination was powerful, sensitive and fast, and required less amounts of reagents compared with PAGE. It be employed as a reliable alternative method and further applied in other relative studies of nucleoside analogue substrates and DNA polymerases or RTs.
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