Reversible lysine fatty acylation of an anchoring protein mediates adipocyte adrenergic signaling.

N-myristoylation on glycine is an irreversible modification that has long been recognized to govern protein localization and function. In contrast, the biological roles of lysine myristoylation remain ill-defined. We demonstrate that the cytoplasmic scaffolding protein, gravin-α/A kinase-anchoring protein 12, is myristoylated on two lysine residues embedded in its carboxyl-terminal protein kinase A (PKA) binding domain. Histone deacetylase 11 (HDAC11) docks to an adjacent region of gravin-α and demyristoylates these sites. In brown and white adipocytes, lysine myristoylation of gravin-α is required for signaling via ß2- and ß3-adrenergic receptors (ß-ARs), which are G protein-coupled receptors (GPCRs). Lysine myristoylation of gravin-α drives ß-ARs to lipid raft membrane microdomains, which results in PKA activation and downstream signaling that culminates in protective thermogenic gene expression. These findings define reversible lysine myristoylation as a mechanism for controlling GPCR signaling and highlight the potential of inhibiting HDAC11 to manipulate adipocyte phenotypes for therapeutic purposes.

c-Abl stabilizes HDAC2 levels by tyrosine phosphorylation repressing neuronal gene expression in Alzheimer's disease.

In Alzheimer's disease (AD), there is a decrease in neuronal gene expression induced by HDAC2 increase; however, the mechanisms involved are not fully elucidated. Here, we described how the tyrosine kinase c-Abl increases HDAC2 levels, inducing transcriptional repression of synaptic genes. Our data demonstrate that (1) in neurons, c-Abl inhibition with Imatinib prevents the AßO-induced increase in HDAC2 levels; (2) c-Abl knockdown cells show a decrease in HDAC2 levels, while c-Abl overexpression increases them; (3) c-Abl inhibition reduces HDAC2-dependent repression activity and HDAC2 recruitment to the promoter of several synaptic genes, increasing their expression; (4) c-Abl induces tyrosine phosphorylation of HDAC2, a posttranslational modification, affecting both its stability and repression activity; and (5) treatment with Imatinib decreases HDAC2 levels in a transgenic mice model of AD. Our results support the participation of the c-Abl/HDAC2 signaling pathway in the epigenetic blockade of gene expression in AD pathology.

HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSα.

MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSß) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.

Regulation of histone deacetylase activities and functions by phosphorylation and its physiological relevance.

Histone deacetylases (HDACs) are conserved enzymes that regulate many cellular processes by catalyzing the removal of acetyl groups from lysine residues on histones and non-histone proteins. As appropriate for proteins that occupy such an essential biological role, HDAC activities and functions are in turn highly regulated. Overwhelming evidence suggests that the dysregulation of HDACs plays a major role in many human diseases. The regulation of HDACs is achieved by multiple different mechanisms, including posttranslational modifications. One of the most common posttranslational modifications on HDACs is reversible phosphorylation. Many HDAC phosphorylations are context-dependent, occurring in specific tissues or as a consequence of certain stimuli. Additionally, whereas phosphorylation can regulate some HDACs in a non-specific manner, many HDAC phosphorylations result in specific consequences. Although some of these modifications support normal HDAC function, aberrations can contribute to disease development. Here we review and critically evaluate how reversible phosphorylation activates or deactivates HDACs and, thereby, regulates their many functions under various cellular and physiological contexts.

HDAC11 regulates type I interferon signaling through defatty-acylation of SHMT2.

The smallest histone deacetylase (HDAC) and the only class IV HDAC member, HDAC11, is reported to regulate immune activation and tumorigenesis, yet its biochemical function is largely unknown. Here we identify HDAC11 as an efficient lysine defatty-acylase that is >10,000-fold more efficient than its deacetylase activity. Through proteomics studies, we hypothesized and later biochemically validated SHMT2 as a defatty-acylation substrate of HDAC11. HDAC11-catalyzed defatty-acylation did not affect the enzymatic activity of SHMT2. Instead, it affects the ability of SHMT2 to regulate type I IFN receptor ubiquitination and cell surface level. Correspondingly, HDAC11 depletion increased type I IFN signaling in both cell culture and mice. This study not only demonstrates that HDAC11 has an activity that is much more efficient than the corresponding deacetylase activity, but also expands the physiological functions of HDAC11 and protein lysine fatty acylation, which opens up opportunities to develop HDAC11-specific inhibitors as therapeutics to modulate immune responses.

Regulation of poly(a)-specific ribonuclease activity by reversible lysine acetylation.

Poly(A)-specific ribonuclease (PARN) is a 3'-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Specifically, PARN cleaves the 3'-end of immature, polyadenylated hTR to form the mature, nonpolyadenylated template. Despite PARN's critical role in mediating telomere maintenance, little is known about how PARN's function is regulated by post-translational modifications. In this study, using shRNA- and CRISPR/Cas9-mediated gene silencing and knockout approaches, along with 3'-exoribonuclease activity assays and additional biochemical methods, we examined whether PARN is post-translationally modified by acetylation and what effect acetylation has on PARN's activity. We found PARN is primarily acetylated by the acetyltransferase p300 at Lys-566 and deacetylated by sirtuin1 (SIRT1). We also revealed how acetylation of PARN can decrease its enzymatic activity both in vitro, using a synthetic RNA probe, and in vivo, by quantifying endogenous levels of adenylated hTR. Furthermore, we also found that SIRT1 can regulate levels of adenylated hTR through PARN. The findings of our study uncover a mechanism by which PARN acetylation and deacetylation regulate its enzymatic activity as well as levels of mature hTR. Thus, PARN's acetylation status may play a role in regulating telomere length.

The histone deacetylase HDAC11 regulates the expression of interleukin 10 and immune tolerance.

Antigen-presenting cells (APCs) induce T cell activation as well as T cell tolerance. The molecular basis of the regulation of this critical 'decision' is not well understood. Here we show that HDAC11, a member of the HDAC histone deacetylase family with no prior defined physiological function, negatively regulated expression of the gene encoding interleukin 10 (IL-10) in APCs. Overexpression of HDAC11 inhibited IL-10 expression and induced inflammatory APCs that were able to prime naive T cells and restore the responsiveness of tolerant CD4+ T cells. Conversely, disruption of HDAC11 in APCs led to upregulation of expression of the gene encoding IL-10 and impairment of antigen-specific T cell responses. Thus, HDAC11 represents a molecular target that influences immune activation versus immune tolerance, a critical 'decision' with substantial implications in autoimmunity, transplantation and cancer immunotherapy.

Emerging roles of histone modifications and HDACs in RNA splicing.

Histone modifications and RNA splicing, two seemingly unrelated gene regulatory processes, greatly increase proteome diversity and profoundly influence normal as well as pathological eukaryotic cellular functions. Like many histone modifying enzymes, histone deacetylases (HDACs) play critical roles in governing cellular behaviors and are indispensable in numerous biological processes. While the association between RNA splicing and histone modifications is beginning to be recognized, a lack of knowledge exists regarding the role of HDACs in splicing. Recent studies however, reveal that HDACs interact with spliceosomal and ribonucleoprotein complexes, actively control the acetylation states of splicing-associated histone marks and splicing factors, and thereby unexpectedly could modulate splicing. Here, we review the role of histone/protein modifications and HDACs in RNA splicing and discuss the convergence of two parallel fields, which supports the argument that HDACs, and perhaps most histone modifying enzymes, are much more versatile and far more complicated than their initially proposed functions. Analogously, an HDAC-RNA splicing connection suggests that splicing is regulated by additional upstream factors and pathways yet to be defined or not fully characterized. Some human diseases share common underlying causes of aberrant HDACs and dysregulated RNA splicing and, thus, further support the potential link between HDACs and RNA splicing.

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

Sirtuin 6 Attenuates Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Suppressing Ori-Lyt Activity and Expression of RTA.

Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8 [HHV-8]), upon being reactivated, causes serious diseases in immunocompromised individuals. Its reactivation, especially how the cellular regulating mechanisms play roles in KSHV gene expression and viral DNA replication, is not fully understood. In searching for the cellular factors that regulate KSHV gene expression, we found that several histone deacetylases (HDACs) and sirtuins (SIRTs), including HDACs 2, 7, 8, and 11 and SIRTs 4 and 6, repress KSHV ori-Lyt promoter activity. Interestingly, the nuclear protein SIRT6 presents the greatest inhibitory effect on ori-Lyt promoter activity. A more detailed investigation revealed that SIRT6 exerts repressive effects on multiple promoters of KSHV. As a consequence of inhibiting the KSHV promoters, SIRT6 not only represses viral protein production but also inhibits viral DNA replication, as investigated in a KSHV-containing cell line, SLK-iBAC-gfpK52. Depletion of the SIRT6 protein using small interfering RNA could not directly reactivate KSHV from SLK-iBAC-gfpK52 cells but made the reactivation of KSHV by use of a small amount of the reactivator (doxycycline) more effective and enhanced viral DNA replication in the KSHV infection system. We performed DNA chromatin immunoprecipitation (ChIP) assays for SIRT6 in the SLK-iBAC-gfpK52 cell line to determine whether SIRT6 interacts with the KSHV genome in order to exhibit regulatory effects. Our results suggest that SIRT6 interacts with KSHV ori-Lyt and ORF50 promoters. Furthermore, the SIRT6-KSHV DNA interaction is significantly negated by reactivation. Therefore, we identified a cellular regulator, SIRT6, that represses KSHV replication by interacting with KSHV DNA and inhibiting viral gene expression.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogen causing cancer in the immune-deficient population. The reactivation of KSHV from latency is important for it to be carcinogenic. Our finding that SIRT6 has inhibitory effects on KSHV reactivation by interacting with the viral genome and suppressing viral gene expression is important because it might lead to a strategy of interfering with KSHV reactivation. Overexpression of SIRT6 repressed the activities of several KSHV promoters, leading to reduced gene expression and DNA replication by KSHV in a KSHV bacterial artificial chromosome-containing cell line. Depletion of SIRT6 favored reactivation of KSHV from SLK-iBACV-gfpK52 cells. More importantly, we reveal that SIRT6 interacts with KSHV DNA. Whether the interaction of SIRT6 with KSHV DNA occurs at a global level will be further studied in the future.

The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men.

Protein lysine deacetylases have a pivotal role in numerous biological processes and can be divided into the Rpd3/Hda1 and sirtuin families, each having members in diverse organisms including prokaryotes. In vertebrates, the Rpd3/Hda1 family contains 11 members, traditionally referred to as histone deacetylases (HDAC) 1-11, which are further grouped into classes I, II and IV. Whereas most class I HDACs are subunits of multiprotein nuclear complexes that are crucial for transcriptional repression and epigenetic landscaping, class II members regulate cytoplasmic processes or function as signal transducers that shuttle between the cytoplasm and the nucleus. Little is known about class IV HDAC11, although its evolutionary conservation implies a fundamental role in various organisms.

T cells lacking HDAC11 have increased effector functions and mediate enhanced alloreactivity in a murine model.

Histone acetylation and the families of enzymes responsible for controlling these epigenetic marks have been implicated in regulating T-cell maturation and phenotype. Here, we demonstrate a previously undefined role of histone deacetylase 11 (HDAC11) in regulating T-cell effector functions. Using EGFP-HDAC11 transgenic reporter mice, we found that HDAC11 expression was lower in effector relative to naive and central memory T-cell populations, and activation of resting T cells resulted in its decreased expression. Experiments using HDAC11 knockout (KO) mice revealed that T cells from these mice displayed enhanced proliferation, proinflammatory cytokine production, and effector molecule expression. In addition, HDAC11KO T cells had increased expression of Eomesodermin (Eomes) and TBX21 (Tbet), transcription factors previously shown to regulate inflammatory cytokine and effector molecule production. Conversely, overexpression of HDAC11 resulted in decreased expression of these genes. Chromatin immunoprecipitation showed the presence of HDAC11 at the Eomes and Tbet gene promoters in resting T cells, where it rapidly disassociated following T-cell activation. In vivo, HDAC11KO T cells were refractory to tolerance induction. HDAC11KO T cells also mediated accelerated onset of acute graft-versus-host disease (GVHD) in a murine model, characterized by increased proliferation of T cells and expression of interferon-γ, tumor necrosis factor, and EOMES. In addition, adoptive transfer of HDAC11KO T cells resulted in significantly reduced tumor burden in a murine B-cell lymphoma model. Taken together, these data demonstrate a previously unknown role of HDAC11 as a negative epigenetic regulator of T-cell effector phenotype and function.

Neuronal gene repression in Niemann-Pick type C models is mediated by the c-Abl/HDAC2 signaling pathway.

BACKGROUND: Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of histone deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDAC activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models. RESULTS: Our data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1(-/-) mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-ß-cyclodextrin and vitamin E decreases the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1(-/-) mice; and vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression. CONCLUSION: Our data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease.

Epigenetic regulation of soluble guanylate cyclase (sGC) ß1 in breast cancer cells.

Soluble guanylate cyclase (sGC) is a heterodimer composed of α and ß subunits. The loss of sGCß1 has been implicated in several vascular and nonvascular diseases. Our analysis showed that higher levels of sGCß1 in breast cancer tissues are correlated with greater survival probability than lower sGCß1 levels. However, there is no information on sGC regulation by epigenetic mechanisms. We examined the role of histone deacetylase (HDAC) inhibitors in regulating sGCα1 and -ß1 expression in human breast cancer MDA-MB-231 and MDA-MB-468 cell lines. The class I HDAC inhibitors increased the expression of sGCß1 more than sGCα1. Transient overexpression of HDAC3, but not HDAC1 or HDAC2, significantly reduced sGCß1 mRNA. Chromatin immunoprecipitation assay confirmed an enhanced binding of HDAC3 to the sGCß1 proximal promoter, which could be reversed by panobinostat (LBH-589) treatment. Mutations at the CCAAT binding sequence, a major element regulating sGCß1 expression, markedly reduced the efficacy of LBH-589 in augmenting sGCß1 promoter activity. LBH-589 markedly enhanced the binding of nuclear transcription factor Y, subunit α, to the sGCß1 promoter (CCAAT binding sequence). In summary, HDAC3 is an endogenous antagonist of sGCß1 expression. Inhibition of HDAC3 with targeted therapy could benefit treatment of the diseases associated with sGCß1 down-regulation and/or deficiency such as cancer and several vascular-related diseases.-Sotolongo, A., Mónica, F. Z., Kots, A., Xiao, H., Liu, J., Seto, E., Bian, K., Murad, F. Epigenetic regulation of soluble guanylate cyclase (sGC) ß1 in breast cancer cells.

MPP8 and SIRT1 crosstalk in E-cadherin gene silencing and epithelial-mesenchymal transition.

As a critical developmental process, epithelial-mesenchymal transition (EMT) involves complex transcriptional reprogramming and has been closely linked to malignant progression. Although various epigenetic modifications, such as histone deacetylation and H3K9 methylation, have been implicated in this process, how they are coordinated remains elusive. We recently revealed that MPP8 couples H3K9 methylation and DNA methylation for E-cadherin gene silencing and promotes tumor cell migration, invasion, and EMT. Here, we show that MPP8 cooperates with the class III HDAC SIRT1 in this process through their physical interaction. SIRT1 antagonizes PCAF-catalyzed MPP8-K439 acetylation to protect MPP8 from ubiquitin-proteasome-mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl-H3K9 on target promoters. Consequently, disabling either MPP8 methyl-H3K9 binding or SIRT1 interaction de-represses E-cadherin and reduces EMT phenotypes, as does knockdown of MPP8 or SIRT1 in prostate cancer cells. These results illustrate how SIRT1 and MPP8 reciprocally promote each other's function and coordinate epithelial gene silencing and EMT.

Histone deacetylase 10 regulates DNA mismatch repair and may involve the deacetylation of MutS homolog 2.

MutS homolog 2 (MSH2) is an essential DNA mismatch repair (MMR) protein. It interacts with MSH6 or MSH3 to form the MutSα or MutSß complex, respectively, which recognize base-base mispairs and insertions/deletions and initiate the repair process. Mutation or dysregulation of MSH2 causes genomic instability that can lead to cancer. MSH2 is acetylated at its C terminus, and histone deacetylase (HDAC6) deacetylates MSH2. However, whether other regions of MSH2 can be acetylated and whether other histone deacetylases (HDACs) and histone acetyltransferases (HATs) are involved in MSH2 deacetylation/acetylation is unknown. Here, we report that MSH2 can be acetylated at Lys-73 near the N terminus. Lys-73 is highly conserved across many species. Although several Class I and II HDACs interact with MSH2, HDAC10 is the major enzyme that deacetylates MSH2 at Lys-73. Histone acetyltransferase HBO1 might acetylate this residue. HDAC10 overexpression in HeLa cells stimulates cellular DNA MMR activity, whereas HDAC10 knockdown decreases DNA MMR activity. Thus, our study identifies an HDAC10-mediated regulatory mechanism controlling the DNA mismatch repair function of MSH2.

Ubiquitinated sirtuin 1 (SIRT1) function is modulated during DNA damage-induced cell death and survival.

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR.

A novel role for histone deacetylase 6 in the regulation of the tolerogenic STAT3/IL-10 pathway in APCs.

APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4(+) T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising amino acids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are both recruited to the same DNA sequence within the Il10 gene promoter. Of note, disruption of this complex by knocking down HDAC6 resulted in decreased STAT3 phosphorylation--but no changes in STAT3 acetylation--as well as diminished recruitment of STAT3 to the Il10 gene promoter region. The additional demonstration that a selective HDAC6 inhibitor disrupts this STAT3/IL-10 tolerogenic axis points to HDAC6 as a novel molecular target in APCs to overcome immune tolerance and tips the balance toward T cell immunity.

Lysine acetylation: codified crosstalk with other posttranslational modifications.

Lysine acetylation has emerged as a major posttranslational modification for histones. Crossregulation between this and other modifications is crucial in modulating chromatin-based transcriptional control and shaping inheritable epigenetic programs. In addition to histones, many other nuclear proteins and various cytoplasmic regulators are subject to lysine acetylation. This review focuses on recent findings pertinent to acetylation of nonhistone proteins and emphasizes how this modification might crosstalk with phosphorylation, methylation, ubiquitination, sumoylation, and others to form code-like multisite modification programs for dynamic control of cellular signaling under diverse conditions.