HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes.

Histone deacetylases (HDACs) play a crucial role in transcriptional regulation and are implicated in various diseases, including cancer. They are involved in histone tail deacetylation and canonically linked to transcriptional repression. Previous studies suggested that HDAC recruitment to cell-cycle gene promoters via the retinoblastoma (RB) protein or the DREAM complex through SIN3B is essential for G1/S and G2/M gene repression during cell-cycle arrest and exit. Here we investigate the interplay among DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. Knockout of SIN3B does not globally derepress cell-cycle genes in non-proliferating HCT116 and C2C12 cells. Loss of SIN3A/B moderately upregulates several cell-cycle genes in HCT116 cells but does so independently of DREAM/RB. HDAC inhibition does not induce general upregulation of RB/DREAM target genes in arrested transformed or non-transformed cells. Our findings suggest that E2F:RB and DREAM complexes can repress cell-cycle genes without relying on HDAC activity.

Oxidative stress promotes liver fibrosis by modulating the microRNA-144 and SIN3A-p38 pathways in hepatic stellate cells.

Background & Aims: Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro. Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A, and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis.

A capped Tudor domain within a core subunit of the Sin3L/Rpd3L histone deacetylase complex binds to nucleic acid G-quadruplexes.

Chromatin-modifying complexes containing histone deacetylase (HDAC) activities play critical roles in the regulation of gene transcription in eukaryotes. These complexes are thought to lack intrinsic DNA-binding activity, but according to a well-established paradigm, they are recruited via protein-protein interactions by gene-specific transcription factors and posttranslational histone modifications to their sites of action on the genome. The mammalian Sin3L/Rpd3L complex, comprising more than a dozen different polypeptides, is an ancient HDAC complex found in diverse eukaryotes. The subunits of this complex harbor conserved domains and motifs of unknown structure and function. Here, we show that Sds3, a constitutively-associated subunit critical for the proper functioning of the Sin3L/Rpd3L complex, harbors a type of Tudor domain that we designate the capped Tudor domain. Unlike canonical Tudor domains that bind modified histones, the Sds3 capped Tudor domain binds to nucleic acids that can form higher-order structures such as G-quadruplexes and shares similarities with the knotted Tudor domain of the Esa1 histone acetyltransferase that was previously shown to bind single-stranded RNA. Our findings expand the range of macromolecules capable of recruiting the Sin3L/Rpd3L complex and draw attention to potentially new biological roles for this HDAC complex.

Structural Insight into the Binding of TGIF1 to SIN3A PAH2 Domain through a C-Terminal Amphipathic Helix.

TGIF1 is a transcriptional repressor playing crucial roles in human development and function and is associated with holoprosencephaly and various cancers. TGIF1-directed transcriptional repression of specific genes depends on the recruitment of corepressor SIN3A. However, to date, the exact region of TGIF1 binding to SIN3A was not clear, and the structural basis for the binding was unknown. Here, we demonstrate that TGIF1 utilizes a C-terminal domain (termed as SIN3A-interacting domain, SID) to bind with SIN3A PAH2. The TGIF1 SID adopts a disordered structure at the apo state but forms an amphipathic helix binding into the hydrophobic cleft of SIN3A PAH2 through the nonpolar side at the holo state. Residues F379, L382 and V383 of TGIF1 buried in the hydrophobic core of the complex are critical for the binding. Moreover, homodimerization of TGIF1 through the SID and key residues of F379, L382 and V383 was evidenced, which suggests a dual role of TGIF1 SID and a correlation between dimerization and SIN3A-PAH2 binding. This study provides a structural insight into the binding of TGIF1 with SIN3A, improves the knowledge of the structure-function relationship of TGIF1 and its homologs and will help in recognizing an undiscovered SIN3A-PAH2 binder and developing a peptide inhibitor for cancer treatment.

miR-138-5p inhibits proliferation and invasion in kidney renal clear cell carcinoma by targeting SINA3 and regulation of the Notch signaling pathway.

BACKGROUND: The function of miR-138-5p as an oncogenic factor has been reported in certain cancers. This study was performed to analyze the potential involvement of miR-138-5p in kidney renal clear cell carcinoma (KIRC). METHODS: The Cancer Genome Atlas (TCGA) database was used to explain the expression of miR-138-5p in cancer and paired non-cancer tissues of KIRC patients. Subsequently, miR-138-5p expression in KIRC tissues and cell lines, as well as that in normal tissues and normal renal tubular epithelial cell line, was detected. Artificial overexpressing of miR-138-5p was applied to observe its effect on the biological behaviors of KIRC cells. The target mRNA of miR-138-5p, SIN3A, was predicted and validated. Altered expression of miR-138-5p and SIN3A was introduced to confirm their functions in KIRC proliferation and invasion. RESULTS: We showed that miR-138-5p was down-regulated in tumor tissues of KIRC patients comparing to adjacent healthy tissues and linked to dismal prognosis in patients. miR-138-5p could hinder KIRC proliferation and invasion, while artificial overexpression of SIN3A led to reversed trends. SIN3A was a target mRNA of miR-138-5p. miR-138-5p and SIN3A together affect the activation of the Notch signaling pathway. CONCLUSION: This study evidenced that up-regulated miR-138-5p inhibits proliferation and invasion of KIRC cells involving the transcription of SIN3A and the following regulation of the Notch signaling pathway.

Regulation of the Methylation and Expression Levels of the BMPR2 Gene by SIN3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension.

BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

Fusion transcripts FYN-TRAF3IP2 and KHDRBS1-LCK hijack T cell receptor signaling in peripheral T-cell lymphoma, not otherwise specified.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify FYN-TRAF3IP2, KHDRBS1-LCK and SIN3A-FOXO1 as new in-frame fusion transcripts, with FYN-TRAF3IP2 as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that FYN-TRAF3IP2 and KHDRBS1-LCK activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs.

Transcription Repressor Protein ZBTB25 Associates with HDAC1-Sin3a Complex in Mycobacterium tuberculosis-Infected Macrophages, and Its Inhibition Clears Pathogen by Autophagy.

Downregulation of host gene expression is a key strategy employed by intracellular pathogens for their survival in macrophages and subsequent pathogenesis. In a previous study, we have shown that histone deacetylase 1 (HDAC1) levels go up in macrophages infected with Mycobacterium tuberculosis, and it hypoacetylates histone H3 at the promoter of IL-12B gene, leading to its downregulation. We now show that after infection with M. tuberculosis, HDAC1 is phosphorylated, and the levels of phosphorylated HDAC1 (pHDAC1) increase significantly in macrophages. We found that transcriptional repressor protein zinc finger and BTB domain 25 (ZBTB25) and transcriptional corepressor Sin3a associate with the HDAC1 silencing complex, which is recruited to the promoter of IL-12B to downregulate its expression in infected macrophages. Knocking down of ZBTB25 enhanced release of IL-12p40 from infected macrophages. Inhibition of HDAC1 and ZBTB25 promoted colocalization of M. tuberculosis and LC3 (microtubule-associated protein 1A/1B-light chain 3) in autophagosomes. Induction of autophagy resulted in the killing of intracellular M. tuberculosis Enhanced phosphorylation of JAK2 and STAT4 was observed in macrophages upon treatment with HDAC1 and ZBTB inhibitors, and inhibition of JAK2/STAT4 negated the killing of the intracellular pathogen, suggesting their role in the autophagy-mediated killing of intracellular M. tuberculosis In view of the emergence of drug resistance in M. tuberculosis, host-directed therapy is an attractive alternative strategy to combat tuberculosis (TB). HDACs have been proposed to be host targets for TB treatment. Our study indicates that ZBTB25, a functional subunit of the HDAC1/Sin3a repressor complex involved in IL-12B suppression, could be an alternative target for host-directed anti-TB therapy.IMPORTANCE Following infection with M. tuberculosis, levels of HDAC1 go up in macrophages, and it is recruited to the promoter of IL-12B where it hypoacetylates histone H3, leading to the downregulation of the gene. Here, we show that host transcriptional repressor protein ZBTB25 and transcriptional corepressor Sin3a associate with HDAC1 in the silencing complex. Knocking down of ZBTB25 prevented the recruitment of the complex to the promoter and consequently enhanced the gene expression and the release of IL-12p40 from infected macrophages. Pharmacological inhibition of ZBTB25 in infected macrophages resulted in the induction of autophagy and killing of intracellular M. tuberculosis Drug-resistant TB is a serious challenge to TB control programs all over the world which calls for finding alternative therapeutic methods. Host-directed therapy is gaining significant momentum in treating infectious diseases. We propose that ZBTB25 is a potential target for host-directed treatment of TB.

Expression of the Neural REST/NRSF-SIN3 Transcriptional Corepressor Complex as a Target for Small-Molecule Inhibitors.

The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factor and the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacetylase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF-SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.

CaMKII exacerbates heart failure progression by activating class I HDACs.

BACKGROUND: Persistent cardiac Ca2+/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling. Class I HDACs, including HDAC1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. While it is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, the role of CaMKII in regulating class I HDACs during heart failure progression is unclear. METHODS AND RESULTS: CaMKII increases the deacetylase activity of recombinant HDAC1, HDAC2 and HDAC3 via in vitro phosphorylation assays. Phosphorylation sites on HDAC1 and HDAC3 are identified with mass spectrometry. HDAC1 activity is also increased in cardiac-specific CaMKIIδC transgenic mice (CaMKIIδC-tg). Beyond post-translational modifications, CaMKII induces HDAC1 and HDAC3 expression. HDAC1 and HDAC3 expression are significantly increased in CaMKIIδC-tg mice. Inhibition of CaMKII by overexpression of the inhibitory peptide AC3-I in the heart attenuates the upregulation of HDAC1 after myocardial infarction surgery. Importantly, a potent HDAC1 inhibitor Quisinostat improves downregulated autophagy genes and cardiac dysfunction in CaMKIIδC-tg mice. In addition to Quisinostat, selective class I HDACs inhibitors, Apicidin and Entinostat, HDAC3 specific inhibitor RGFP966, as well as HDAC1 and HDAC3 siRNA prevent CaMKII overexpression induced cardiac myocyte hypertrophy. CONCLUSION: CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression. Selective class I HDACs inhibition may be a novel therapeutic avenue to alleviate CaMKII hyperactivity induced cardiac dysfunction.

Berberine chloride suppresses non-small cell lung cancer by deregulating Sin3A/TOP2B pathway in vitro and in vivo.

PURPOSE: Berberine chloride (BBC) is a well-known plant isoquinoline alkaloid derived from Berberis aristata. In this study, we aim to explore the effect of BBC on non-small cell lung cancer (NSCLC), and further expound the underlying mechanism of BBC induces NSCLC cell death in vitro and in vivo. METHODS: CCK-8 assay and colony formation assay were used to test the viability and colony formation ability of NSCLC cells. Apoptosis analysis was used to analyze the apoptotic cells. siRNAs were utilized to disturb the expression of Sin3A. qPCR and Western blot analysis were employed to determine mRNA and protein levels of related genes and proteins. Tumor xenografts model was used for in vivo detection. RESULTS: BBC inhibited the proliferation and colony formation of human NSCLC cells in a dose- and time-dependent manner. In addition, BBC induced DNA double-stranded breaks (DSBs) through downregulating TOP2B level, leading to apoptosis in human NSCLC cells. The Chip-seq data of A549 cells obtained from the ENCODE consortium indicate that Sin3A binds on the promoters of TOP2B. Knockdown of Sin3A led to downregulation of TOP2B in human NSCLC cells. Furthermore, BBC decreased Sin3A expression and shortened the half-life of Sin3A, results in downregulation of TOP2B in human NSCLC cells. CONCLUSION: In this study, we demonstrated a new mechanism that BBC suppresses human NSCLC by deregulating Sin3A/TOP2B pathway, leading to DNA damage and apoptosis in human NSCLC in vitro and in vivo.

PAK4 phosphorylating RUNX1 promotes ERα-positive breast cancer-induced osteolytic bone destruction.

The biological function of nuclear PAK4 in ERα-positive breast cancer osteolytic bone destruction remains unclear. Here, we find that the nuclear PAK4 promotes osteoclastogenesis and tumor-induced osteolysis via phosphorylating RUNX1. We show that nuclear PAK4 interacts with and phosphorylates RUNX1 at Thr-207, which induces its localization from the nucleus to the cytoplasm and influences direct interaction with SIN3A/HDAC1 and PRMT1. Furthermore, we reveal that RUNX1 phosphorylation by PAK4 at Thr-207 promotes osteolytic bone destruction via targeting downstream genes related to osteoclast differentiation and maturation. Importantly, we verify changes in RUNX1 subcellular localization when nuclear PAK4 is positive in breast cancer bone metastasis tissues. Functionally, we demonstrate that RUNX1 phosphorylation promotes osteolytic bone maturation and ERα-positive breast cancer-induced osteolytic bone damage in the mouse model of orthotopic breast cancer bone metastasis. Our results suggest PAK4 can be a therapeutic target for ERα-positive breast cancer osteolytic bone destruction.

A complex interplay between SAM synthetase and the epigenetic regulator SIN3 controls metabolism and transcription.

The SIN3 histone-modifying complex regulates the expression of multiple methionine catabolic genes, including SAM synthetase (Sam-S), as well as SAM levels. To further dissect the relationship between methionine catabolism and epigenetic regulation by SIN3, we sought to identify genes and metabolic pathways controlled by SIN3 and SAM synthetase (SAM-S) in Drosophila melanogaster Using several approaches, including RNAi-mediated gene silencing, RNA-Seq- and quantitative RT-PCR-based transcriptomics, and ultra-high-performance LC-MS/MS- and GC/MS-based metabolomics, we found that, as a global transcriptional regulator, SIN3 impacted a wide range of genes and pathways. In contrast, SAM-S affected only a narrow range of genes and pathways. The expression and levels of additional genes and metabolites, however, were altered in Sin3A+Sam-S dual knockdown cells. This analysis revealed that SIN3 and SAM-S regulate overlapping pathways, many of which involve one-carbon and central carbon metabolisms. In some cases, the factors acted independently; in some others, redundantly; and for a third set, in opposition. Together, these results, obtained from experiments with the chromatin regulator SIN3 and the metabolic enzyme SAM-S, uncover a complex relationship between metabolism and epigenetic regulation.

Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex.

At face value, the Sin3 histone deacetylase (HDAC) complex appears to be a prototypical co-repressor complex, that is, a multi-protein complex recruited to chromatin by DNA bound repressor proteins to facilitate local histone deacetylation and transcriptional repression. While this is almost certainly part of its role, Sin3 stubbornly refuses to be pigeon-holed in quite this way. Genome-wide mapping studies have found that Sin3 localises predominantly to the promoters of actively transcribed genes. While Sin3 knockout studies in various species result in a combination of both up- and down-regulated genes. Furthermore, genes such as the stem cell factor, Nanog, are dependent on the direct association of Sin3 for active transcription to occur. Sin3 appears to have properties of a co-repressor, co-activator and general transcription factor, and has thus been termed a co-regulator complex. Through a series of unique domains, Sin3 is able to assemble HDAC1/2, chromatin adaptors and transcription factors in a series of functionally and compositionally distinct complexes to modify chromatin at both gene-specific and global levels. Unsurprisingly, therefore, Sin3/HDAC1 have been implicated in the regulation of numerous cellular processes, including mammalian development, maintenance of pluripotency, cell cycle regulation and diseases such as cancer.

ROS is the major player in regulating altered autophagy and lifespan in sin-3 mutants of C. elegans.

SIN3, a transcriptional corepressor has been implicated in varied functions both as transcription activator and repressor. Recent studies associated Sin3 with the macroautophagic/autophagic process as a negative regulator of Atg8 and Atg32. Though the role of SIN3 in autophagy is being explored, little is known about the overall effect of SIN3 deletion on the survival of an organism. In this study using a Caenorhabditis elegans sin-3(tm1279);him-5(e1490) strain, we demonstrate that under in vivo conditions SIN-3 differentially modulates autophagy and lifespan. We provide evidence that the enhanced autophagy and decreased lifespan observed in sin-3 deletion mutants is dependent on ROS and intracellular oxidative stress. Inability of the mutant worms to maintain redox balance along with dysregulation of enzymatic antioxidants, depletion of GSH and NADP reserves and elevation of ROS markers compromises the longevity of the worms. It is possible that the enhanced autophagic process observed in sin-3(tm1279);him-5(e1490) worms is required to compensate for oxidative stress generated in these worms. ABBREVIATIONS: cat: catalase; DCFDA: 2',7'-dichlorodihydrofluoroscein diacetate; GSH: reduced glutathione; GSSG: oxidized glutathione; H2O2: hydrogen peroxide; HDAC: Histone deacetylase; HID: HDAC interacting domain; him-5: high incidence of males; HLH-30: Helix Loop Helix-30; HNE: 4-hydroxyl-2-noneal; LIPL: LIPase Like; MDA: malondialdehyde; NGM: nematode growth medium; PAH: paired amphipathic α-helix; PE: phosphatidylethanolamine; RFU: relative fluorescence unit; ROS: reactive oxygen species; sin-3/SIN3: yeast Switch Independent; SOD: superoxide dismutase; NADP: nicotinamide adenine dinucleotide phosphate; SQST-1: SeQueSTosome related-1; ATG: AuTophaGy related.

Epigenetic regulation of uterine biology by transcription factor KLF11 via posttranslational histone deacetylation of cytochrome p450 metabolic enzymes.

Endocrine regulation of uterine biology is critical for embryo receptivity and human reproduction. Uterine endometrium depends on extrinsic sex steroid input and hence likely has mechanisms that enable adaptation to hormonal variation. Emerging evidence suggests that sex steroid bioavailability in the endometrium is determined by adjusting their metabolic rate and fate via regulation of cytochrome (CYP) p450 enzymes. The CYP enzymes are targeted by ubiquitously expressed Sp/Krüppel-like (Sp/KLF) transcription factors. Specifically, KLF11 is highly expressed in reproductive tissues, regulates an array of endocrine/metabolic pathways via epigenetic histone-based mechanisms and, when aberrantly expressed, is associated with diabetes and reproductive tract diseases, such as leiomyoma and endometriosis. Using KLF11 as a model to investigate epigenetic regulation of endometrial first-pass metabolism, we evaluated the expression of a comprehensive array of metabolic enzymes in Ishikawa cells. KLF11 repressed most endometrial CYP enzymes. To characterize KLF11-recruited epigenetic regulatory mechanisms, we focused on the estrogen-metabolizing enzyme CYP3A4. KLF11 expression declined in secretory phase endometrial epithelium associated with increased CYP3A4 expression. Additionally, KLF11 bound to CYP3A4 promoter GC elements and thereby repressed promoter, message, protein as well as enzymatic function. This repression was epigenetically mediated, because KLF11 colocalized with and recruited the corepressor SIN3A/histone deacetylase resulting in selective deacetylation of the CYP3A4 promoter. Repression was reversed by a mutation in KLF11 that abrogated cofactor recruitment and binding. This repression was also pharmacologically reversible with an histone deacetylase inhibitor. Pharmacological alteration of endometrial metabolism could have long-term translational implications on human reproduction and uterine disease.

Suberoylanilide hydroxamic acid (SAHA)-induced dynamics of a human histone deacetylase protein interaction network.

Histone deacetylases (HDACs) are targets for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma. To obtain a better mechanistic understanding of the Sin3/HDAC complex in cancer, we extended its protein-protein interaction network and identified a mutually exclusive pair within the complex. We then assessed the effects of SAHA on the disruption of the complex network through six homologous baits. SAHA perturbs multiple protein interactions and therefore compromises the composition of large parts of the Sin3/HDAC network. A comparison of the effect of SAHA treatment on gene expression in breast cancer cells to a knockdown of the ING2 subunit indicated that a portion of the anticancer effects of SAHA may be attributed to the disruption of ING2's association with the complex. Our dynamic protein interaction network resource provides novel insights into the molecular mechanism of SAHA action and demonstrates the potential for drugs to rewire networks.

Sin3: insight into its transcription regulatory functions.

Sin3, a large acidic protein, shares structural similarity with the helix-loop-helix dimerization domain of proteins of the Myc family of transcription factors. Sin3/HDAC corepressor complex functions in transcriptional regulation of several genes and is therefore implicated in the regulation of key biological processes. Knockdown studies have confirmed the role of Sin3 in cellular proliferation, differentiation, apoptosis and cell cycle regulation, emphasizing Sin3 as an essential regulator of critical cellular events in normal and pathological processes. The present review covers the diverse functions of this master transcriptional regulator as well as illustrates the redundant and distinct functions of its two mammalian isoforms.

Human family with sequence similarity 60 member A (FAM60A) protein: a new subunit of the Sin3 deacetylase complex.

Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration.

Family with sequence similarity 60A (FAM60A) protein is a cell cycle-fluctuating regulator of the SIN3-HDAC1 histone deacetylase complex.

The SIN3A-HDAC complex deacetylates histones thereby repressing gene transcription. Here we describe family with sequence similarity 60A (FAM60A), a cell cycle-regulated protein that binds to the SIN3-HDAC complex. FAM60A expression peaks during G(1) and S phases of the cell cycle in U2OS cells, in a manner similar to the G(1) regulator cyclin D1, which is a known target of SIN3-HDAC. In this light we found that FAM60A binds to SIN3-HDAC-regulated promoters such as cyclin D1 in G(1) and S phases. Cells depleted of FAM60A show increased histone acetylation at the cyclin D1 promoter and elevated levels of cyclin D1 mRNA and protein. Furthermore, depletion of FAM60A altered the periodic association of HDAC1 with the cyclin D1 promoter, increased cyclin D1 expression at all cell cycle phases, and caused premature S phase entry. The data in this study introduce FAM60A as a novel regulator of SIN3-HDAC function and gene expression.