Browsing by Subject "Histone Deacetylases"

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CAMTA: A Signal-Responsive Transcription Factor That Promotes Cardiac Growth by Opposing Class II Histone Deacetylases
(2007-05-23) Song, Kunhua; Olson, Eric N.
Cardiac growth is finely regulated by transcriptional circuits. In an effort to discover new regulators of cardiac growth, I performed a eukaryotic expression screen for activators of the atrial natriuretic factor (ANF) gene, a cardiac-specific marker of hypertrophic signaling and embryonic development. I discovered that a family of transcription factors, called CAMTAs, regulate the ANF promoter. CAMTA proteins were first discovered in plants, however, little was known of the mechanism of their action and biological function and virtually nothing was known about mammalian CAMTA proteins, CAMTA1 and CAMTA2. CAMTA1 and CAMTA2 are enriched in embryonic and adult hearts, skeletal muscle at the embryonic stage, and brain. To define the mechanism whereby CAMTA2 activates the ANF promoter, I used a series of promoter deletion mutants to map the cis-regulatory sequences that confer responsiveness to CAMTA2. I found that CAMTA activates the ANF gene, at least in part, by associating with Nkx2-5, a cardiac transcription factor. CAMTA proteins also activate promoters of myogenin and beta myosin heavy chain via direct DNA binding. Therefore, CAMTAs activate target genes through diverse mechanisms. Over-expression of CAMTA2 in vitro and in vivo promotes cardiac growth. Based on the ability of CAMTA2 to induce hypertrophy, I tested whether signaling molecules implicated in cardiac hypertrophy might enhance the activity of CAMTA2. I discovered that the transcriptional activity of CAMTAs is governed by association with class II histone deacetylases (HDACs), which negatively regulate cardiac growth. Mice homozygous for a mutation in the CAMTA2 gene are defective in cardiac growth in response to pressure overload and neurohumoral signaling, whereas mice lacking HDAC5, a class II HDAC are sensitized to the pro-hypertrophic actions of CAMTA. CAMTA proteins are also required for embryonic heart development, as demonstrated by heart defects in mice with low dosage of CAMTA1. These findings reveal a transcriptional regulatory mechanism that modulates cardiac growth and gene expression by linking cardiac growth signals to the cardiac genome.
Characterization of Histone Deacetylase 4 and Histone Deacetylase 5 in Cocaine-Related Behaviors
(2016-04-15) Carreira Franceschi, Maria Beatriz; Self, David W.; Cowan, Christopher W.; Huber, Kimberly M.; Rothenfluh, Adrian
In recent years a focus on epigenetic mechanisms as mediators of cocaine-related behavioral, structural and functional plasticity has developed. One family of epigenetic molecules that may underlie cocaine behavioral and functional changes is the histone deacetylase family that acts to mediate transcriptional repression. The class IIa subgroup of histone deacetylases is poised as an intracellular signal detector and effector by virtue of their ability to shuttle subcellularly in a dynamic and activity-dependent manner primarily driven by phosphorylation status of the protein at multiple residues. The overlying goal of this thesis was to two-faceted: to characterize the regulation of two class IIa members, HDAC4 and HDAC5, by cocaine-mediated signaling and to characterize the role of HDAC4 and HDAC5 in cocaine-associated behavioral plasticity. We report the regulation of phosphorylation and localization of HDAC4 and HDAC5 is in opposition. HDAC5 is dephosphorylated and accumulated in the nuclear compartment in response to cocaine, dopamine dependent signaling and cAMP activity. Meanwhile, we observe that HDAC4 is weakly dephosphorylated by cAMP activity in culture but weakly phosphorylated in vivo. These findings encouraged the analysis of function of these highly homologous class members. We assessed the function of HDAC4 and HDAC5 in the nucleus accumbens, a critical region for reward, by over-expressing wildtype and nuclear variants by targeted viral-mediated gene transfer. We report an attenuation of cocaine reward learning by nuclear HDAC5 but not wildtype or HDAC4 over-expression. We further analyzed the role of HDAC5 in self-administration behavior and report an effect of nuclear HDAC5, but not wildtype, on models of reinstatement of seeking, a preclinical model of relapse. These effects were observed in the absence of an effect on intake, sensitivity or motivation to self-administer. Because HDAC4 and HDAC5 bind nuclear transcriptional regulators to exert transcriptional repression of target genes we analyzed the dependence of nuclear HDAC5 on interacting with MEF2, a primary binding partners, and report that this interaction is likely required for modulating reinstatement of seeking but dispensable for cocaine reward. Taken together, these findings highlight the role of nuclear HDAC5 but not HDAC4 to limit cocaine reward and aspects of cocaine addiction-like behavior.
Histone Deacetylase 7 and Transcriptional Regulatory Networks of the Vascular Endothelium
(2010-05-14) Young, Bryan Daniel; Olson, Eric N.
Cells respond to stimuli in part through the modulation of gene expression. Signal transduction from the environment to the nucleus culminates in the activation of factors that modify chromatin structure to either facilitate or inhibit gene transcription. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two such classes of enzymes that regulate the epigenetic code. Their opposing actions – to activate transcription by histone acetylation and to inhibit transcription by deacetylation – are tightly regulated to coordinate the vast gene programs required for cellular growth and differentiation. The class II HDACs are restricted in their expression patterns, and each have unique developmental and physiological functions. The studies described here focus on HDAC7, a class II HDAC that is expressed in vascular endothelial cells and whose function is essential for the maintenance of vascular integrity during embryogenesis. Mice lacking HDAC7 die by e11.5 with complex cardiovascular malformations including endothelial, vascular smooth muscle, and myocardial defects. By generating HDAC7 conditional knockout mice, it was observed that all of these defects are recapitulated in mice bearing an endothelial-specific deletion of HDAC7, but no defects are observed upon deletion of HDAC7 in the other cell types that were affected in the HDAC7 nulls. This in vivo evidence demonstrated that HDAC7 acts cell autonomously to maintain normal vascular development, and lead to the identification of the genetic abnormalities and mechanism leading to cardiovascular failure in the HDAC7 knockout. Further, this work begins the investigation of HDAC7 in adult vascular physiology, the findings of which will reveal new mechanisms whereby the vasculature responds to stress signals or disease. To this end, methods have been developed for the deletion of HDAC7 in the adult mouse using an inducible cre recombinase system together with the HDAC7 conditional allele. Additionally, these studies present progress toward the identification of the enhancer elements driving the endothelial-specific expression pattern of HDAC7. Detailed characterization of this enhancer is likely to implicate new signaling pathways as being involved in the genetic regulation of vascular development and maintenance. Finally, this work investigates the role of microRNAmediated gene silencing in the vascular system by identifying microRNAs involved in MEF2-dependent signaling in endothelial cells.
Inhibition of Class I HDACs Blunts Cardiac Hypertrophy via TSC2-Dependent mTOR Repression
(2014-11-21) Morales Medina, Cyndi Raquel; Rothermel, Beverly A.; Levine, Beth; Yin, Helen L.; Turer, Aslan T.; Hill, Joseph A.
Stress-induced pathological hypertrophy is observed in most forms of heart disease. If left unchecked, pathological remodeling can lead to heart failure. Histone deacetylases (HDACs) participate in the progression of pathological cardiac growth, and small molecule inhibitors of HDACs can both reduce and regress pathological hypertrophy. The mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of cell growth. It has been shown that mTORC1 is active during cardiac hypertrophy, leading to increased protein synthesis. Inhibiting mTORC1 can repress pathological remodeling. Interestingly, pan-HDAC inhibitors target mTOR activity in some cancer models. Therefore, we hypothesized that class I HDACs regulate cardiac hypertrophy in an mTOR-dependent manner. To test this hypothesis, neonatal rat ventricular myocytes (NRVMs) were exposed to a variety of growth stimuli, and class I HDACs were inhibited by either pharmacological means or by knockdown of individual HDAC isoforms. We found that HDAC1, HDAC2 and HDAC3 act together to facilitate pathological and physiological cardiomyocyte hypertrophy. In addition, inhibition of class I HDACs decreases mTOR activation by hypertrophic growth stimuli. HDAC inhibition also decreased mTOR activity in the setting of pressure overload using an in vivo surgical model of transverse aortic constriction (TAC). Adult mice with conditional cardiomyocyte-specific knockout of both HDAC1 and HDAC2 together had improved function following TSC surgery as well as decreased mTOR activity. Tuberin (TSC2) is a component of the tuberin-hamartin complex, which inhibits mTOR. We found that inhibition of class I HDACs by either genetic knockdown or using small molecules increased expression of TSC2 in both NRVMs and embryonic stem cell-derived cardiomyocytes. Furthermore, using siRNA we observed that TSC2 is required for HDAC-dependent inhibition of mTOR in NRVMs. These findings point to mTOR, and TSC2-dependent control of mTOR, as critical components of the mechanism through which HDAC inhibitors blunt pathological cardiac growth. Together, these results enhance our understanding of the function of HDACs in cardiac pathology and facilitate the ultimate translational application of HDAC inhibitors in the treatment of heart disease.
Postnatal Role for Histone Deacetylase 1 and 2 in Behavioral and Neuronal Homeostasis
(2014-11-20) Mahgoub, Melissa; Zigman, Jeffrey M.; Monteggia, Lisa; Kavalali, Ege T.; Self, David W.
Epigenetics is a dynamic process that can change gene expression without alterations in the DNA sequence. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) can influence gene activity by inducing either an active or inactive chromatin state, respectively. Accumulating in vitro data has demonstrated a crucial function for histone acetylation and deacetylation in regulating the cellular and behavioral mechanisms underlying synaptic plasticity and learning and memory. In trying to delineate the roles of individual HDACs, genetic tools have been used to manipulate HDAC expression in rodents, uncovering distinct contributions of separate HDACs in regulating the processes of memory formation. Moreover, recent findings have suggested an important role for inhibitors of HDACs in enhancing learning and memory as well as ameliorating symptoms related to neurodegenerative diseases with recent attention focused on HDAC1 and HDAC2. The overlying goal of my Ph.D. thesis has been to further delineate how the loss of the HDAC1 and HDAC2 genes affects learning and memory and other complex behaviors. We accomplished this in three separate studies. First, we examined whether the individual loss of HDAC1 or HDAC2 postnatally could recapitulate the memory enhancements observed in previous pharmacological studies. We found that a conditional postnatal deletion of HDAC2 improves learning and memory behavior, while no effects were observed in HDAC1 knockout mice. Next, since HDAC1 and HDAC2 share a high degree of sequence homology we examined whether the simultaneous deletion of both genes from the postnatal brain would result in beneficial effects on learning and memory compared to the loss of the individual genes. We found that the loss of both HDAC1 and HDAC2 leads to early lethality in conditional double knockout mice, suggesting redundant functions of these HDACs in postmitotic neurons. Finally, after observing and characterizing an excessive grooming phenotype in conditional HDAC1/2 double knockout mice we mechanistically attributed this phenotype to dysregulation of SAP90/PSD-95-associated protein 3 (SAPAP3), a key protein linked to the development of obsessive-compulsive disorder (OCD). In summary, we have characterized important roles for HDAC1 and HDAC2 in mechanisms underlying learning and memory, and have uncovered a novel role for HDAC1/2 in mediating obsessive-compulsive-like behaviors.
The Role of Class I Histone Deacetylases in Cardiovascular Development and Disease
(2008-05-13) Montgomery, Rusty Lee; Olson, Eric N.
Histone acetylation/deacetylation is a dynamic process that coordinates proper gene expression through the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibitors continue to show promise in a multitude of pathological settings such as cancer and heart disease, however the role of individual HDACs remains largely unexplored. Genetic studies have shown class II HDACs regulate developmental and physiological processes through the interaction with and repression of myocyte enhancer factor 2, MEF2, however the biological functions of class I HDACs have not been determined. To potentially understand the role of individual class I HDACs in development and disease, we generated conditional knockout alleles for HDAC1, HDAC2, and HDAC3. Through global and tissue specific analyses, we hope to identify specific roles of these enzymes in developmental, physiological, and pathological settings. Here I show that HDAC1 and HDAC2 act redundantly in controlling cardiac growth, morphogenesis, and contractility. Mice with cardiac-specific deletion of either HDAC1 or HDAC2 are viable and lack obvious phenotypes, however cardiac-specific deletion of both HDAC1 and HDAC2 results in lethality by two weeks of age. These mice show cardiac arrhythmias, dilated cardiomyopathy, and increased expression of calcium channels and skeletal muscle-specific contractile proteins. HDAC3 is an independent regulator of cardiac development. Global deletion of HDAC3 results in embryonic lethality, whereas cardiac-specific deletion of HDAC3 results in massive cardiac hypertrophy by 3 months of age and lethality by 16 weeks. These mice show metabolic abnormalities including up-regulation of genes involved in fatty acid uptake and oxidation, down-regulation of the glucose utilization pathway, and ligand induced myocardial lipid accumulation. Additionally, these hearts show mitochondrial dysfunction and decreased cardiac efficiency. These studies have identified HDAC3 as a central regulator of myocardial energy metabolism. In addition to cardiac studies, tissue-specific deletions in multiple cell-types have led to the discovery that functional redundancy of HDAC1 and HDAC2 is not restricted to postnatal cardiomyocytes, but extends to early cardiomyocytes, endothelial cells, smooth muscle cells, chondrocytes, and neurons. Deletion of HDAC1 or HDAC2 individually in these cell types does not evoke a phenotype, however deletion of both HDAC1 and HDAC2 results in embryonic lethality or neonatal lethality. Taken together, these studies identify HDAC1 and HDAC2 as redundant regulators of multiple cell types during development. Collectively, these studies have identified distinct and specific roles for HDAC1, HDAC2, and HDAC3 during development and disease. Furthermore, these genetic studies have provided mechanistic insight into the pathways regulated by each enzyme. Additional analyses on these mice will prove instrumental to the development of more specific inhibitors for the treatment of a wide array of pathological conditions.
Roles of Class II Histone Deacetylases in the Cardiovascular System
(2005-12-19) Chang, Shurong; Olson, Eric N.
Histone acetylation/deacetylation, which is orchestrated by two opposing families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), represents one of the fundamental mechanisms to control gene transcription. Class II histone deacetylases regulate developmental and physiological processes through interaction with and repression of a variety of transcription factors, including myocyte enhancer factor 2 (MEF2). Using gene targeting combined with biochemical assays, the function and regulation of class II HDACs are being elucidated. Here I show that in the absence of HDAC5, the heart becomes profoundly enlarged in response to calcineurin signaling and pressure overload. The cardiac phenotype of HDAC5 mutant mice is remarkably similar to that of HDAC9 mutant mice, strongly suggesting that these two HDACs play comparable roles in the control of cardiac growth. HDAC 5 and 9 also appear to play overlapping roles during heart development, as evidenced by cardiac malformations that occur in mice lacking both genes. Histone deacetylase 7 (HDAC7) is specifically expressed in the endothelium during early embryogenesis. Disruption of the HDAC7 gene in mice results in embryonic lethality due to a failure in endothelial cell-cell adhesion and consequent dilatation and rupture of blood vessels. HDAC7 represses MMP10 gene transcription by associating with MEF2, a direct activator of MMP10 transcription and essential regulator of blood vessel development. By in vitro kinase assays, I showed that class II HDACs are substrates for a novel stressresponsive kinase(s) specific for conserved serines that regulate MEF2-HDAC interactions. A eukaryotic expression screen revealed a remarkable variety of signaling pathways that converge on the signal-responsive phosphorylation sites in HDAC5, thereby enabling HDAC5 to connect extracellular signals to the genome. Microarray analysis was performed to provide a genome-wide molecular description of the target genes of the HDAC5/MEF2 complex in the muscle differentiation pathway. This approach was validated by characterizing the transcriptional regulatory element of a novel gene identified in the microarray analysis, which was confirmed as a direct target of MEF2. Taken together, this study provided mechanistic insights into the regulatory pathways for class II HDACs and the biological functions of these histone modifying enzymes.
Roles of HDACs and MEF2 in Adult Hippocampal Neurogenesis
(2014-11-17) Jiang, Yindi; Zhang, Chun-Li; Olson, Eric N.; Johnson, Jane E.; Hsieh, Jenny
The maintenance of the resident adult neural stem/progenitor cell (NSPC) pool depends on the precise balance of proliferation, differentiation, and maintenance of the undifferentiated state. Identifying the mechanisms that regulate this balance in adult hippocampal NSPCs can provide insight into basic neurogenesis principles important for tissue homeostasis and preventing tumor formation. Pharmacological inhibition of histone deacetylases (HDACs), a class of histone-modifying enzymes, have promising effects in cancer cells, yet the specific roles of individual HDACs in adult NSPCs are unclear. In this dissertation, I focus on dissecting the roles of two different HDACs in adult hippocampal neurogenesis: the Class I HDAC, HDAC3 and the Class IIa HDAC, HDAC5 as well as the Class IIa HDAC binding partner, myocyte enhancer factor 2 (MEF2). Using conditional knockout (cKO) mice and in vitro cell culture, I show that histone deacetylase 3 (HDAC3) is required for the proliferation of adult NSPCs. Detailed cell cycle analysis of NSPCs from Hdac3 cKO mice reveals a defect in cell cycle progression through G2/M phase, but not S phase. Moreover, HDAC3 controls G2/M phase progression mainly through post-translational stabilization of the G2/M cyclin- dependent kinase-1 (CDK1). These results demonstrate that HDAC3 plays a critical role in NSPC proliferation. HDAC5 is the most abundant Class IIa HDAC in adult dentate gyrus. HDAC5 is only expressed in immature and mature neurons. Using Hdac5 knockout mice and in vitro cell culture, I show that HDAC5 is necessary and sufficient to restrict the neuronal differentiation of NSPCs. However, the detailed mechanisms are yet to be determined. Class IIa HDACs bind to myocyte enhancer factor 2 (MEF2) in the nucleus to repress transcription of pro-neuronal genes. Thus, we also examined the function of Mef2 genes in adult hippocampal neurogenesis. In adult hippocampus, the three most highly expressed MEF2 proteins are MEF2A, 2C, and 2D, which are expressed in immature and mature neurons similar to HDAC5. We have shown that one synthetic small molecule, Isoxazole-9 (Isx-9) could trigger neuronal differentiation robustly in vitro and in vivo. Inducible knockout of all three Mef2 genes specifically in NSPCs and their progeny revealed their critical roles in mediating Isx-9 induced neurogenesis and baseline neurogenesis. In summary, these results demonstrate that HDACs and MEF2 control different stages of adult hippocampal neurogenesis and suggest that strategies aimed at pharmacological modulation of these proteins may be beneficial for tissue regeneration and controlling tumor cell growth in mammalian brain.
[Southwestern News]
(2004-11-11) Siegfried, Amanda
[Southwestern News]
(2002-08-23) Carter, Wayne
[UT Southwestern Medical Center News]
(2011-05-31) Ladson, LaKisha