Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary - albeit often ...ineffective - treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity.
Drug resistance is a major obstacle for the successful treatment of many malignancies, including neuroblastoma, the most common extracranial solid tumor in childhood. Therefore, current attempts to ...improve the survival of neuroblastoma patients, as well as those with other cancers, largely depend on strategies to counter cancer cell drug resistance; hence, it is critical to understand the molecular mechanisms that mediate resistance to chemotherapeutics. The levels of LIM-kinase 2 (LIMK2) are increased in neuroblastoma cells selected for their resistance to microtubule-targeted drugs, suggesting that LIMK2 might be a possible target to overcome drug resistance. Here, we report that depletion of LIMK2 sensitizes SHEP neuroblastoma cells to several microtubule-targeted drugs, and that this increased sensitivity correlates with enhanced cell cycle arrest and apoptosis. Furthermore, we show that LIMK2 modulates microtubule acetylation and the levels of tubulin Polymerization Promoting Protein 1 (TPPP1), suggesting that LIMK2 may participate in the mitotic block induced by microtubule-targeted drugs through regulation of the microtubule network. Moreover, LIMK2-depleted cells also show an increased sensitivity to certain DNA-damage agents, suggesting that LIMK2 might act as a general pro-survival factor. Our results highlight the exciting possibility of combining specific LIMK2 inhibitors with anticancer drugs in the treatment of multi-drug resistant cancers.
Rod and cone photoreceptor neurons in the mammalian retina possess specialized cellular architecture and functional features for converting light to a neuronal signal. Establishing and maintaining ...these characteristics requires appropriate expression of a specific set of genes, which is tightly regulated by a network of photoreceptor transcription factors centered on the cone-rod homeobox protein CRX. CRX recruits transcription coactivators p300 and CBP to acetylate promoter-bound histones and activate transcription of target genes. To further elucidate the role of these two coactivators, we conditionally knocked out Ep300 and/or CrebBP in differentiating rods or cones, using opsin-driven Cre recombinase. Knockout of either factor alone exerted minimal effects, but loss of both factors severely disrupted target cell morphology and function: the unique nuclear chromatin organization seen in mouse rods was reversed, accompanied by redistribution of nuclear territories associated with repressive and active histone marks. Transcription of many genes including CRX targets was severely impaired, correlating with reduced histone H3/H4 acetylation (the products of p300/CBP) on target gene promoters. Interestingly, the presence of a single wild-type allele of either coactivator prevented many of these defects, with Ep300 more effective than Cbp. These results suggest that p300 and CBP play essential roles in maintaining photoreceptor-specific structure, function and gene expression.
LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe ...pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1 beta /SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1 beta that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1 beta are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1 beta levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex.
Background Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is ...hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development. Methods and Findings NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers. Conclusion/Interpretation This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.
In mammalian germ cells, meiotic commitment requires the expression of Stimulated by retinoic acid gene 8 (Stra8), which is transcriptionally activated by retinoic acid (RA). However, little is known ...about the epigenetic mechanism by which RA induces Stra8 expression. Utilizing a chromatin immunoprecipitation assay (ChIP), we showed that RA increases histone acetylation at the Stra8 promoter in murine embryonic stem cells (ESCs), a model for germ cell differentiation. Furthermore, we explored whether two coregulators with histone acetyltransferase (HAT) activity, Creb-binding protein (CBP) and p300, are involved in the activation of Stra8. The lentiviral shRNA knockdown of endogenous CBP led to Stra8 repression, while the overexpression of CBP enhanced Stra8 expression at both the mRNA and protein levels. ChIP analysis confirmed that CBP is the crucial coactivator for RA-mediated Stra8 transcription and that it enhances the level of histone acetylation and recruits RNA polymerase II to establish transcriptionally active chromatin. Furthermore, shRNA of p300 enhanced Stra8 expression, and the overexpression of p300 reduced Stra8 expression, independently of its HAT activity. ChIP showed that the knockdown of p300 significantly increased the level of CBP at the Stra8 promoter. These findings demonstrate that CBP and p300 play distinct roles in RA-mediated Stra8 gene transcription.
The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate ...transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.
The multi-potentiality of mesenchymal stem cells makes them excellent options for future tissue engineering and clinical therapy, including liver injury. In this study, we investigated the effects of ...valproic acid (VPA), a direct inhibitor of histone deacetylase (HDAC), on the hepatic differentiation of human bone marrow mesenchymal stem cells (BMMSCs). The cells were found to differentiate into a more homogeneous hepatocyte-like population when pretreated with 5 mM VPA for 72 h. The expression of liver-specific markers was significantly upregulated in the VPA-treated group at the mRNA and protein levels. VPA treatment also significantly enhanced the hepatic functions of the differentiated cells, including glycogen storage, cytochrome P450 activity, AFP and ALB synthesis, and urea production. Further analysis showed that treatment with 5 mM of VPA for 72 h greatly improved the histones H3 and H4 acetylation. These results demonstrated that VPA could considerably improve the hepatic differentiation of human BMMSCs, probably because the chromatin-acetylated state changes upon VPA treatment through its HDAC inhibitory effect. Thus, this study provides a direct research model for producing human hepatocytes for clinical purposes.
The epithelial to mesenchymal transition (EMT) has been well recognized for many decades as an essential early step in the progression of primary tumors towards metastases. Widespread epigenetic ...reprogramming of DNA and histone modifications tightly regulates gene expression and cellular activity during carcinogenesis, and epigenetic therapy has been developed to design efficient strategies for cancer treatment. As the first oral agent approved for the clinical treatment of cancer, sorafenib has significant inhibitory effects on tumor growth and EMT. However, a detailed understanding of the underlying epigenetic mechanism remains elusive. In this manuscript, we performed a ChIP-seq assay to evaluate the activity of sorafenib on the genome-wide profiling of histone modifications. We demonstrate that sorafenib largely reverses the changes in histone modifications that occur during EMT in A549 alveolar epithelial cells. Sorafenib also significantly reduces the coordinated epigenetic switching of critical EMT-associated genes in accordance with their expression levels. Furthermore, we show that sorafenib potentiates histone acetylation by regulating the expression levels of histone-modifying enzymes. Collectively, these findings provide the first evidence that sorafenib inhibits the EMT process through an epigenetic mechanism, which holds enormous promise for identifying novel epigenetic candidate diagnostic markers and drug targets for the treatment of human malignancies.
Hypokalemia is a common electrolyte disturbance frequently encountered in clinical medicine. Animals with hypokalemia had a significant arginine vasopressin (AVP)‐resistant polyuria with reduced ...expression of aquaporin 2 (AQP2) levels in the collecting ducts of the kidneys. The precise molecular mechanisms of hypokalemia‐induced nephrogenic diabetes insipidus remain unresolved. AVP induced a marked increase in histone H3K27 acetylation (H3K27ac) across the body of the AQP2 gene, in the promoter and in upstream of the AQP2 transcriptional start site in mouse mpkCCD cell line (PMID: 29572403). The purpose of the present study was to investigate whether enhanced histone acetylation prevented decreased AQP2 expression and improved urinary concentrating defect in rats with hypokalemia. Male Wistar rats were fed potassium‐free diet with or without 4‐phenylbutyric acid (4‐PBA) or a selective inhibitor of histone deacetylase 3 (HDAC3) RGFP966 for 4 days. 4‐PBA or RGFP966 markedly increased AQP2 mRNA and protein levels in kidney inner medulla of rats with hypokalemia, which was associated with decreased urine output and increased urinary osmolality. Protein expression of H3K27ac was markedly decreased in kidney inner medulla of hypokalemic rats, which was significantly prevented by 4‐PBA or RGFP966. Expression of H3K27ac was decreased in primary cultured inner medullary collecting duct cells (IMCD) or mpkCCD cells cultured in potassium‐deprivation media, in particular, H3K27ac in the promoter region of AQP2 was markedly decreased, which was associated with reduced mRNA levels of AQP2. Protein expression of HDAC3 was significantly upregulated in both mpkCCD and IMCD cells in response to potassium deprivation; the binding of HDAC3 to the promoter of AQP2 was also dramatically increased. RGFP966 significantly increased expression of H3K27ac and AQP2 protein and enhanced the binding of H3K27ac to AQP2 in mpkCCD cells. In conclusion, HDAC3 inhibition prevented downregulation of AQP2 and improved urinary concentrating defect induced by potassium deprivation, likely through enhancing acetylation of H3K27 and transcription of AQP2.
PDF
