Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

Histone methyltransferase TXR1 is required for both H3 and H3.3 lysine 27 methylation in the well-known ciliated protist <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

DNA replication elongation is tightly controlled by histone-modifying enzymes. Our previous studies showed that the histone methytransferase TXR1 (Tetrahymena Trithorax related protein 1) specifically catalyzes H3K27 monomethylation and affects DNA replication elongation in Tetrahymena thermophila. In this study, we investigated whether TXR1 has a substrate preference to the canonical H3 over the replacement variant H3.3. We demonstrated by histone mutagenesis that K27Q mutation in H3.3 further aggravated the replication stress phenotype of K27Q mutation in canonical H3, supporting H3.3 as a physiologically relevant substrate of TXR1. This result is in apparent contrast to the strong preference for canonical H3 recently reported in Arabidopsis homologues ATXR5 and ATXR6, and further corroborates the role of TXR1 in DNA replication.     

Human testis–specific Y-encoded protein-like protein 5 is a histone H3/H4-specific chaperone that facilitates histone deposition in vitro

DNA and core histones are hierarchically packaged into a complex organization called chromatin. The nucleosome assembly protein (NAP) family of histone chaperones is involved in the deposition of histone complexes H2A/H2B and H3/H4 onto DNA and prevents nonspecific aggregation of histones. Testis-specific Y-encoded protein (TSPY)–like protein 5 (TSPYL5) is a member of the TSPY-like protein family, which has been previously reported to interact with ubiquitin-specific protease USP7 and regulate cell proliferation and is thus implicated in various cancers, but its interaction with chromatin has not been investigated. In this study, we characterized the chromatin association of TSPYL5 and found that it preferentially binds histone H3/H4 via its C-terminal NAP-like domain both in vitro and ex vivo. We identified the critical residues involved in the TSPYL5–H3/H4 interaction and further quantified the binding affinity of TSPYL5 toward H3/H4 using biolayer interferometry. We then determined the binding stoichiometry of the TSPYL5–H3/H4 complex in vitro using a chemical cross-linking assay and size-exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that a TSPYL5 dimer binds to either two histone H3/H4 dimers or a single tetramer. We further demonstrated that TSPYL5 has a specific affinity toward longer DNA fragments and that the same histone-binding residues are also critically involved in its DNA binding. Finally, employing histone deposition and supercoiling assays, we confirmed that TSPYL5 is a histone chaperone responsible for histone H3/H4 deposition and nucleosome assembly. We conclude that TSPYL5 is likely a new member of the NAP histone chaperone family.     

Structural insights for MPP8 chromodomain interaction with histone H3 lysine 9: potential effect of phosphorylation on methyl-lysine binding

M-phase phosphoprotein 8 (MPP8) harbors an N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/H3K9me2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine-9-containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80 and Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side-chain hydroxyl oxygen of Tyr83 suggests that the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).     

The bromodomain of Gcn5p interacts in vitro with specific residues in the N terminus of histone H4

Whereas the histone acetyltransferase activity of yeast Gcn5p has been widely studied, its structural interactions with the histones and the role of the carboxy-terminal bromodomain are still unclear. Using a glutathione S-transferase pull down assay we show that Gcn5p binds the amino-terminal tails of histones H3 and H4, but not H2A and H2B. The deletion of bromodomain abolishes this interaction and bromodomain alone is able to interact with the H3 and H4 N termini. The amino acid residues of the H4 N terminus involved in the binding with Gcn5p have been studied by site-directed mutagenesis. The substitution of amino acid residues R19 or R23 of the H4 N terminus with a glutamine (Q) abolishes the interaction with Gcn5p and the bromodomain. These residues differ from those known to be acetylated or to be involved in binding the SIR proteins. This evidence and the known dispensability of the bromodomain for Gcn5p acetyltransferase activity suggest a new structural role for the highly evolutionary conserved bromodomain.     

Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.     

Molecular basis for Gcn5/PCAF histone acetyltransferase selectivity for histone and nonhistone substrates

Histone acetyltransferase (HAT) proteins often exhibit a high degree of specificity for lysine-bearing protein substrates. We have previously reported on the structure of the Tetrahymena Gcn5 HAT protein (tGcn5) bound to its preferred histone H3 substrate, revealing the mode of substrate binding by the Gcn5/PCAF family of HAT proteins. Interestingly, the Gcn5/PCAF HAT family has a remarkable ability to acetylate lysine residues within diverse cognate sites such as those found around lysines 14, 8, and 320 of histones H3, H4, and p53, respectively. To investigate the molecular basis for this, we now report on the crystal structures of tGcn5 bound to 19-residue histone H4 and p53 peptides. A comparison of these structures with tGcn5 bound to histone H3 reveals that the Gcn5/PCAF HATs can accommodate divergent substrates by utilizing analogous interactions with the lysine target and two C-terminal residues with a related chemical nature, suggesting that these interactions play a general role in Gcn5/PCAF substrate binding selectivity. In contrast, while the histone H3 complex shows extensive interactions with tGcn5 and peptide residues N-terminal to the target lysine, the corresponding residues in histone H4 and p53 are disordered, suggesting that the N-terminal substrate region plays an important role in the enhanced affinity of the Gcn5/PCAF HAT proteins for histone H3. Together, these studies provide a framework for understanding the substrate selectivity of HAT proteins.     

Loss of a DNA binding site within the tail of prelamin A contributes to altered heterochromatin anchorage by progerin

Mutations in the lamin A/C (LMNA) gene that cause Hutchinson-Gilford progeria syndrome (HGPS) lead to expression of a protein called progerin with 50 amino acids deleted from the tail of prelamin A. In cells from patients with HGPS, both the amount and distribution of heterochromatin are altered. We designed in vitro assays to ask whether such alterations might reflect changes in chromatin, DNA and/or histone binding properties of progerin compared to wild-type lamin C-terminal tails. We show that progerin tail has a reduced DNA/chromatin binding capacity and modified trimethylated H3K27 binding pattern, offering a molecular mechanism for heterochromatin alterations related to HGPS.

Structured summary

MINT-7893924, MINT-7893941, MINT-7893990, MINT-7894005, MINT-7894023, MINT-7894038: H3 (uniprotkb:Q71DI3) binds (MI:0407) to LaminA (uniprotkb:P02545) by surface plasmon resonance (MI:0107)MINT-7893957, MINT-7893974, MINT-7894055: H3 (uniprotkb:Q71DI3) binds (MI:0407) to progerin (uniprotkb:Q6UYC3) by surface plasmon resonance (MI:0107)     

Insights into the Specificity of Lysine Acetyltransferases

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. Here we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.     

The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9

Proteins containing the evolutionarily conserved SET domain are involved in regulation of eukaryotic gene expression and chromatin structure through their histone lysine methyltransferase (HMTase) activity. The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization. In Arabidopsis there are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far. A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.     

A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties

A comparison of the globular domain sequences of the somatic H1d and testis-specific H1t revealed a single substitution of lysine 52 in H1d to glutamine 54 in H1t, which is one of the three crucial residues within the second DNA binding site. The globular domains of both histones were modeled using the crystal structure of chicken GH5 as a template and was also docked onto the nucleosome structure. The glutamine residue in histone H1t forms a hydrogen bond with main chain carbonyl of methionine-52 (in H1t) and is spatially oriented away from the nucleosome dyad axis. A consequence of this change was a lower affinity of recombinant histone H1t towards Four-way junction DNA and reconstituted 5S mononucleosomes. When Gln-54 in Histone H1t was mutated to lysine, its binding affinity towards DNA substrates was comparable to that of histone H1d. The differential binding of histones H1d and H1t towards reconstituted mononucleosomes was also reflected in the chromatosome-stop assay.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.