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

BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a "BCL-XL-addiction" model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models

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

Insights into the recruitment of class IIa Histone Deacetylases (HDACs) to the SMRT/NCoR transcriptional repression complex

Class IIa histone deacetylases repress transcription of target genes. However their mechanism of action is poorly understood since they exhibit very low levels of deacetylase activity. The class IIa HDACs are associated with the SMRT / NCoR re pression complexes and this may, at least in part, a ccount for their repressive activity. However, the molecular mechanism of recruitment to co - repressor proteins has yet to be established. Here we show that a repeated peptide motif present in both SMRT and NCoR is sufficient to mediate specific interaction , with micromolar affinity, with all the class IIa HDACs (HDACs 4, 5, 7 & 9). Mutations in the consensus motif abrogate binding. Mutational analysis of HDAC4 suggests that the peptide interacts in the vicinity of the active site of the enzyme and requires the “closed” conformation of the zinc - binding loop on the surface of the enzyme. Together these findings represent the first insights into the molecular mechanism of recruitment of class IIa HDACs to the SMRT / NCoR repression complexes

Lysine-14 acetylation of histone H3 in chromatin confers resistance to the deacetylase and demethylase activities of an epigenetic silencing complex.

The core CoREST complex (LHC) contains histone deacetylase HDAC1 and histone demethylase LSD1 held together by the scaffold protein CoREST. Here, we analyze the purified LHC with modified peptide and reconstituted semisynthetic mononucleosome substrates. LHC demethylase activity toward methyl-Lys4 in histone H3 is strongly inhibited by H3 Lys14 acetylation, and this appears to be an intrinsic property of the LSD1 subunit. Moreover, the deacetylase selectivity of LHC unexpectedly shows a marked preference for H3 acetyl-Lys9 versus acetyl-Lys14 in nucleosome substrates but this selectivity is lost with isolated acetyl-Lys H3 protein. This diminished activity of LHC to Lys-14 deacetylation in nucleosomes is not merely due to steric accessibility based on the pattern of sensitivity of the LHC enzymatic complex to hydroxamic acid-mediated inhibition. Overall, these studies have revealed how a single Lys modification can confer a composite of resistance in chromatin to a key epigenetic enzyme complex involved in gene silencing

Backbone resonance assignment of the BCL6-BTB/POZ domain

BCL6 is a transcriptional repressor. Two domains of the protein, the N-terminal BTB-POZ domain and the RD2 domain are responsible for recruitment of co-repressor molecules and histone deacetylases. The BTB-POZ domain is found in a large and diverse range of proteins that play important roles in development, homeostasis and neoplasia. Crystal structures of several BTB-POZ domains, including BCL6 have been determined. The BTB-POZ domain of BCL6 not only mediates dimerisation but is also responsible for recruitment of co-repressors such as SMRT, NCOR and BCOR. Interestingly both SMRT and BCOR bind to the same site within the BCL6 BTB-POZ domain despite having very different primary sequences. Since both peptides and small molecules have been shown to bind to the co-repressor binding site it would suggest that the BTB_POZ domain is a suitable target for drug discovery. Here we report near complete backbone 15N, 13C and 1H assignments for the BTB-POZ domain of BCL6 to assist in the analysis of binding modes for small molecules

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells

The structure of the core NuRD 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

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

Structural and functional characterisation 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 aminoterminal region forms a tight dimerisation domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxyterminal 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