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.

A role for WDR5 in integrating threonine 11 phosphorylation to lysine 4 methylation on histone H3 during androgen signaling and in prostate cancer.

Upon androgen stimulation, PKN1-mediated histone H3 threonine 11 phosphorylation (H3T11P) promotes AR target gene activation. However, the underlying mechanism is not completely understood. Here, we show that WDR5, a subunit of the SET1/MLL complex, interacts with H3T11P, and this interaction facilitates the recruitment of the MLL1 complex and subsequent H3K4 tri-methylation (H3K4me3). Using ChIP-seq, we find that androgen stimulation results in a 6-fold increase in the number of H3T11P-marked regions and induces WDR5 colocalization to one third of H3T11P-enriched promoters, thus establishing a genome-wide relationship between H3T11P and recruitment of WDR5. Accordingly, PKN1 knockdown or chemical inhibition severely blocks WDR5 chromatin association and H3K4me3 on AR target genes. Finally, WDR5 is critical in prostate cancer cell proliferation and is hyperexpressed in human prostate cancers. Together, these results identify WDR5 as a critical epigenomic integrator of histone phosphorylation and methylation and as a major driver of androgen-dependent prostate cancer cell proliferation.

Inositol phosphates and core subunits of the Sin3L/Rpd3L histone deacetylase (HDAC) complex up-regulate deacetylase activity.

The constitutively nuclear histone deacetylases (HDACs) 1, 2, and 3 erase acetyl marks on acetyllysine residues, alter the landscape of histone modifications, and modulate chromatin structure and dynamics and thereby crucially regulate gene transcription in higher eukaryotes. Nuclear HDACs exist as at least six giant multiprotein complexes whose nonenzymatic subunits confer genome targeting specificity for these enzymes. The deacetylase activity of HDACs has been shown previously to be enhanced by inositol phosphates, which also bridge the catalytic domain in protein-protein interactions with SANT (Swi3, Ada2, N-Cor, and TFIIIB) domains in all HDAC complexes except those that contain the Sin3 transcriptional corepressors. Here, using purified recombinant proteins, coimmunoprecipitation and HDAC assays, and pulldown and NMR experiments, we show that HDAC1/2 deacetylase activity in one of the most ancient and evolutionarily conserved Sin3L/Rpd3L complexes is inducibly up-regulated by inositol phosphates but involves interactions with a zinc finger motif in the Sin3-associated protein 30 (SAP30) subunit that is structurally unrelated to SANT domains, indicating convergent evolution at the functional level. This implies that this mode of regulation has evolved independently multiple times and provides an evolutionary advantage. We also found that constitutive association with another core subunit, Rb-binding protein 4 chromatin-binding factor (RBBP4), further enhances deacetylase activity, implying both inducible and constitutive regulatory mechanisms within the same HDAC complex. Our results indicate that inositol phosphates stimulate HDAC activity and that the SAP30 zinc finger motif performs roles similar to that of the unrelated SANT domain in promoting the SAP30-HDAC1 interaction and enhancing HDAC activity.

Solution Nuclear Magnetic Resonance Studies of the Ligand-Binding Domain of an Orphan Nuclear Receptor Reveal a Dynamic Helix in the Ligand-Binding Pocket.

The ligand-binding domains (LBDs) of the NR5A subfamily of nuclear receptors activate transcription via ligand-dependent and ligand-independent mechanisms. The Drosophila Ftz-F1 receptor (NR5A3) belongs to the latter category, and its ligand independence is attributed to a short helical segment (α6) within the protein that resides in the canonical ligand-binding pocket (LBP) in the crystalline state. Here, we show that the α6 helix is dynamic in solution when Ftz-F1 is bound to the LxxLL motif of its cofactor Ftz, undergoing motions on the fast (picosecond to nanosecond) as well as slow (microsecond to millisecond) time scales. Motions on the slow time scale (∼10-3 s) appear to pervade throughout the domain, most prominently in the LBP and residues at or near the cofactor-binding site. We ascribe the fast time scale motions to a solvent-accessible conformation for the α6 helix akin to those described for its orthologs in higher organisms. We assign this conformation where the LBP is "open" to a lowly populated species, while the major conformer bears the properties of the crystal structure where the LBP is "closed". We propose that these conformational transitions could allow binding to small molecule ligands and/or play a role in dissociation of the cofactor from the binding site. Indeed, we show that the Ftz-F1 LBD can bind phospholipids, not unlike its orthologs. Our studies provide the first detailed insights into intrinsic motions occurring on a variety of time scales in a nuclear receptor LBD and reveal that potentially functionally significant motions pervade throughout the domain in solution, despite evidence to the contrary implied by the crystal structure.

Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex.

Acetylation is correlated with chromatin decondensation and transcriptional activation, but its regulation by histone deacetylase (HDAC)-bearing corepressor complexes is poorly understood. Here, we describe the mechanism of assembly of the mammalian Sin3L/Rpd3L complex facilitated by Sds3, a conserved subunit deemed critical for proper assembly. Sds3 engages a globular, helical region of the HDAC interaction domain (HID) of the scaffolding protein Sin3A through a bipartite motif comprising a helix and an adjacent extended segment. Sds3 dimerizes through not only one of the predicted coiled-coil motifs but also, the segment preceding it, forming an ∼ 150-Å-long antiparallel dimer. Contrary to previous findings in yeast, Sin3A rather than Sds3 functions in recruiting HDAC1 into the complex by engaging the latter through a highly conserved segment adjacent to the helical HID subdomain. In the resulting model for the ternary complex, the two copies of the HDACs are situated distally and dynamically because of a natively unstructured linker connecting the dimerization domain and the Sin3A interaction domain of Sds3; these features contrast with the static organization described previously for the NuRD (nucleosome remodeling and deacetylase) complex. The Sds3 linker features several conserved basic residues that could potentially maintain the complex on chromatin by nonspecific interactions with DNA after initial recruitment by sequence-specific DNA-binding repressors.

Molecular Basis for the Mechanism of Constitutive CBP/p300 Coactivator Recruitment by CRTC1-MAML2 and Its Implications in cAMP Signaling.

The cyclic AMP response element-binding protein (CREB) is a signal-dependent transcription factor that exerts its positive effects on gene transcription of a broad range of genes by recruiting coactivators, including CREB-binding protein (CBP), its paralog, p300, and the family of CRTC (CREB-regulated transcriptional coactivators) proteins. Whereas recruitment of CBP/p300 is dependent on CREB phosphorylation at Ser133, recruitment of CRTCs is not. Here we describe how both mechanisms could concurrently drive transcription of CREB targets in a subset of head and neck cancers featuring chromosomal translocations that fuse portions of CRTC1 and CRTC3 genes with that of the Mastermind-like transcriptional coactivator MAML2. We show that a peptide derived from transactivation domain 1 (TAD1) of MAML2 binds to the CBP KIX domain with micromolar affinity. An ∼20-residue segment within this peptide, conserved in MAML2 orthologs and paralogs, binds directly to a KIX surface previously shown to bind to MLL1. The 20-residue MAML2 segment shares sequence similarity with MLL1, especially at those positions in direct contact with KIX, and like MLL1, the segment is characterized by the presence of an ∼10-residue helix. Because CRTC1/3-MAML2 fusion proteins are constitutively nuclear, like CREB, our results suggest constitutive recruitment of CBP/p300 to CREB targets that could be further enhanced by signals that cause CREB Ser133 phosphorylation.

Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2.

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

Per-ARNT-Sim (PAS) Domains in Basic Helix-Loop-Helix (bHLH)-PAS Transcription Factors and Coactivators: Structures and Mechanisms.

PAS domains are ubiquitous in biology. They perform critically important roles in sensing and transducing a wide variety of environmental signals, and through their ability to bind small-molecule ligands, have emerged as targets for therapeutic intervention. Here, we discuss our current understanding of PAS domain structure and function in the context of basic helix-loop-helix (bHLH)-PAS transcription factors and coactivators. Unlike the bHLH-PAS domains of transcription factors, those of the steroid receptor coactivator (SRC) family are poorly characterized. Recent progress for this family and for the broader bHLH-PAS proteins suggest that these domains are ripe for deeper structural and functional studies.

Histone acetylation and deacetylation - Mechanistic insights from structural biology.

Histones are subject to a diverse array of post-translational modifications. Among them, lysine acetylation is not only the most pervasive and dynamic modification but also highly consequential for regulating gene transcription. Although enzymes responsible for the addition and removal of acetyl groups were discovered almost 30 years ago, high-resolution structures of the enzymes in the context of their native complexes are only now beginning to become available, thanks to revolutionary technologies in protein structure determination and prediction. Here, we will review our current understanding of the molecular mechanisms of acetylation and deacetylation engendered by chromatin-modifying complexes, compare and contrast shared features, and discuss some of the pressing questions for future studies.

Prostaglandins as Candidate Ligands for a Per-ARNT-Sim (PAS) Domain of Steroid Receptor Coactivator 1 (SRC1).

Steroid receptor coactivators (SRCs) comprise a family of three paralogous proteins commonly recruited by eukaryotic transcription factors. Each SRC harbors two tandem Per-ARNT-Sim (PAS) domains that are broadly distributed that bind small molecules and regulate interactions. Using computational docking, solution NMR, mass spectrometry, and molecular dynamics simulations, we show that the SRC1 PAS-B domain can bind to certain prostaglandins (PGs) either non-covalently to a surface that overlaps with the site used to engage transcription factors or covalently to a single, specific, conserved cysteine residue next to a solvent accessible hydrophobic pocket. This pocket is in proximity to the canonical transcription factor binding site, but on the opposite side of the domain, suggesting a potential mode of regulating transcriptional activator-coactivator interactions.

Cryo-EM structure of the Saccharomyces cerevisiae Rpd3L histone deacetylase complex.

The Rpd3L histone deacetylase (HDAC) complex is an ancient 12-subunit complex conserved in a broad range of eukaryotes that performs localized deacetylation at or near sites of recruitment by DNA-bound factors. Here we describe the cryo-EM structure of this prototypical HDAC complex that is characterized by as many as seven subunits performing scaffolding roles for the tight integration of the only catalytic subunit, Rpd3. The principal scaffolding protein, Sin3, along with Rpd3 and the histone chaperone, Ume1, are present in two copies, with each copy organized into separate lobes of an asymmetric dimeric molecular assembly. The active site of one Rpd3 is completely occluded by a leucine side chain of Rxt2, while the tips of the two lobes and the more peripherally associated subunits exhibit varying levels of flexibility and positional disorder. The structure reveals unexpected structural homology/analogy between unrelated subunits in the fungal and mammalian complexes and provides a foundation for deeper interrogations of structure, biology, and mechanism of these complexes, as well as for the discovery of HDAC complex-specific inhibitors.

Structure of the 30-kDa Sin3-associated protein (SAP30) in complex with the mammalian Sin3A corepressor and its role in nucleic acid binding.

The ∼2-megadalton evolutionarily conserved histone deacetylase-associated Rpd3L/Sin3L complex plays critical roles in altering the histone code and repressing transcription of a broad range of genes involved in many aspects of cellular physiology. Targeting of this complex to specific regions of the genome is presumed to rely on interactions involving one or more of at least 10 distinct subunits in the complex. Here we describe the solution structure of the complex formed by the interacting domains of two constitutively associated subunits, mSin3A and SAP30. The mSin3A paired amphipathic helix 3 (PAH3) domain in the complex adopts the left-handed four-helix bundle structure characteristic of PAH domains. The SAP30 Sin3 interaction domain (SID) binds to PAH3 via a tripartite structural motif, including a C-terminal helix that targets the canonical PAH hydrophobic cleft while two other helices and an N-terminal extension target a discrete surface formed largely by the PAH3 α2, α3, and α3' helices. The protein-protein interface is extensive (∼1400 Å(2)), accounting for the high affinity of the interaction and the constitutive association of the SAP30 subunit with the Rpd3L/Sin3L complex. We further show using NMR that the mSin3A PAH3-SAP30 SID complex can bind to nucleic acids, hinting at a role for a nucleolar localization sequence in the SID αA helix in targeting the Rpd3L/Sin3L complex for silencing ribosomal RNA genes.

A Novel Mechanism of Coactivator Recruitment by the Nurr1 Nuclear Receptor.

Nuclear receptors constitute one of the largest families of transcription factors that regulate genes in metazoans in response to small molecule ligands. Many receptors harbor two transactivation domains, one at each end of the protein sequence. Whereas the molecular mechanisms of transactivation mediated by the ligand-binding domain at the C-terminus of the protein are generally well established, the mechanism involving the N-terminal domain called activation function 1 (AF1) has remained elusive. Previous studies implicated the AF1 domain as a significant contributor towards the overall transcriptional activity of the NR4A family of nuclear receptors and suggested that the steroid receptor coactivators (SRCs) play an important role in this process. Here we show that a short segment within the AF1 domain of the NR4A receptor Nurr1 can directly engage with the SRC1 PAS-B domain. We also show that this segment forms a helix upon binding to a largely hydrophobic groove on PAS-B, overlapping with the surface engaged by the STAT6 transcription factor, suggesting that this mode of recruitment could be shared by diverse transcription factors including other nuclear receptors.

Solution structure of a novel zinc finger motif in the SAP30 polypeptide of the Sin3 corepressor complex and its potential role in nucleic acid recognition.

Giant chromatin-modifying complexes regulate gene transcription in eukaryotes by acting on chromatin substrates and 'setting' the histone code. The histone deacetylase (HDAC)-associated mammalian Sin3 corepressor complex regulates a wide variety of genes involved in all aspects of cellular physiology. The recruitment of the corepressor complex by transcription factors to specific regions of the genome is mediated by Sin3 as well as 10 distinct polypeptides that comprise the corepressor complex. Here we report the solution structure of a novel CCCH zinc finger (ZnF) motif in the SAP30 polypeptide, a key component of the corepressor complex. The structure represents a novel fold comprising two beta-strands and two alpha-helices with the zinc organizing center showing remote resemblance to the treble clef motif. In silico analysis of the structure revealed a highly conserved surface that is dominated by basic residues. NMR-based analysis of potential ligands for the SAP30 ZnF motif indicated a strong preference for nucleic acid substrates. We propose that the SAP30 ZnF functions as a double-stranded DNA-binding motif, thereby expanding the known functions of both SAP30 and the mammalian Sin3 corepressor complex. Our results also call into question the common assumption about the exclusion of DNA-binding core subunits within chromatin-modifying/remodeling complexes.

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.

HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.

Recruitment of the histone deacetylase (HDAC)-associated Sin3 corepressor is an obligatory step in many eukaryotic gene silencing pathways. Here we show that HBP1, a cell cycle inhibitor and regulator of differentiation, represses transcription in a HDAC/Sin3-dependent manner by targeting the mammalian Sin3A (mSin3A) PAH2 domain. HBP1 is unrelated to the Mad1 repressor for which high-resolution structures in complex with PAH2 have been described. We show that like Mad1, the HBP1 transrepression domain binds through a helical structure to the hydrophobic cleft of mSin3A PAH2. Notably, the HBP1 helix binds PAH2 in a reversed orientation relative to Mad1 and, equally unexpectedly, this is correlated with a chain reversal of the minimal Sin3 interaction motifs. These results not only provide insights into how multiple, unrelated transcription factors recruit the same coregulator, but also have implications for how sequence similarity searches are conducted.

Solution NMR studies of apo-mSin3A and -mSin3B reveal that the PAH1 and PAH2 domains are structurally independent.

The evolutionarily conserved mammalian Sin3 (mSin3) transcriptional corepressor interacts with a diverse array of transcription factors mainly through two PAH (paired amphipathic helix) domains located near the N terminus. Previous studies suggested the possibility of interdomain interactions involving the PAH domains. Here, we show that the domains are structurally independent and the properties of the individual domains, such as the conformational heterogeneity and the ability of mSin3A PAH2 to homodimerize, are preserved in constructs that span both PAH domains. Our results thus suggest that the N-terminal segments of the Sin3 proteins are broadly available for interactions with other proteins and that the PAH domains are organized into structurally independent modules. Our data also rule out any heterotypic association between the paralogous mSin3A and mSin3B proteins via interactions involving the mSin3A PAH2 domain.

Structural basis for ubiquitin recognition by SH3 domains.

The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function.

Structural basis for monoubiquitin recognition by the Ede1 UBA domain.

Monoubiquitination is a general mechanism for downregulating the activity of cell surface receptors by consigning these proteins for lysosome-mediated degradation through the endocytic pathway. The yeast Ede1 protein functions at the internalization step of endocytosis and binds monoubiquitinated proteins through a ubiquitin associated (UBA) domain. UBA domains are found in a broad range of cellular proteins but previous studies have suggested that the mode of ubiquitin recognition might not be universally conserved. Here we present the solution structure of the Ede1 UBA domain in complex with monoubiquitin. The Ede1 UBA domain forms a three-helix bundle structure and binds ubiquitin through a largely hydrophobic surface in a manner reminiscent of the Dsk2 UBA and the remotely homologous Cue2 CUE domains, for which high-resolution structures have been described. However, the interaction is dissimilar to the molecular models proposed for the hHR23A UBA domains bound to either monoubiquitin or Lys48-linked diubiquitin. Our mutational analyses of the Ede1 UBA domain-ubiquitin interaction reveal several key affinity determinants and, unexpectedly, a negative affinity determinant in the wild-type Ede1 protein, implying that high-affinity interactions may not be the sole criterion for optimal function of monoubiquitin-binding endocytic proteins.

Coupled unfolding and dimerization by the PAH2 domain of the mammalian Sin3A corepressor.

Coregulator recruitment by sequence-specific DNA binding transcription factors constitutes an important step in many eukaryotic transcription regulatory pathways. The Sin3 corepressor is an evolutionarily conserved protein and a key component of a large histone deacetylase-associated corepressor complex. The Sin3 corepressor contains four imperfect repeats of a domain called PAH (paired amphipathic helix) that serve as docking sites for a variety of sequence-specific DNA binding factors and coregulators. At least two closely related Sin3 proteins designated Sin3A and Sin3B have been described in higher organisms and although functional differences between these paralogs are only beginning to be appreciated, differences at the structural level are poorly understood. Here we analyze the conformational properties of the apo form of the mammalian Sin3A (mSin3A) PAH2 domain. At low micromolar concentrations, the domain is predominantly monomeric and folded in a conformation similar to those found in complexes with the Mad1 and HBP1 repressors. Unexpectedly, at higher concentrations, the domain dimerizes with concomitant population of a partially unfolded conformer. These findings are in contrast to those reported for the mSin3B PAH2 domain and may have implications for the manner in which these paralogous domains interact with their targets.