Regulation of SIK1 abundance and stability is critical for myogenesis.

cAMP signaling can both promote and inhibit myogenic differentiation, but little is known about the mechanisms mediating promyogenic effects of cAMP. We previously demonstrated that the cAMP response element-binding protein (CREB) transcriptional target salt-inducible kinase 1 (SIK1) promotes MEF2 activity in myocytes via phosphorylation of class II histone deacetylase proteins (HDACs). However, it was unknown whether SIK1 couples cAMP signaling to the HDAC-MEF2 pathway during myogenesis and how this response could specifically occur in differentiating muscle cells. To address these questions, we explored SIK1 regulation and function in muscle precursor cells before and during myogenic differentiation. We found that in primary myogenic progenitor cells exposed to cAMP-inducing agents, Sik1 transcription is induced, but the protein is rapidly degraded by the proteasome. By contrast, sustained cAMP signaling extends the half-life of SIK1 in part by phosphorylation of Thr475, a previously uncharacterized site that we show can be phosphorylated by PKA in cell-free assays. We also identified a functional PEST domain near Thr475 that contributes to SIK1 degradation. During differentiation of primary myogenic progenitor cells, when PKA activity has been shown to increase, we observe elevated Sik1 transcripts as well as marked accumulation and stabilization of SIK1 protein. Depletion of Sik1 in primary muscle precursor cells profoundly impairs MEF2 protein accumulation and myogenic differentiation. Our findings support an emerging model in which SIK1 integrates cAMP signaling with the myogenic program to support appropriate timing of differentiation.

Glycine receptors containing α2 or α3 subunits regulate specific ethanol-mediated behaviors.

Glycine receptors (GlyRs) are broadly expressed in the central nervous system. Ethanol enhances the function of brain GlyRs, and the GlyRα1 subunit is associated with some of the behavioral actions of ethanol, such as loss of righting reflex. The in vivo role of GlyRα2 and α3 subunits in alcohol responses has not been characterized despite high expression levels in the nucleus accumbens and amygdala, areas that are important for the rewarding properties of drugs of abuse. We used an extensive panel of behavioral tests to examine ethanol actions in mice lacking Glra2 (the gene encoding the glycine receptor alpha 2 subunit) or Glra3 (the gene encoding the glycine receptor alpha 3 subunit). Deletion of Glra2 or Glra3 alters specific ethanol-induced behaviors. Glra2 knockout mice demonstrate reduced ethanol intake and preference in the 24-hour two-bottle choice test and increased initial aversive responses to ethanol and lithium chloride. In contrast, Glra3 knockout mice show increased ethanol intake and preference in the 24-hour intermittent access test and increased development of conditioned taste aversion to ethanol. Mutants and wild-type mice consumed similar amounts of ethanol in the limited access drinking in the dark test. Other ethanol effects, such as anxiolysis, motor incoordination, loss of righting reflex, and acoustic startle response, were not altered in the mutants. The behavioral changes in mice lacking GlyRα2 or α3 subunits were distinct from effects previously observed in mice with knock-in mutations in the α1 subunit. We provide evidence that GlyRα2 and α3 subunits may regulate ethanol consumption and the aversive response to ethanol.

Synaptic microRNAs Coordinately Regulate Synaptic mRNAs: Perturbation by Chronic Alcohol Consumption.

Local translation of mRNAs in the synapse has a major role in synaptic structure and function. Chronic alcohol use causes persistent changes in synaptic mRNA expression, possibly mediated by microRNAs localized in the synapse. We profiled the transcriptome of synaptoneurosomes (SN) obtained from the amygdala of mice that consumed 20% ethanol (alcohol) in a 30-day continuous two-bottle choice test to identify the microRNAs that target alcohol-induced mRNAs. SN are membrane vesicles containing pre- and post-synaptic compartments of neurons and astroglia and are a unique model for studying the synaptic transcriptome. We previously showed that chronic alcohol regulates mRNA expression in a coordinated manner. Here, we examine microRNAs and mRNAs from the same samples to define alcohol-responsive synaptic microRNAs and their predicted interactions with targeted mRNAs. The aim of the study was to identify the microRNA-mRNA synaptic interactions that are altered by alcohol. This was accomplished by comparing the effect of alcohol in SN and total homogenate preparations from the same samples. We used a combination of unbiased bioinformatic methods (differential expression, correlation, co-expression, microRNA-mRNA target prediction, co-targeting, and cell type-specific analyses) to identify key alcohol-sensitive microRNAs. Prediction analysis showed that a subset of alcohol-responsive microRNAs was predicted to target many alcohol-responsive mRNAs, providing a bidirectional analysis for identifying microRNA-mRNA interactions. We found microRNAs and mRNAs with overlapping patterns of expression that correlated with alcohol consumption. Cell type-specific analysis revealed that a significant number of alcohol-responsive mRNAs and microRNAs were unique to glutamate neurons and were predicted to target each other. Chronic alcohol consumption appears to perturb the coordinated microRNA regulation of mRNAs in SN, a mechanism that may explain the aberrations in synaptic plasticity affecting the alcoholic brain.

GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

Systematic identification of functional residues in mammalian histone H2AX.

The histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells.