The topology of chromatin-binding domains in the NuRD deacetylase complex.

Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Class I HDACs share a common mechanism of regulation by inositol phosphates.

Class I histone deacetylases (HDAC1, HDAC2, and HDAC3) are recruited by cognate corepressor proteins into specific transcriptional repression complexes that target HDAC activity to chromatin resulting in chromatin condensation and transcriptional silencing. We previously reported the structure of HDAC3 in complex with the SMRT corepressor. This structure revealed the presence of inositol-tetraphosphate [Ins(1,4,5,6)P4] at the interface of the two proteins. It was previously unclear whether the role of Ins(1,4,5,6)P4 is to act as a structural cofactor or a regulator of HDAC3 activity. Here we report the structure of HDAC1 in complex with MTA1 from the NuRD complex. The ELM2-SANT domains from MTA1 wrap completely around HDAC1 occupying both sides of the active site such that the adjacent BAH domain is ideally positioned to recruit nucleosomes to the active site of the enzyme. Functional assays of both the HDAC1 and HDAC3 complexes reveal that Ins(1,4,5,6)P4 is a bona fide conserved regulator of class I HDAC complexes.

The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.

We previously identified the E3 ubiquitin ligase IDOL as a sterol-dependent regulator of the LDL receptor (LDLR). The molecular pathway underlying IDOL action, however, remains to be determined. Here we report the identification and biochemical and structural characterization of an E2-E3 ubiquitin ligase complex for LDLR degradation. We identified the UBE2D family (UBE2D1-4) as E2 partners for IDOL that support both autoubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1 Å crystal structure of the IDOL RING domain-UBE2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL-UBE2D1 interface also defined the stereochemical basis for the selectivity of IDOL for UBE2Ds over other E2 ligases. Structure-based mutations that inhibit IDOL dimerization or IDOL-UBE2D interaction block IDOL-dependent LDLR ubiquitination and degradation. Furthermore, expression of a dominant-negative UBE2D enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOL-UBE2D complex as an important determinant of LDLR activity, and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake.

Structural and functional characterization of a cell cycle associated HDAC1/2 complex reveals the structural basis for complex assembly and nucleosome targeting.

Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1. The amino-terminal region forms a tight dimerization domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxy-terminal domain of DNTTIP1 has a structure related to the SKI/SNO/DAC domain, despite lacking obvious sequence homology. We show that this domain in DNTTIP1 mediates interaction with both DNA and nucleosomes. Thus, DNTTIP1 acts as a dimeric chromatin binding module in the HDAC1:MIDEAS corepressor complex.

Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells.

Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2-deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.

Towards an understanding of the structure and function of MTA1.

Gene expression is controlled through the recruitment of large coregulator complexes to specific gene loci to regulate chromatin structure by modifying epigenetic marks on DNA and histones. Metastasis-associated protein 1 (MTA1) is an essential component of the nucleosome remodelling and deacetylase (NuRD) complex that acts as a scaffold protein to assemble enzymatic activity and nucleosome targeting proteins. MTA1 consists of four characterised domains, a number of interaction motifs, and regions that are predicted to be intrinsically disordered. The ELM2-SANT domain is one of the best-characterised regions of MTA1, which recruits histone deacetylase 1 (HDAC1) and activates the enzyme in the presence of inositol phosphate. MTA1 is highly upregulated in several types of aggressive tumours and is therefore a possible target for cancer therapy. In this review, we summarise the structure and function of the four domains of MTA1 and discuss the possible functions of less well-characterised regions of the protein.

Identification and structural analysis of type I collagen sites in complex with fibronectin fragments.

Collagen and fibronectin are major components of vertebrate extracellular matrices. Their association and distribution control the development and properties of diverse tissues, but thus far no structural information has been available for the complex formed. Here, we report binding of a peptide, derived from the alpha(1) chain of type I collagen, to the gelatin-binding domain of human fibronectin and present the crystal structure of this peptide in complex with the (8-9)FnI domain pair. Both gelatin-binding domain subfragments, (6)FnI(1-2)FnII(7)FnI and (8-9)FnI, bind the same specific sequence on D-period 4 of collagen I alpha(1), adjacent to the MMP-1 cleavage site. (8-9)FnI also binds the equivalent sequence of the alpha(2) chain. The collagen peptide adopts an antiparallel beta-strand conformation, similar to structures of proteins from pathogenic bacteria bound to FnI domains. Analysis of the type I collagen sequence suggests multiple putative fibronectin-binding sites compatible with our structural model. We demonstrate, by kinetic unfolding experiments, that the triple-helical collagen state is destabilized by (8-9)FnI. This finding suggests a role for fibronectin in collagen proteolysis and tissue remodeling.

Hydroxamic Acid-Modified Peptide Library Provides Insights into the Molecular Basis for the Substrate Selectivity of HDAC Corepressor Complexes.

Targeting the lysine deacetylase activity of class I histone deacetylases (HDACs) is potentially beneficial for the treatment of several diseases including human immunodeficiency virus (HIV) infection, Alzheimer's disease, and various cancers. It is therefore important to understand the function and mechanism of action of these enzymes. Class I HDACs act as catalytic components of seven large, multiprotein corepressor complexes. Different HDAC corepressor complexes have specific, nonredundant roles in the cell. It is likely that their specific functions are at least partly influenced by the substrate specificity of the complexes. To address this, we developed chemical tools to probe the specificity of HDAC complexes. We assessed a library of acetyl-lysine-containing substrate peptides and hydroxamic acid-containing inhibitor peptides against the full range of class I HDAC corepressor complexes. The results suggest that site-specific HDAC corepressor complex activity is driven in part by the recognition of the primary amino acid sequence surrounding a particular lysine position in the histone tail.

Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3.

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.

The streptococcal binding site in the gelatin-binding domain of fibronectin is consistent with a non-linear arrangement of modules.

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the (8)F1(9)F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to (8)F1(9)F1 and that UR binding to (8)F1 is likely to occur through anti-parallel ß-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem ß-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.

PROTAC-mediated degradation of class I histone deacetylase enzymes in corepressor complexes.

We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.

Diverse nucleosome Site-Selectivity among histone deacetylase complexes.

Histone acetylation regulates chromatin structure and gene expression and is removed by histone deacetylases (HDACs). HDACs are commonly found in various protein complexes to confer distinct cellular functions, but how the multi-subunit complexes influence deacetylase activities and site-selectivities in chromatin is poorly understood. Previously we reported the results of studies on the HDAC1 containing CoREST complex and acetylated nucleosome substrates which revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al, 2018). Here we analyze the enzymatic properties of five class I HDAC complexes: CoREST, NuRD, Sin3B, MiDAC and SMRT with site-specific acetylated nucleosome substrates. Our results demonstrate that these HDAC complexes show a wide variety of deacetylase rates in a site-selective manner. A Gly13 in the histone H3 tail is responsible for a sharp reduction in deacetylase activity of the CoREST complex for H3K14ac. These studies provide a framework for connecting enzymatic and biological functions of specific HDAC complexes.

The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure.

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.

Preparation of recombinant fibronectin fragments for functional and structural studies.

Fibronectin, an ubiquitous extracellular matrix (ECM) glycoprotein, plays a major role in fundamental biological processes such as cell adhesion and migration, maintenance of normal cell morphology, cytoskeletal organization, and cell differentiation. Fibronectin is constructed from three types of independently folding protein module (Fn1, Fn2, and Fn3) and is found as a Fibrillar network in the ECM where it interacts with other ECM components and provides anchorage sites for cell surface integrin receptors. The mosaic nature of fibronectin permits it to be analyzed by a "dissection" strategy, where protein fragments generated by recombinant expression in E. coli, P. pastoris, and human cell lines are employed in structural and functional investigations. We describe methods suitable for the production of various fibronectin fragments for study by a variety of techniques including crystallography and electron microscopy but special mention is made of methods suitable for the production of samples for NMR studies.

Synthesis of HDAC Substrate Peptidomimetic Inhibitors Using Fmoc Amino Acids Incorporating Zinc-Binding Groups.

Syntheses of Fmoc amino acids having zinc-binding groups were prepared and incorporated into substrate inhibitor H3K27 peptides using Fmoc/tBu solid-phase peptide synthesis (SPPS). Peptide 11, prepared using Fmoc-Asu(NHOtBu)-OH, is a potent inhibitor (IC50 = 390 nM) of the core NuRD corepressor complex (HDAC1-MTA1-RBBP4). The Fmoc amino acids have the potential to facilitate the rapid preparation of substrate peptidomimetic inhibitor (SPI) libraries in the search for selective HDAC inhibitors.

A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes.

Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, here we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/ß, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3 containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.

Targeting Class I Histone Deacetylases in a "Complex" Environment.

Histone deacetylase (HDAC) inhibitors are proven anticancer therapeutics and have potential in the treatment of many other diseases including HIV infection, Alzheimer's disease, and Friedreich's ataxia. A problem with the currently available HDAC inhibitors is that they have limited specificity and target multiple deacetylases. Designing isoform-selective inhibitors has proven challenging due to similarities in the structure and chemistry of HDAC active sites. However, the fact that HDACs 1, 2, and 3 are recruited to several large multi-subunit complexes, each with particular biological functions, raises the possibility of specifically inhibiting individual complexes. This may be assisted by recent structural and functional information about the assembly of these complexes. Here, we review the available structural information and discuss potential targeting strategies.

The structure of the core NuRD repression complex provides insights into its interaction with chromatin.

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin.

Insights into the activation mechanism of class I HDAC complexes by inositol phosphates.

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.