Rusty L. Montgomery

The many roles of histone deacetylases in development and physiology: implications for disease and therapy.

Histone deacetylases (HDACs) are part of a vast family of enzymes that have crucial roles in numerous biological processes, largely through their repressive influence on transcription. The expression of many HDAC isoforms in eukaryotic cells raises questions about their possible specificity or redundancy, and whether they control global or specific programmes of gene expression. Recent analyses of HDAC knockout mice have revealed highly specific functions of individual HDACs in development and disease. Mutant mice lacking individual HDACs are a powerful tool for defining the functions of HDACs in vivo and the molecular targets of HDAC inhibitors in disease.

Therapeutic Inhibition of miR-208a Improves Cardiac Function and Survival During Heart Failure

Background— Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown. Methods and Results— Here, we show that systemic delivery of an antisense oligonucleotide induces potent and sustained silencing of miR-208a in the heart. Therapeutic inhibition of miR-208a by subcutaneous delivery of antimiR-208a during hypertension-induced heart failure in Dahl hypertensive rats dose-dependently prevents pathological myosin switching and cardiac remodeling while improving cardiac function, overall health, and survival. Transcriptional profiling indicates that antimiR-208a evokes prominent effects on cardiac gene expression; plasma analysis indicates significant changes in circulating levels of miRNAs on antimiR-208a treatment. Conclusions— These studies indicate the potential of oligonucleotide-based therapies for modulating cardiac miRNAs and validate miR-208 as a potent therapeutic target for the modulation of cardiac function and remodeling during heart disease progression.

HDAC1 and HDAC2 regulate oligodendrocyte differentiation by disrupting the β-catenin–TCF interaction

Oligodendrocyte development is regulated by the interaction of repressors and activators in a complex transcriptional network. We found that two histone-modifying enzymes, HDAC1 and HDAC2, were required for oligodendrocyte formation. Genetic deletion of both Hdac1 and Hdac2 in oligodendrocyte lineage cells resulted in stabilization and nuclear translocation of β-catenin, which negatively regulates oligodendrocyte development by repressing Olig2 expression. We further identified the oligodendrocyte-restricted transcription factor TCF7L2/TCF4 as a bipartite co-effector of β-catenin for regulating oligodendrocyte differentiation. Targeted disruption of Tcf7l2 in mice led to severe defects in oligodendrocyte maturation, whereas expression of its dominant-repressive form promoted precocious oligodendrocyte specification in developing chick neural tube. Transcriptional co-repressors HDAC1 and HDAC2 compete with β-catenin for TCF7L2 interaction to regulate downstream genes involved in oligodendrocyte differentiation. Thus, crosstalk between HDAC1/2 and the canonical Wnt signaling pathway mediated by TCF7L2 serves as a regulatory mechanism for oligodendrocyte differentiation.

Inhibition of miR-15 Protects Against Cardiac Ischemic Injury

Rationale: Myocardial infarction (MI) is a leading cause of death worldwide. Because endogenous cardiac repair mechanisms are not sufficient for meaningful tissue regeneration, MI results in loss of cardiac tissue and detrimental remodeling events. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in a sequence dependent manner. Our previous data indicate that miRNAs are dysregulated in response to ischemic injury of the heart and actively contribute to cardiac remodeling after MI. Objective: This study was designed to determine whether miRNAs are dysregulated on ischemic damage in porcine cardiac tissues and whether locked nucleic acid (LNA)-modified anti-miR chemistries can target cardiac expressed miRNAs to therapeutically inhibit miR-15 on ischemic injury. Methods and Results: Our data indicate that the miR-15 family, which includes 6 closely related miRNAs, is regulated in the infarcted region of the heart in response to ischemia-reperfusion injury in mice and pigs. LNA-modified chemistries can effectively silence miR-15 family members in vitro and render cardiomyocytes resistant to hypoxia-induced cardiomyocyte cell death. Correspondingly, systemic delivery of miR-15 anti-miRs dose-dependently represses miR-15 in cardiac tissue of both mice and pigs, whereas therapeutic targeting of miR-15 in mice reduces infarct size and cardiac remodeling and enhances cardiac function in response to MI. Conclusions: Oligonucleotide-based therapies using LNA-modified chemistries for modulating cardiac miRNAs in the setting of heart disease are efficacious and validate miR-15 as a potential therapeutic target for the manipulation of cardiac remodeling and function in the setting of ischemic injury.

Maintenance of cardiac energy metabolism by histone deacetylase 3 in mice

Histone deacetylase (HDAC) inhibitors show remarkable therapeutic potential for a variety of disorders, including cancer, neurological disease, and cardiac hypertrophy. However, the specific HDAC isoforms that mediate their actions are unclear, as are the physiological and pathological functions of individual HDACs in vivo. To explore the role of Hdac3 in the heart, we generated mice with a conditional Hdac3 null allele. Although global deletion of Hdac3 resulted in lethality by E9.5, mice with a cardiac-specific deletion of Hdac3 survived until 3-4 months of age. At this time, they showed massive cardiac hypertrophy and upregulation of genes associated with fatty acid uptake, fatty acid oxidation, and electron transport/oxidative phosphorylation accompanied by fatty acid-induced myocardial lipid accumulation and elevated triglyceride levels. These abnormalities in cardiac metabolism can be attributed to excessive activity of the nuclear receptor PPARalpha. The phenotype associated with cardiac-specific Hdac3 gene deletion differs from that of all other Hdac gene mutations. These findings reveal a unique role for Hdac3 in maintenance of cardiac function and regulation of myocardial energy metabolism.

Histone deacetylases 1 and 2 control the progression of neural precursors to neurons during brain development

The molecular mechanism by which neural progenitor cells commit to a specified lineage of the central nervous system remains unknown. We show that HDAC1 and HDAC2 redundantly control neuronal development and are required for neuronal specification. Mice lacking HDAC1 or HDAC2 in neuronal precursors show no overt histoarchitectural phenotypes, whereas deletion of both HDAC1 and HDAC2 in developing neurons results in severe hippocampal abnormalities, absence of cerebellar foliation, disorganization of cortical neurons, and lethality by postnatal day 7. These abnormalities in brain formation can be attributed to a failure of neuronal precursors to differentiate into mature neurons and to excessive cell death. These results reveal redundant and essential roles for HDAC1 and HDAC2 in the progression of neuronal precursors to mature neurons in vivo.

ΔFosB Mediates Epigenetic Desensitization of the c-fos Gene After Chronic Amphetamine Exposure

The molecular mechanisms underlying the transition from recreational drug use to chronic addiction remain poorly understood. One molecule implicated in this process is ΔFosB, a transcription factor that accumulates in striatum after repeated drug exposure and mediates sensitized behavioral responses to psychostimulants and other drugs of abuse. The downstream transcriptional mechanisms by which ΔFosB regulates drug-induced behaviors are incompletely understood. We reported previously the chromatin remodeling mechanisms by which ΔFosB activates the expression of certain genes; however, the mechanisms underlying ΔFosB-mediated gene repression remain unknown. Here, we identify c-fos, an immediate early gene rapidly induced in striatum after acute psychostimulant exposure, as a novel downstream target that is repressed chronically by ΔFosB. We show that accumulation of ΔFosB in striatum after chronic amphetamine treatment desensitizes c-fos mRNA induction to a subsequent drug dose. ΔFosB desensitizes c-fos expression by recruiting histone deacetylase 1 (HDAC1) to the c-fos gene promoter, which, in turn, deacetylates surrounding histones and attenuates gene activity. Accordingly, local knock-out of HDAC1 in striatum abolishes amphetamine-induced desensitization of the c-fos gene. In concert, chronic amphetamine increases histone H3 methylation on the c-fos promoter, a chromatin modification also known to repress gene activity, as well as expression levels of the H3 histone methyltransferase, KMT1A (lysine methyltransferase 1A, formerly SUV39H1). This study reveals a novel epigenetic pathway through which ΔFosB mediates distinct transcriptional programs that may ultimately alter behavioral plasticity to chronic amphetamine exposure.

Epigenetic control of skull morphogenesis by histone deacetylase 8

Histone deacetylases (Hdacs) are transcriptional repressors with crucial roles in mammalian development. Here we provide evidence that Hdac8 specifically controls patterning of the skull by repressing a subset of transcription factors in cranial neural crest cells. Global deletion of Hdac8 in mice leads to perinatal lethality due to skull instability, and this is phenocopied by conditional deletion of Hdac8 in cranial neural crest cells. Hdac8 specifically represses the aberrant expression of homeobox transcription factors such as Otx2 and Lhx1. These findings reveal how the identity and patterning of vertebrate-specific portions of the skull are epigenetically controlled by a histone deacetylase.

Histone Deacetylases 1 and 2 Form a Developmental Switch That Controls Excitatory Synapse Maturation and Function

The structural assembly of synapses can be accomplished in a rapid time frame, although most nascent synapses formed during early development are not fully functional and respond poorly to presynaptic action potentials. The mechanisms that are responsible for this delay in synapse maturation are unknown. Histone deacetylases (HDACs) regulate the activity state of chromatin and repress gene expression through the removal of acetyl groups from histones. Class I HDACs, which include HDAC1 and HDAC2, are expressed in the CNS, although their specific role in neuronal function has not been studied. To delineate the contribution of HDAC1 and HDAC2 in the brain, we have used pharmacological inhibitors of HDACs and mice with conditional alleles to HDAC1 and HDAC2. We found that a decrease in the activities of both HDAC1 and HDAC2 during early synaptic development causes a robust facilitation of excitatory synapse maturation and a modest increase in synapse numbers. In contrast, in mature neurons a decrease in HDAC2 levels alone was sufficient to attenuate basal excitatory neurotransmission without a significant change in the numbers of detectable nerve terminals. Therefore, we propose that HDAC1 and HDAC2 form a developmental switch that controls synapse maturation and function acting in a manner dependent on the maturational states of neuronal networks.

MicroRNA mimicry blocks pulmonary fibrosis

Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR‐29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast‐enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR‐29 levels in vivo for several days. Moreover, therapeutic delivery of these miR‐29 mimics during bleomycin‐induced pulmonary fibrosis restores endogenous miR‐29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR‐29 to be a potent therapeutic miRNA for treating pulmonary fibrosis.