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.

Analysis of the chromatin landscape and RNA polymerase II binding at SIN3-regulated genes.

The chromatin environment has a significant impact on gene expression. Chromatin structure is highly regulated by histone modifications and RNA polymerase II binding dynamics. The SIN3 histone modifying complex regulates the chromatin environment leading to changes in gene expression. In Drosophila melanogaster, the Sin3A gene is alternatively spliced to produce different protein isoforms, two of which include SIN3 220 and SIN3 187. Both SIN3 isoforms are scaffolding proteins that interact with several other factors to regulate the chromatin landscape. The mechanism through which the SIN3 isoforms regulate chromatin is not well understood. Here, we analyze publicly available data sets to allow us to ask specific questions on how SIN3 isoforms regulate chromatin and gene activity. We determined that genes repressed by the SIN3 isoforms exhibited enrichment in histone H3K4me2, H3K4me3, H3K14ac and H3K27ac near the transcription start site. We observed an increase in the amount of paused RNA polymerase II on the promoter of genes repressed by the isoforms as compared to genes that require SIN3 for maximum activation. Furthermore, we analyzed a subset of genes regulated by SIN3 187 that suggest a mechanism in which SIN3 187 might exhibit hard regulation as well as soft regulation. Data presented here expand our knowledge of how the SIN3 isoforms regulate the chromatin environment and RNA polymerase II binding dynamics.

SIN-3 acts in distinct complexes to regulate the germline transcriptional program in Caenorhabditis elegans.

The transcriptional co-regulator SIN3 influences gene expression through multiple interactions that include histone deacetylases. Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability and autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here, we show that loss of SIN-3, the single SIN3 homolog in Caenorhabditis elegans, results in maternal-effect sterility associated with de-regulation of the germline transcriptome, including de-silencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific histone deacetylases and show that they differentially contribute to fertility. Single-cell, single-molecule fluorescence in situ hybridization reveals that in sin-3 mutants the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells, suggesting a role for SIN-3 in its silencing. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN-3. Together, this work provides a powerful framework for the in vivo study of SIN3 and associated proteins.

SIN-3 acts in distinct complexes to regulate the germline transcriptional program in Caenorhabditis elegans.

The transcriptional co-regulator SIN3 influences gene expression through multiple interactions that include histone deacetylases. Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability and autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here, we show that loss of SIN-3, the single SIN3 homolog in Caenorhabditis elegans, results in maternal-effect sterility associated with de-regulation of the germline transcriptome, including de-silencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific histone deacetylases and show that they differentially contribute to fertility. Single-cell, single-molecule fluorescence in situ hybridization reveals that in sin-3 mutants the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells, suggesting a role for SIN-3 in its silencing. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN-3. Together, this work provides a powerful framework for the in vivo study of SIN3 and associated proteins.

Transcription suppression is mediated by the HDAC1-Sin3 complex in Xenopus nucleoplasmic extract.

Modification of histones provides a dynamic mechanism to regulate chromatin structure and access to DNA. Histone acetylation, in particular, plays a prominent role in controlling the interaction between DNA, histones, and other chromatin-associated proteins. Defects in histone acetylation patterns interfere with normal gene expression and underlie a wide range of human diseases. Here, we utilize Xenopus egg extracts to investigate how changes in histone acetylation influence transcription of a defined gene construct. We show that inhibition of histone deacetylase 1 and 2 (HDAC1/2) specifically counteracts transcription suppression by preventing chromatin compaction and deacetylation of histone residues H4K5 and H4K8. Acetylation of these sites supports binding of the chromatin reader and transcription regulator BRD4. We also identify HDAC1 as the primary driver of transcription suppression and show that this activity is mediated through the Sin3 histone deacetylase complex. These findings highlight functional differences between HDAC1 and HDAC2, which are often considered to be functionally redundant, and provide additional molecular context for their activity.

Isoforms of the transcriptional cofactor SIN3 differentially regulate genes necessary for energy metabolism and cell survival.

The SIN3 scaffolding protein is a conserved transcriptional regulator known to fine-tune gene expression. In Drosophila, there are two major isoforms of SIN3, SIN3 220 and SIN3 187, which each assemble into multi-subunit histone modifying complexes. The isoforms have distinct developmental expression patterns and non-redundant functions. Gene regulatory network analyses indicate that both isoforms affect genes encoding proteins in pathways such as the cell cycle and cell morphogenesis. Interestingly, the SIN3 187 isoform uniquely regulates a subset of pathways including post-embryonic development, phosphate metabolism and apoptosis. Target genes in the phosphate metabolism pathway include nuclear-encoded mitochondrial genes coding for proteins responsible for oxidative phosphorylation. Here, we investigate the physiological effects of SIN3 isoforms on energy metabolism and cell survival. We find that ectopic expression of SIN3 187 represses expression of several nuclear-encoded mitochondrial genes affecting production of ATP and generation of reactive oxygen species (ROS). Forced expression of SIN3 187 also activates several pro-apoptotic and represses a few anti-apoptotic genes. In the SIN3 187 expressing cells, these gene expression patterns are accompanied with an increased sensitivity to paraquat-mediated oxidative stress. These findings indicate that SIN3 187 influences the regulation of mitochondrial function, apoptosis and oxidative stress response in ways that are dissimilar from SIN3 220. The data suggest that the distinct SIN3 histone modifying complexes are deployed in different cellular contexts to maintain cellular homeostasis.

The additional PRC2 subunit and Sin3 histone deacetylase complex are required for the normal distribution of H3K27me3 occupancy and transcriptional silencing in Magnaporthe oryzae.

Development in higher organisms requires proper gene silencing, partially achieved through trimethylation of lysine 27 on histone H3 (H3K27me3). However, how the normal distribution of this modification is established and maintained and how it affects gene expression remains unclear, especially in fungi. Polycomb repressive complex 2 (PRC2) catalyses H3K27me3 to assemble transcriptionally repressed facultative heterochromatin and is crucial in animals, plants, and fungi. Here, we report on the critical role of an additional PRC2 subunit in the normal distribution of H3K27me3 occupancy and the stable maintenance of gene repression in the rice fungal pathogen Magnaporthe oryzae. P55, identified as an additional PRC2 subunit, is physically associated with core subunits of PRC2 and is required for a complete level of H3K27me3 modification. Loss of P55 caused severe global defects in the normal distribution of H3K27me3 and transcriptional reprogramming on the H3K27me3-occupied genes. Furthermore, we found that the Sin3 histone deacetylase complex was required to sustain H3K27me3 occupancy and stably maintain gene repression by directly interacting with P55. Our results revealed a novel mechanism by which P55 and Sin3 participate in the normal distribution of facultative heterochromatic modifications and the stable maintenance of gene repression in eukaryotes.

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.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

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.

Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models.

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer's disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration.

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.

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.

Overlapping and non-overlapping roles of the class-I histone deacetylase-1 corepressors LET-418, SIN-3, and SPR-1 in Caenorhabditis elegans embryonic development.

BACKGROUND: Histone deacetylase (HDAC)-1, a Class-I HDAC family member, forms three types of complexes, the nucleosome remodeling deacetylase, Sin3, and CoREST complexes with the specific corepressor components chromodomain-helicase-DNA-binding protein 3 (Mi2/CHD-3), Sin3, and REST corepressor 1 (RCOR1), respectively, in humans. OBJECTIVE: To elucidate the functional relationships among the three transcriptional corepressors during embryogenesis. METHODS: The activities of HDA-1, LET-418, SIN-3, and SPR-1, the homologs of HDAC-1, Mi2, Sin3, and RCOR1 in Caenorhabditis elegans during embryogenesis were investigated through measurement of relative mRNA expression levels and embryonic lethality given either gene knockdown or deletion. Additionally, the terminal phenotypes of each knockdown and mutant embryo were observed using a differential-interference contrast microscope. Finally, the functional relationships among the three corepressors were examined through genetic interactions and transcriptome analyses. RESULTS: Here, we report that each of the corepressors LET-418, SIN-3, and SPR-1 are expressed and have essential roles in C. elegans embryonic development. Our terminal phenotype observations of single mutants further implied that LET-418, SIN-3, and SPR-1 play similar roles in promoting advancement to the middle and late embryonic stages. Combined analysis of genetic interactions and gene ontology of these corepressors indicate a prominent overlapping role among SIN-3, SPR-1, and LET-418 and between SIN-3 and SPR-1. CONCLUSION: Our findings suggest that the class-I HDAC-1 corepressors LET-418, SIN-3, and SPR-1 may cooperatively regulate the expression levels of some genes during C. elegans embryogenesis or may have some similar roles but functioning independently within a specific cell.

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.

αα-Hub domains and intrinsically disordered proteins: A decisive combo.

Hub proteins are central nodes in protein-protein interaction networks with critical importance to all living organisms. Recently, a new group of folded hub domains, the αα-hubs, was defined based on a shared αα-hairpin supersecondary structural foundation. The members PAH, RST, TAFH, NCBD, and HHD are found in large proteins such as Sin3, RCD1, TAF4, CBP, and harmonin, which organize disordered transcriptional regulators and membrane scaffolds in interactomes of importance to human diseases and plant quality. In this review, studies of structures, functions, and complexes across the αα-hubs are described and compared to provide a unified description of the group. This analysis expands the associated molecular concepts of "one domain-one binding site", motif-based ligand binding, and coupled folding and binding of intrinsically disordered ligands to additional concepts of importance to signal fidelity. These include context, motif reversibility, multivalency, complex heterogeneity, synergistic αα-hub:ligand folding, accessory binding sites, and supramodules. We propose that these multifaceted protein-protein interaction properties are made possible by the characteristics of the αα-hub fold, including supersite properties, dynamics, variable topologies, accessory helices, and malleability and abetted by adaptability of the disordered ligands. Critically, these features provide additional filters for specificity. With the presentations of new concepts, this review opens for new research questions addressing properties across the group, which are driven from concepts discovered in studies of the individual members. Combined, the members of the αα-hubs are ideal models for deconvoluting signal fidelity maintained by folded hubs and their interactions with intrinsically disordered ligands.

Haploinsufficiency of RREB1 causes a Noonan-like RASopathy via epigenetic reprogramming of RAS-MAPK pathway genes.

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.

Epigenetic marks and their relationship with BDNF in the brain of suicide victims.

BACKGROUND: Suicide is a common phenomenon affecting people of all ages. There is a strong relationship between suicidal ideation and depressive disorders. Increasing number of studies suggest that epigenetic modifications in certain brain areas are the main mechanism through which environmental and genetic factors interact with each other contributing to the development of mental disorders. To verify this hypothesis, some epigenetic marks: H3K9/14ac, HDAC2/3, H3K27me2 and Sin3a, as well as p-S421-MeCP2/MeCP2 were examined. On the other hand, BDNF protein level were studied. MATERIALS AND METHODS: Western blot analysis were performed in the frontal cortex (FCx) and hippocampus (HP) of suicide victims (n = 14) and non-suicidal controls (n = 8). The differences between groups and correlations between selected proteins were evaluated using Mann-Whitney U-test and Spearman's rank correlation. RESULTS: Statistically significant decrease in H3K9/14ac (FCx:↓~23%;HP:↓~33%) combined with increase in HDAC3 (FCx:↑~103%;HP:↑~85% in HP) protein levels in suicides compared to the controls was shown. These alterations were accompanied by an increase in H3K27me2 (FCx:↑45%;HP:↑~59%) and Sin3a (HP:↑50%) levels and decrease in p-S421-MeCP2/MeCP2 protein ratio (HP:↓~55%;FCx:↓~27%). Moreover, reduced BDNF protein level (FCx:↓~43%;HP:↓~28%) in suicides was observed. On the other hand, some significant correlations (e.g. between H3K9/14ac and HDAC2 or between BDNF and p-S421-MeCP2/MeCP2) were demonstrated. CONCLUSIONS: Our findings confirm the role of epigenetic component and BDNF protein in suicidal behavior. Lowered BDNF protein level in suicides is probably due to decrease in histone acetylation and increased level of factors related with deacetylation and methylation processes, including MeCP2 factor, which may operate bidirectionally (an activator or inhibitor of transcription).