Multisite Substrate Recognition in Asf1-Dependent Acetylation of Histone H3 K56 by Rtt109.

Rtt109 is a unique histone acetyltransferase acetylating histone H3 lysine 56 (H3K56), a modification critical for DNA replication-coupled nucleosome assembly and genome stability. In cells, histone chaperone Asf1 is essential for H3K56 acetylation, yet the mechanisms for H3K56 specificity and Asf1 requirement remain unknown. We have determined the crystal structure of the Rtt109-Asf1-H3-H4 complex and found that unwinding of histone H3 αN, where K56 is normally located, and stabilization of the very C-terminal ß strand of histone H4 by Asf1 are prerequisites for H3K56 acetylation. Unexpectedly, an interaction between Rtt109 and the central helix of histone H3 is also required. The observed multiprotein, multisite substrate recognition mechanism among histone modification enzymes provides mechanistic understandings of Rtt109 and Asf1 in H3K56 acetylation, as well as valuable insights into substrate recognition by histone modification enzymes in general.

Topography of histone H3-H4 interaction with the Hat1-Hat2 acetyltransferase complex.

Chaperones influence histone conformation and intermolecular interaction in multiprotein complexes, and the structures obtained with full-length histones often provide more accurate and comprehensive views. Here, our structure of the Hat1-Hat2 acetyltransferase complex bound to Asf1-H3-H4 shows that the core domains of H3 and H4 are involved in binding Hat1 and Hat2, and the N-terminal tail of H3 makes extensive interaction with Hat2. These findings expand the knowledge about histone-protein interaction and implicate a function of Hat2/RbAp46/48, which is a versatile histone chaperone found in many chromatin-associated complexes, in the passing of histones between chaperones.

Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase.

Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification1-3. The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome4, counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1)5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6-9). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3-H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A-H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1.

Distinct histone H3-H4 binding modes of sNASP reveal the basis for cooperation and competition of histone chaperones.

Chromosomal duplication requires de novo assembly of nucleosomes from newly synthesized histones, and the process involves a dynamic network of interactions between histones and histone chaperones. sNASP and ASF1 are two major histone H3-H4 chaperones found in distinct and common complexes, yet how sNASP binds H3-H4 in the presence and absence of ASF1 remains unclear. Here we show that, in the presence of ASF1, sNASP principally recognizes a partially unfolded Nα region of histone H3, and in the absence of ASF1, an additional sNASP binding site becomes available in the core domain of the H3-H4 complex. Our study also implicates a critical role of the C-terminal tail of H4 in the transfer of H3-H4 between sNASP and ASF1 and the coiled-coil domain of sNASP in nucleosome assembly. These findings provide mechanistic insights into coordinated histone binding and transfer by histone chaperones.

Structural Basis for pri-miRNA Recognition by Drosha.

A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.

The postsynaptic density proteins Homer and Shank form a polymeric network structure.

The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of N-terminal EVH1 domains at each end of the coiled coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.

Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5.

Assembly of the spliceosomal small nuclear ribonucleoparticle (snRNP) core requires the participation of the multisubunit SMN (survival of motor neuron) complex, which contains SMN and several Gemin proteins. The SMN and Gemin2 subunits directly bind Sm proteins, and Gemin5 is required for snRNP biogenesis and has been implicated in snRNA recognition. The RNA sequence required for snRNP assembly includes the Sm site and an adjacent 3' stem-loop, but a precise understanding of Gemin5's RNA-binding specificity is lacking. Here we show that the N-terminal half of Gemin5, which is composed of two juxtaposed seven-bladed WD40 repeat domains, recognizes the Sm site. The tandem WD40 repeat domains are rigidly held together to form a contiguous RNA-binding surface. RNA-contacting residues are located mostly on loops between ß strands on the apical surface of the WD40 domains. Structural and biochemical analyses show that base-stacking interactions involving four aromatic residues and hydrogen bonding by a pair of arginines are crucial for specific recognition of the Sm sequence. We also show that an adenine immediately 5' to the Sm site is required for efficient binding and that Gemin5 can bind short RNA oligos in an alternative mode. Our results provide mechanistic understandings of Gemin5's snRNA-binding specificity as well as valuable insights into the molecular mechanism of RNA binding by WD40 repeat proteins in general.

Energy landscape quantifications of histone H3.3 recognition by chaperone DAXX reveal an uncoupled binding specificity and affinity.

Histone variant H3.3 differs from the canonical histone H3.1 by only five amino acids, yet its chaperone death domain-associated protein (DAXX) can specifically recognize H3.3 over H3.1, despite having a large DAXX-interacting surface on the H3.3-H4 heterodimer common to that on the H3.1-H4 complex. This observation gives rise to the question of, from the binding energy point view, how high binding specificity may be achieved with small differences of the overall binding energy for protein-protein interactions in general. Here we investigate the mechanism of coupling of binding specificity and affinity in protein-protein interactions using the DAXX-H3.3-H4 complex as a model. Using a multi-scale method, we found that the hydrophobic interactions between DAXX and the H3.3-specific region contributed to their initial binding process. And the structural flexibility of the interacting partners contributed to the binding affinity after their encounter. By quantifying the free energy landscape, we revealed that the interaction between the specific residues of H3.3 and DAXX decreased the encounter barrier height while the folding of H3.3-H4 and DAXX increased the depth of the free energy basin of the final binding state. The encounter barrier height, which is not coupled to the thermodynamic stability of the final binding state, had a marked effect on the initial binding rate of flexible histones and chaperones. Based on the energy landscape theory, we found that the intrinsic binding energy funnel of this uncoupled recognition process was affected by the structural flexibility and the flexibility modulated the degree of coupling between binding specificity and affinity. Our work offers a biophysical explanation of the specific recognition between the histones and their chaperones, and also extends the use of energy landscape theory for understanding molecular recognitions in general.

Structure specific DNA recognition by the SLX1-SLX4 endonuclease complex.

The SLX1-SLX4 structure-specific endonuclease complex is involved in processing diverse DNA damage intermediates, including resolution of Holliday junctions, collapse of stalled replication forks and removal of DNA flaps. The nuclease subunit SLX1 is inactive on its own, but become activated upon binding to SLX4 via its conserved C-terminal domain (CCD). Yet, how the SLX1-SLX4 complex recognizes specific DNA structure and chooses cleavage sites remains unknown. Here we show, through a combination of structural, biochemical and computational analyses, that the SAP domain of SLX4 is critical for efficient and accurate processing of 5'-flap DNA. It binds the minor groove of DNA about one turn away from the flap junction, and the 5'-flap is implicated in binding the core domain of SLX1. This binding mode accounts for specific recognition of 5'-flap DNA and specification of cleavage site by the SLX1-SLX4 complex.

Structural basis for allosteric, substrate-dependent stimulation of SIRT1 activity by resveratrol.

Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.

Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N.

Specific recognition of centromere-specific histone variant CENP-A-containing chromatin by CENP-N is an essential process in the assembly of the kinetochore complex at centromeres prior to mammalian cell division. However, the mechanisms of CENP-N recruitment to centromeres/kinetochores remain unknown. Here, we show that a CENP-A-specific RG loop (Arg80/Gly81) plays an essential and dual regulatory role in this process. The RG loop assists the formation of a compact "ladder-like" structure of CENP-A chromatin, concealing the loop and thus impairing its role in recruiting CENP-N. Upon G1/S-phase transition, however, centromeric chromatin switches from the compact to an open state, enabling the now exposed RG loop to recruit CENP-N prior to cell division. Our results provide the first insights into the mechanisms by which the recruitment of CENP-N is regulated by the structural transitions between compaction and relaxation of centromeric chromatin during the cell cycle.

Structural basis for hydroxymethylcytosine recognition by the SRA domain of UHRF2.

Methylated cytosine of CpG dinucleotides in vertebrates may be oxidized by Tet proteins, a process that can lead to DNA demethylation. The predominant oxidation product, 5-hydroxymethylcytosine (5hmC), has been implicated in embryogenesis, cell differentiation, and human diseases. Recently, the SRA domain of UHRF2 (UHRF2-SRA) has been reported to specifically recognize 5hmC, but how UHRF2 recognizes this modification is unclear. Here we report the structure of UHRF2-SRA in complex with a 5hmC-containing DNA. The structure reveals that the conformation of a phenylalanine allows the formation of an optimal 5hmC binding pocket, and a hydrogen bond between the hydroxyl group of 5hmC and UHRF2-SRA is critical for their preferential binding. Further structural and biochemical analyses unveiled the role of SRA domains as a versatile reader of modified DNA, and the knowledge should facilitate further understanding of the biological function of UHRF2 and the comprehension of DNA hydroxymethylation in general.

A higher-order configuration of the heterodimeric DOT1L-AF10 coiled-coil domains potentiates their leukemogenenic activity.

Chromosomal translocations of MLL1 (Mixed Lineage Leukemia 1) yield oncogenic chimeric proteins containing the N-terminal portion of MLL1 fused with distinct partners. The MLL1-AF10 fusion causes leukemia through recruiting the H3K79 histone methyltransferase DOT1L via AF10's octapeptide and leucine zipper (OM-LZ) motifs. Yet, the precise interaction sites in DOT1L, detailed interaction modes between AF10 and DOT1L, and the functional configuration of MLL1-AF10 in leukeomogenesis remain unknown. Through a combined approach of structural and functional analyses, we found that the LZ domain of AF10 interacts with the coiled-coil domains of DOT1L through a conserved binding mode and discovered that the C-terminal end of the LZ domain and the OM domain of AF10 mediate the formation of a DOT1L-AF10 octamer via tetramerization of the binary complex. We reveal that the oligomerization ability of the DOT1L-AF10 complex is essential for MLL1-AF10's leukemogenic function. These findings provide insights into the molecular basis of pathogenesis by MLL1 rearrangements.

Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex.

Post-translational modifications of histones are significant regulators of replication, transcription, and DNA repair. Particularly, newly synthesized histone H4 in H3/H4 heterodimers becomes acetylated on N-terminal lysine residues prior to its incorporation into chromatin. Previous studies have established that the histone acetyltransferase (HAT) complex Hat1p/Hat2p medicates this modification. However, the mechanism of how Hat1p/Hat2p recognizes and facilitates the enzymatic activities on the newly assembled H3/H4 heterodimer remains unknown. Furthermore, Hat2p is a WD40 repeat protein, which is found in many histone modifier complexes. However, how the WD40 repeat proteins facilitate enzymatic activities of histone modification enzymes is unclear. In this study, we first solved the high-resolution crystal structure of a Hat1p/Hat2p/CoA/H4 peptide complex and found that the H4 tail interacts with both Hat1p and Hat2p, by which substrate recruitment is facilitated. We further discovered that H3 N-terminal peptides can bind to the Hat2p WD40 domain and solved the structure of the Hat1p/Hat2p/CoA/H4/H3 peptide complex. Moreover, the interaction with Hat2p requires unmodified Arg2/Lys4 and Lys9 on the H3 tail, suggesting a novel model to specify the activity of Hat1p/Hat2p toward newly synthesized H3/H4 heterodimers. Together, our study demonstrated the substrate recognition mechanism by the Hat1p/Hat2p complex, which is critical for DNA replication and other chromatin remodeling processes.

Structural basis for allosteric stimulation of Sir2 activity by Sir4 binding.

The budding yeast Sir2 (silent information regulator 2) protein is the founding member of the sirtuin family of NAD-dependent histone/protein deacetylases. Its function in transcriptional silencing requires both the highly conserved catalytic domain and a poorly understood N-terminal regulatory domain (Sir2N). We determined the structure of Sir2 in complex with a fragment of Sir4, a component of the transcriptional silencing complex in Saccharomyces cerevisiae. The structure shows that Sir4 is anchored to Sir2N and contacts the interface between the Sir2N and the catalytic domains through a long loop. We discovered that the interaction between the Sir4 loop and the interdomain interface in Sir2 is critical for allosteric stimulation of the deacetylase activity of Sir2. These results bring to light the structure and function of the regulatory domain of Sir2, and the knowledge should be useful for understanding allosteric regulation of sirtuins in general.

Stella protein facilitates DNA demethylation by disrupting the chromatin association of the RING finger-type E3 ubiquitin ligase UHRF1.

Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger-type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1.

UBN1/2 of HIRA complex is responsible for recognition and deposition of H3.3 at cis-regulatory elements of genes in mouse ES cells.

BACKGROUND: H3.3 is an ancient and conserved H3 variant and plays essential roles in transcriptional regulation. HIRA complex, which is composed of HIRA, UBN1 or UBN2, and Cabin1, is a H3.3 specific chaperone complex. However, it still remains largely uncharacterized how HIRA complex specifically recognizes and deposits H3.3 to the chromatin, such as promoters and enhancers. RESULTS: In this study, we demonstrate that the UBN1 or UBN2 subunit is mainly responsible for specific recognition and direct binding of H3.3 by the HIRA complex. While the HIRA subunit can enhance the binding affinity of UBN1 toward H3.3, Cabin1 subunit cannot. We also demonstrate that both Ala87 and Gly90 residues of H3.3 are required and sufficient for the specific recognition and binding by UBN1. ChIP-seq studies reveal that two independent HIRA complexes (UBN1-HIRA and UBN2-HIRA) can cooperatively deposit H3.3 to cis-regulatory regions, including active promoters and active enhancers in mouse embryonic stem (mES) cells. Importantly, disruption of histone chaperone activities of UBN1 and UBN2 by FID/AAA mutation results in the defect of H3.3 deposition at promoters of developmental genes involved in neural differentiation, and subsequently causes the failure of activation of these genes during neural differentiation of mES cells. CONCLUSION: Together, our results provide novel insights into the mechanism by which the HIRA complex specifically recognizes and deposits H3.3 at promoters and enhancers of developmental genes, which plays a critical role in neural differentiation of mES cells.

Structure of a CENP-A-histone H4 heterodimer in complex with chaperone HJURP.

In higher eukaryotes, the centromere is epigenetically specified by the histone H3 variant Centromere Protein-A (CENP-A). Deposition of CENP-A to the centromere requires histone chaperone HJURP (Holliday junction recognition protein). The crystal structure of an HJURP-CENP-A-histone H4 complex shows that HJURP binds a CENP-A-H4 heterodimer. The C-terminal ß-sheet domain of HJURP caps the DNA-binding region of the histone heterodimer, preventing it from spontaneous association with DNA. Our analysis also revealed a novel site in CENP-A that distinguishes it from histone H3 in its ability to bind HJURP. These findings provide key information for specific recognition of CENP-A and mechanistic insights into the process of centromeric chromatin assembly.

Structure of Drosophila Oskar reveals a novel RNA binding protein.

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix-fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3'UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3'UTRs, and provide structural insights into a novel protein-RNA interaction motif involving a hydrolase-related domain.

Structural basis for methylarginine-dependent recognition of Aubergine by Tudor.

Piwi proteins are modified by symmetric dimethylation of arginine (sDMA), and the methylarginine-dependent interaction with Tudor domain proteins is critical for their functions in germline development. Cocrystal structures of an extended Tudor domain (eTud) of Drosophila Tudor with methylated peptides of Aubergine, a Piwi family protein, reveal that sDMA is recognized by an asparagine-gated aromatic cage. Furthermore, the unexpected Tudor-SN/p100 fold of eTud is important for sensing the position of sDMA. The structural information provides mechanistic insights into sDMA-dependent Piwi-Tudor interaction, and the recognition of sDMA by Tudor domains in general.