07-732 Sigma-AldrichAnti-HDAC6 Antibody, CT

Repeated exposure to ethanol followed by withdrawal leads to alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc) in both clinical and preclinical models of ethanol exposure. Homer2 is a member of a family of postsynaptic density (PSD) scaffolding proteins that functions in part to cluster N-methyl-D-aspartate (NMDA) signaling complexes in the PSD, and has been shown to be critically important for plasticity in multiple models of drug and alcohol abuse. Here we used Homer2 knockout (KO) mice and a chronic intermittent intraperitoneal (IP) ethanol injection model to investigate a potential role for the protein in ethanol-induced adaptations in dendritic spine morphology and PSD protein expression. While deletion of Homer2 was associated with increased density of long spines on medium spiny neurons of the NAc core of saline treated mice, ethanol exposure had no effect on dendritic spine morphology in either wild-type (WT) or Homer2 KO mice. Western blot analysis of tissue samples from the NAc enriched for PSD proteins revealed a main effect of ethanol treatment on the expression of GluN2B, but there was no effect of genotype or treatment on the expression other glutamate receptor subunits or PSD95. These data indicate that the global deletion of Homer2 leads to aberrant regulation of dendritic spine morphology in the NAc core that is associated with an increased density of long, thin spines. Unexpectedly, intermittent IP ethanol did not affect spine morphology in either WT or KO mice. Together these data implicate Homer2 in the formation of long, thin spines and further supports its role in neuronal structure.

Alcohol use disorders (AUDs) are associated with functional and morphological changes in subfields of the prefrontal cortex. Clinical and preclinical evidence indicates that the orbitofrontal cortex (OFC) is critical for controlling impulsive behaviors, representing the value of a predicted outcome, and reversing learned associations. Individuals with AUDs often demonstrate deficits in OFC-dependent tasks, and rodent models of alcohol exposure show that OFC-dependent behaviors are impaired by chronic alcohol exposure. To explore the mechanisms that underlie these impairments, we examined dendritic spine density and morphology, and NMDA-type glutamate receptor expression in the lateral OFC of C57BL/6J mice following chronic intermittent ethanol (CIE) exposure. Western blot analysis demonstrated that NMDA receptors were not altered immediately following CIE exposure or after 7 days of withdrawal. Morphological analysis of basal dendrites of layer II/III pyramidal neurons revealed that dendritic spine density was also not affected immediately after CIE exposure. However, the total density of dendritic spines was significantly increased after a 7-day withdrawal from CIE exposure. The effect of withdrawal on spine density was mediated by an increase in the density of long, thin spines with no change in either stubby or mushroom spines. These data suggest that morphological neuroadaptations in lateral OFC neurons develop during alcohol withdrawal and occur in the absence of changes in the expression of NMDA-type glutamate receptors. The enhanced spine density that follows alcohol withdrawal may contribute to the impairments in OFC-dependent behaviors observed in CIE-treated mice.

In embryonic stem cells (ESCs), the Tip60 histone acetyltransferase activates genes required for proliferation and silences genes that promote differentiation. Here we show that the class II histone deacetylase Hdac6 co-purifies with Tip60-p400 complex from ESCs. Hdac6 is necessary for regulation of most Tip60-p400 target genes, particularly those repressed by the complex. Unlike differentiated cells, where Hdac6 is mainly cytoplasmic, Hdac6 is largely nuclear in ESCs, neural stem cells (NSCs), and some cancer cell lines, and interacts with Tip60-p400 in each. Hdac6 localizes to promoters bound by Tip60-p400 in ESCs, binding downstream of transcription start sites. Surprisingly, Hdac6 does not appear to deacetylate histones, but rather is required for Tip60-p400 binding to many of its target genes. Finally, we find that, like canonical subunits of Tip60-p400, Hdac6 is necessary for robust ESC differentiation. These data suggest that Hdac6 plays a major role in the modulation of Tip60-p400 function in stem cells. DOI: http://dx.doi.org/10.7554/eLife.01557.001.

Mesenchmal stem cells (MSCs) can be differentiated into either adipocytes or osteoblasts, and a reciprocal relationship exists between adipogenesis and osteogenesis. Multiple transcription factors and signaling pathways have been reported to regulate adipogenic or osteogenic differentiation, respectively, yet the molecular mechanism underlying the cell fate alteration between adipogenesis and osteogenesis still remains to be illustrated. MicroRNAs are important regulators in diverse biological processes by repressing protein expression of their targets. Here, miR-22 was found to regulate adipogenic and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hADMSCs) in opposite directions. Our data showed that miR-22 decreased during the process of adipogenic differentiation but increased during osteogenic differentiation. On one hand, overexpression of miR-22 in hADMSCs could inhibit lipid droplets accumulation and repress the expression of adipogenic transcription factors and adipogenic-specific genes. On the other hand, enhanced alkaline phosphatase activity and matrix mineralization, as well as increased expression of osteo-specific genes, indicated a positive role of miR-22 in regulating osteogenic differentiation. Target databases prediction and validation by Dual Luciferase Reporter Assay, western blot, and real-time polymerase chain reaction identified histone deacetylase 6 (HDAC6) as a direct downstream target of miR-22 in hADMSCs. Inhibition of endogenous HDAC6 by small-interfering RNAs suppressed adipogenesis and stimulated osteogenesis, consistent with the effect of miR-22 overexpression in hADMSCs. Together, our results suggested that miR-22 acted as a critical regulator of balance between adipogenic and osteogenic differentiation of hADMSCs by repressing its target HDAC6.

Cardiac hypertrophy and failure are characterized by transcriptional reprogramming of gene expression. Adult cardiomyocytes in mice primarily express alpha-myosin heavy chain (alpha-MHC, also known as Myh6), whereas embryonic cardiomyocytes express beta-MHC (also known as Myh7). Cardiac stress triggers adult hearts to undergo hypertrophy and a shift from alpha-MHC to fetal beta-MHC expression. Here we show that Brg1, a chromatin-remodelling protein, has a critical role in regulating cardiac growth, differentiation and gene expression. In embryos, Brg1 promotes myocyte proliferation by maintaining Bmp10 and suppressing p57(kip2) expression. It preserves fetal cardiac differentiation by interacting with histone deacetylase (HDAC) and poly (ADP ribose) polymerase (PARP) to repress alpha-MHC and activate beta-MHC. In adults, Brg1 (also known as Smarca4) is turned off in cardiomyocytes. It is reactivated by cardiac stresses and forms a complex with its embryonic partners, HDAC and PARP, to induce a pathological alpha-MHC to beta-MHC shift. Preventing Brg1 re-expression decreases hypertrophy and reverses this MHC switch. BRG1 is activated in certain patients with hypertrophic cardiomyopathy, its level correlating with disease severity and MHC changes. Our studies show that Brg1 maintains cardiomyocytes in an embryonic state, and demonstrate an epigenetic mechanism by which three classes of chromatin-modifying factors-Brg1, HDAC and PARP-cooperate to control developmental and pathological gene expression.

The meiotic cell cycle is required for production of fertilization-competent gametes. Germ cell meiotic commitment requires expression of Stimulated by retinoic acid gene 8 (Stra8), which is transcriptionally activated by retinoic acid (RA). Meiotic suppression in embryonic male germ cells is believed to result from sex-specific differences in CYP26B1-catalyzed RA metabolism in the developing gonads. Here we show in mice that RA-induced Stra8 transcription is epigenetically controlled and requires a co-activator that binds proximal to the RA response elements (RAREs) in the Stra8 promoter. Embryonic male germ cells exposed in utero to the class I/II histone deacetylase (HDAC) inhibitor, trichostatin-A (TSA), show premature Stra8 activation and meiotic entry without altered Cyp26b1 expression. We also show that Stra8 expression is detectable and physiologically regulated in adult mouse ovaries. Further, oogenesis induction in adult females using TSA is associated with Stra8 activation, and these events are absent in mice deficient in the RA precursor vitamin A. Finally, all of the actions of TSA in premeiotic germ cells in vitro and in mouse ovaries in vivo can be reproduced with the small molecule HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). Thus, the ability of RA to transcriptionally induce expression of the meiosis-commitment gene, Stra8, is epigenetically controlled and this process involves a novel co-activator that functions upstream of the RAREs. These events not only coordinate the sex-specific timing of meiotic entry during embryogenesis, but also contribute to the regulation of oogenesis in adult female mammals.

Ligand-dependent corepressor LCoR was identified as a protein that interacts with the estrogen receptor alpha (ERalpha) ligand binding domain in a hormone-dependent manner. LCoR also interacts directly with histone deacetylase 3 (HDAC3) and HDAC6. Notably, HDAC6 has emerged as a marker of breast cancer prognosis. However, although HDAC3 is nuclear, HDAC6 is cytoplasmic in many cells. We found that HDAC6 is partially nuclear in estrogen-responsive MCF7 cells, colocalizes with LCoR, represses transactivation of estrogen-inducible reporter genes, and augments corepression by LCoR. In contrast, no repression was observed upon HDAC6 expression in COS7 cells, where it is exclusively cytoplasmic. LCoR binds to HDAC6 in vitro via a central domain, and repression by LCoR mutants lacking this domain was attenuated. Kinetic chromatin immunoprecipitation assays revealed hormone-dependent recruitment of LCoR to promoters of ERalpha-induced target genes in synchrony with ERalpha. HDAC6 was also recruited to these promoters, and repeat chromatin immunoprecipitation experiments confirmed the corecruitment of LCoR with ERalpha and with HDAC6. Remarkably, however, although we find evidence for corecruitment of LCoR and ERalpha on genes repressed by the receptor, LCoR and HDAC6 failed to coimmunoprecipitate, suggesting that they are part of distinct complexes on these genes. Although small interfering RNA-mediated knockdown of LCoR or HDAC6 augmented expression of an estrogen-sensitive reporter gene in MCF7 cells, unexpectedly their ablation led to reduced expression of some endogenous estrogen target genes. Taken together, these data establish that HDAC6 can function as a cofactor of LCoR but suggest that they may act in enhance expressing some target genes.

Reversible acetylation of alpha-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated alpha-tubulin is tightly controlled and stereotypic. Acetylated alpha-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are not known. Here we report that a member of the histone deacetylase family, HDAC6, functions as a tubulin deacetylase. HDAC6 is localized exclusively in the cytoplasm, where it associates with microtubules and localizes with the microtubule motor complex containing p150(glued) (ref. 3). In vivo, the overexpression of HDAC6 leads to a global deacetylation of alpha-tubulin, whereas a decrease in HDAC6 increases alpha-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates alpha-tubulin in assembled microtubules. Furthermore, overexpression of HDAC6 promotes chemotactic cell movement, supporting the idea that HDAC6-mediated deacetylation regulates microtubule-dependent cell motility. Our results show that HDAC6 is the tubulin deacetylase, and provide evidence that reversible acetylation regulates important biological processes beyond histone metabolism and gene transcription.