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.

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.

The neuronal transcription factor Myt1L interacts via a conserved motif with the PAH1 domain of Sin3 to recruit the Sin3L/Rpd3L histone deacetylase complex.

The Sin3L/Rpd3L histone deacetylase (HDAC) complex is one of six major HDAC complexes in the nucleus, and its recruitment by promoter-bound transcription factors is an important step in many gene transcription regulatory pathways. Here, we investigate how the Myt1L zinc finger transcription factor, important for neuronal differentiation and the maintenance of neuronal identity, recruits this complex at the molecular level. We show that Myt1L, through a highly conserved segment shared with its paralogs, interacts directly and specifically with the Sin3 PAH1 domain, binding principally to the canonical hydrophobic cleft found in paired amphipathic helix domain (PAH) domains. Our findings are relevant not only for other members of the Myt family but also for enhancing our understanding of the rules of protein-protein interactions involving Sin3 PAH domains.

ARID4B is critical for mouse embryonic stem cell differentiation towards mesoderm and endoderm, linking epigenetics to pluripotency exit.

Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.

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.

Same agent, different messages: insight into transcriptional regulation by SIN3 isoforms.

SIN3 is a global transcriptional coregulator that governs expression of a large repertoire of gene targets. It is an important player in gene regulation, which can repress or activate diverse gene targets in a context-dependent manner. SIN3 is required for several vital biological processes such as cell proliferation, energy metabolism, organ development, and cellular senescence. The functional flexibility of SIN3 arises from its ability to interact with a large variety of partners through protein interaction domains that are conserved across species, ranging from yeast to mammals. Several isoforms of SIN3 are present in these different species that can perform common and specialized functions through interactions with distinct enzymes and DNA-binding partners. Although SIN3 has been well studied due to its wide-ranging functions and highly conserved interaction domains, precise roles of individual SIN3 isoforms have received less attention. In this review, we discuss the differences in structure and function of distinct SIN3 isoforms and provide possible avenues to understand the complete picture of regulation by SIN3.

Solution NMR Studies of an Alternative Mode of Sin3 Engagement by the Sds3 Subunit in the Histone Deacetylase-Associated Sin3L/Rpd3L Corepressor Complex.

The Sds3 transcriptional corepressor facilitates the assembly of the 1- to 2-MDa histone deacetylase-associated Sin3L/Rpd3L complex by providing a crucial homodimerization activity. Sds3 engages the scaffolding protein Sin3A, via a bipartite motif within the Sin3 interaction domain (SID) comprising a helix and an extended segment. Here, we show that the SID samples two discrete, substantially populated conformations with lifetimes in the tens of milliseconds range. The two conformations differ via a translation of the main chain and the corresponding side chains in the 5- to 7-Å range. Given the close proximity of the SID to other functional motifs in Sds3 at the sequence level, the conformational exchange has the potential to regulate these activities.

Differential coupling of KLF10 to Sin3-HDAC and PCAF regulates the inducibility of the FOXP3 gene.

Inducible gene expression, which requires chromatin remodeling on gene promoters, underlies the epigenetically inherited differentiation program of most immune cells. However, chromatin-mediated mechanisms that underlie these events in T regulatory cells remain to be fully characterized. Here, we report that inducibility of FOXP3, a key transcription factor for the development of T regulatory cells, depends upon Kruppel-like factor 10 (KLF10) interacting with two antagonistic histone-modifying systems. We utilized chromatin immunoprecipitation, genome-integrated reporter assays, and functional domain KLF10 mutant proteins, to characterize reciprocal interactions between this transcription factor and either the Sin3-histone deacetylase complex or the histone acetyltransferase, p300/CBP-associated factor (PCAF). We characterize a Sin3-interacting repressor domain on the NH2 terminus of KLF10, which works to limit the activating function of this transcription factor. Indeed, inactivation of this Sin3-interacting domain renders KLF10 able to physically associate with PCAF as to induce FOXP3 gene transcription. We show that this biochemical data derived from studying our genome-integrated reporter cell system are recapitulated in primary murine lymphocytes. Collectively, these results advance our understanding of how a single transcription factor, namely KLF10, functions as a toggle to integrate antagonistic signals regulating FOXP3 and, thus, immune activation.

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.

Multiple histone deacetylases are recruited by corepressor Sin3 and contribute to gene repression mediated by Opi1 regulator of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae.

Yeast genes of phospholipid biosynthesis are negatively regulated by repressor protein Opi1 when precursor molecules inositol and choline (IC) are available. Opi1-triggered gene repression is mediated by recruitment of the Sin3 corepressor complex. In this study, we systematically investigated the regulatory contribution of subunits of Sin3 complexes and identified Pho23 as important for IC-dependent gene repression. Two non-overlapping regions within Pho23 mediate its direct interaction with Sin3. Previous work has shown that Sin3 recruits the histone deacetylase (HDAC) Rpd3 to execute gene repression. While deletion of SIN3 strongly alleviates gene repression by IC, an rpd3 null mutant shows almost normal regulation. We thus hypothesized that various HDACs may contribute to Sin3-mediated repression of IC-regulated genes. Indeed, a triple mutant lacking HDACs, Rpd3, Hda1 and Hos1, could phenocopy a sin3 single mutant. We show that these proteins are able to contact Sin3 in vitro and in vivo and mapped three distinct HDAC interaction domains, designated HID1, HID2 and HID3. HID3, which is identical to the previously described structural motif PAH4 (paired amphipathic helix), can bind all HDACs tested. Chromatin immunoprecipitation studies finally confirmed that Hda1 and Hos1 are recruited to promoters of phospholipid biosynthetic genes INO1 and CHO2.

Structural basis for molecular interactions involving MRG domains: implications in chromatin biology.

MRG15 is a member of the mortality family of transcription factors that targets a wide variety of multiprotein complexes involved in transcription regulation, DNA repair, and alternative splicing to chromatin. The structure of the apo-MRG15 MRG domain implicated in interactions with diverse proteins has been described, but not in complex with any of its targets. Here, we structurally and functionally characterize the interaction between MRG15 and Pf1, two constitutively associated subunits of the histone deacetylase-associated Rpd3S/Sin3S corepressor complex. The MRG domain adopts a structure reminiscent of the apo state, whereas the Pf1 MRG-binding domain engages two discrete hydrophobic surfaces on the MRG domain via a bipartite motif comprising an α-helix and a segment in an extended conformation, both of which are critical for high-affinity interactions. Multiple MRG15 interactors share an FxLP motif in the extended segment, but equivalent sequence/helical motifs are not readily evident, implying potential diversity in MRG-recognition mechanisms.

Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.

Histone deacetylation constitutes an important mechanism for silencing genes. The histone-deacetylase-associated mammalian Rpd3S/Sin3S corepressor complex plays key roles in repressing aberrant gene transcription from cryptic transcription initiation sites and in mitigating RNA polymerase II progression in intragenic regions of actively transcribed genes. The Sin3 corepressor functions as a molecular adaptor linking histone deacetylases on the one hand, with the chromatin targeting subunits Pf1 and MRG15 on the other. Pf1 also functions as an adaptor by interacting with MRG15 and engaging in multivalent interactions with Sin3 targeting among other domains the two N-terminal paired amphipathic helix (PAH) domains that serve as sites of interaction with sequence-specific DNA-binding transcription factors. Here, we structurally and functionally evaluate the interaction between the PAH2 domain of mSin3A and the Sin3 interaction domain 1 (SID1) motif of Pf1 and find the structural aspects to be reminiscent of the interaction between the Mad1/Mxd1 transcription factor and Sin3. Pf1 residues within a highly conserved sequence motif immediately C-terminal to SID1 appear not to be important for the interaction with Sin3 PAH2. Unexpectedly, the MRG15 subunit competes, rather than collaborates, with Sin3 for the Pf1 segment encompassing the two conserved motifs, implying competition between two subunits for another subunit of the same chromatin-modifying complex.

PAH-domain-specific interactions of the Arabidopsis transcription coregulator SIN3-LIKE1 (SNL1) with telomere-binding protein 1 and ALWAYS EARLY2 Myb-DNA binding factors.

The eukaryotic SIN3 protein is the central component of the evolutionarily conserved multisubunit SIN3 complex that has roles in regulating gene expression and genome stability. Here we characterise the structure of the SIN3 protein in higher plants through the analysis of SNL1 (SIN3-LIKE1), SNL2, SNL3, SNL4, SNL5 and SNL6, a family of six SIN3 homologues in Arabidopsis thaliana. In an Arabidopsis-protoplast beta-glucuronidase reporter gene assay, as well as in a heterologous yeast repression assay, full-length SNL1 was shown to repress transcription in a histone-deacetylase-dependent manner, demonstrating the conserved nature of SIN3 function. Yeast two-hybrid screening identified a number of DNA binding proteins each containing a single Myb domain that included the Arabidopsis ALWAYS EARLY proteins AtALY2 and AtALY3, and two telomere binding proteins AtTBP1 and AtTRP2/TRFL1 as SNL1 partners, suggesting potential functions for SNL1 in development and telomere maintenance. The interaction with telomere-binding protein 1 was found to be mediated through the well-defined paired amphipathic helix domain PAH2. In contrast, the AtALY2 interaction was mediated through the PAH3 domain of SNL1, which is structurally distinct from PAH1 and PAH2, suggesting that evolution of this domain to a more novel structural motif has occurred. These findings support a diverse role of SNL1 in the regulation of transcription and genome stability.