Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated.     

The recognition specificity of the CHD1 chromodomain with modified histone H3 peptides

Histone tail peptides comprise the flexible portion of chromatin, the substance which serves as the packaging for the eukaryotic genome. According to the histone code hypothesis, reader protein domains (chromodomains) can recognize modifications of amino acid residues within these peptides, regulating the expression of genes. We have performed simulations on models of chromodomain helicase DNA-binding protein 1 complexed with a variety of histone H3 modifications. Binding free energies for both the overall complexes and the individual residues within the protein and peptides were computed with molecular mechanics-generalized Born surface area. The simulation results agree well with experimental data and identify several chromodomain helicase DNA-binding protein 1 residues that play key roles in the interaction with each of the H3 modifications. We identified one class of protein residues that bind to H3 in all of the complexes (generally interacting hydrophobically), and a second class of residues that bind only to particular H3 modifications (generally interacting electrostatically). Additionally, we found that modifications of H3R2 and H3T3 have a dominant effect on the binding affinity; methylation of H3K4 has little effect on the interaction strength when H3R2 or H3T3 is modified. Our findings with regard to the specificity shown by the latter class of protein residues in their binding affinity to certain modifications of H3 support the histone code hypothesis.     

Recruitment of histone modifications by USF proteins at a vertebrate barrier element

The chicken beta-globin 5'HS4 insulator element acts as a barrier to the encroachment of chromosomal silencing. Endogenous 5'HS4 sequences are highly enriched with histone acetylation and H3K4 methylation regardless of neighboring gene expression. We report here that 5'HS4 elements recruit these histone modifications when protecting a reporter transgene from chromosomal silencing. Deletion studies identified a single protein binding site within 5'HS4, footprint IV, that is necessary for the recruitment of histone modifications and for barrier activity. We have determined that USF proteins bind to footprint IV. USF1 is present in complexes with histone modifying enzymes in cell extracts, and these enzymes specifically interact with the endogenous 5'HS4 element. Knockdown of USF1 expression leads to a loss of histone modification recruitment and subsequent encroachment of H3K9 methylation. We propose that barrier activity requires the constitutive recruitment of H3K4 methylation and histone acetylation at multiple residues to counteract the propagation of condensed chromatin structures.     

The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus.     

Distinct domains in high mobility group N variants modulate specific chromatin modifications

We have demonstrated that levels of specific modification in histone H3 are modulated by members of the nucleosome-binding high mobility group N (HMGN) protein family in a variant-specific manner. HMGN1 (but not HMGN2) inhibits the phosphorylation of both H3S10 and H3S28, whereas HMGN2 enhances H3K14 acetylation more robustly than HMGN1. Two HMGN domains are necessary for modulating chromatin modifications, a non-modification-specific domain necessary for chromatin binding and a modification-specific domain localized in the C terminus of the HMGNs. Thus, chromatin-binding structural proteins such as HMGNs affect the levels of specific chromatin modifications and therefore may play a role in epigenetic regulation.     

Nucleosome-interacting proteins regulated by DNA and histone methylation

Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify "crosstalk" between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding "profile" for proteins regulated by DNA and histone methylation.     

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.     

植物PHD结构域蛋白的结构与功能特性

植物同源结构域(plant homeodomain,PHD)是锌指结构域家族的一类转录调控因子,其最主要的功能是可以识别各种组蛋白修饰密码,包括组蛋白甲基化和乙酰化等;此外PHD结构域还可以与DNA结合.含有PHD结构域的蛋白,或者本身具有组蛋白修饰酶活性,或者可以与各类组蛋白修饰酶相互作用,还有部分与DNA甲基化相关...     

Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.     

Structural basis of the chromodomain of Cbx3 bound to methylated peptides from histone h1 and G9a

Background

HP1 proteins are highly conserved heterochromatin proteins, which have been identified to be structural adapters assembling a variety of macromolecular complexes involved in regulation of gene expression, chromatin remodeling and heterochromatin formation. Much evidence shows that HP1 proteins interact with numerous proteins including methylated histones, histone methyltransferases and so on. Cbx3 is one of the paralogues of HP1 proteins, which has been reported to specifically recognize trimethylated histone H3K9 mark, and a consensus binding motif has been defined for the Cbx3 chromodomain.

Methodology/Principal Findings

Here, we found that the Cbx3 chromodomain can bind to H1K26me2 and G9aK185me3 with comparable binding affinities compared to H3K9me3. We also determined the crystal structures of the human Cbx3 chromodomain in complex with dimethylated histone H1K26 and trimethylated G9aK185 peptides, respectively. The complex structures unveil that the Cbx3 chromodomain specifically bind methylated histone H1K26 and G9aK185 through a conserved mechanism.

Conclusions/Significance

The Cbx3 chromodomain binds with comparable affinities to all of the methylated H3K9, H1K26 and G9aK185 peptides. It is suggested that Cbx3 may regulate gene expression via recognizing both histones and non-histone proteins.     

Recognition of unmodified histone H3 by the first PHD finger of bromodomain-PHD finger protein 2 provides insights into the regulation of histone acetyltransferases monocytic leukemic zinc-finger protein (MOZ) and MOZ-related factor (MORF)

MOZ (monocytic leukemic zinc-finger protein) and MORF (MOZ-related factor) are histone acetyltransferases important for HOX gene expression as well as embryo and postnatal development. They form complexes with other regulatory subunits through the scaffold proteins BRPF1/2/3 (bromodomain-PHD (plant homeodomain) finger proteins 1, 2, or 3). BRPF proteins have multiple domains, including two PHD fingers, for potential interactions with histones. Here we show that the first PHD finger of BRPF2 specifically recognizes the N-terminal tail of unmodified histone H3 (unH3) and report the solution structures of this PHD finger both free and in complex with the unH3 peptide. Structural analysis revealed that the unH3 peptide forms a third antiparallel β-strand that pairs with the PHD1 two-stranded antiparallel β-sheet. The binding specificity was determined primarily through the recognition of arginine 2 and lysine 4 of the unH3 by conserved aspartic acids of PHD1 and of threonine 6 of the unH3 by a conserved asparagine. Isothermal titration calorimetry and NMR assays showed that post-translational modifications such as H3R2me2as, H3T3ph, H3K4me, H3K4ac, and H3T6ph antagonized the interaction between histone H3 and PHD1. Furthermore, histone binding by PHD1 was important for BRPF2 to localize to the HOXA9 locus in vivo. PHD1 is highly conserved in yeast NuA3 and other histone acetyltransferase complexes, so the results reported here also shed light on the function and regulation of these complexes.     

Engineering human JMJD2A tudor domains for an improved understanding of histone peptide recognition

JMJD2A is a histone lysine demethylase which recognizes and demethylates H3K9me3 and H3K36me3 residues and is overexpressed in various cancers. It utilizes a tandem tudor domain to facilitate its own recruitment to histone sites, recognizing various di- and tri-methyl lysine residues with moderate affinity. In this study, we successfully engineered the tudor domain of JMJD2A to specifically bind to H4K20me3 with a 20-fold increase of affinity and improved selectivity. To reveal the molecular basis, we performed molecular dynamics and free energy decomposition analysis on the human JMJD2A tandem tudor domains bound to H4K20me2, H4K20me3, and H3K23me3 peptides to uncover the residues and conformational changes important for the enhanced binding affinity and selectivity toward H4K20me2/3. These analyses revealed new insights into understanding chromatin reader domains recognizing histone modifications and improving binding affinity and selectivity of these domains. Furthermore, we showed that the tight binding of JMJD2A to H4K20me2/3 is not sufficient to improve the efficiency of CRISPR-CAS9 mediated homology directed repair (HDR), suggesting a complicated relationship between JMJD2A and the DNA damage response beyond binding affinity toward the H4K20me2/3 mark.     

植物组蛋白赖氨酸甲基化建立过程及其遗传性研究进展

表观遗传学主要包括DNA甲基化、组蛋白修饰和非编码RNA,组蛋白甲基化作为组蛋白修饰中的一种重要修饰,在植物体的发育和环境适应中发挥着重要作用。组蛋白甲基化主要发生在赖氨酸残基上,同时根据不同的赖氨酸位点和每个赖氨酸位点甲基化程度的不同,形成了不同的赖氨酸甲基化修饰。根据对基因的不同功能,通常将组蛋白赖氨酸甲基化修饰分为2大类:(1)能够促进基因表达的,如H3K4me3和H3K36me3;(2)能够抑制基因表达的,如H3K9me2和H3K27me3。不同的组蛋白赖氨酸甲基化去甲基化过程需要相应的阅读(reader)、书写(writer)和擦除(eraser)3种蛋白。同时,组蛋白赖氨酸甲基化的遗传性质目前还不是很清楚。综述了植物中组蛋白赖氨酸甲基化建立与去除过程,以及对组蛋白赖氨酸甲基化可遗传性的探讨。     

Structural cooperativity in histone H3 tail modifications

Post-translational modifications of histone H3 tails have crucial roles in regulation of cellular processes. There is cross-regulation between the modifications of K4, K9, and K14 residues. The modifications on these residues drastically promote or inhibit each other. In this work, we studied the structural changes of the histone H3 tail originating from the three most important modifications; tri-methylation of K4 and K9, and acetylation of K14. We performed extensive molecular dynamics simulations of four types of H3 tails: (i) the unmodified H3 tail having no chemical modification on the residues, (ii) the tri-methylated lysine 4 and lysine 9 H3 tail (K4me3K9me3), (iii) the tri-methylated lysine 4 and acetylated lysine 14 H3 tail (K4me3K14ace), and (iv) tri-methylated lysine 9 and acetylated lysine 14 H3 tail (K9me3K14ace). Here, we report the effects of K4, K9, and K14 modifications on the backbone torsion angles and relate these changes to the recognition and binding of histone modifying enzymes. According to the Ramachandran plot analysis; (i) the dihedral angles of K4 residue are significantly affected by the addition of three methyl groups on this residue regardless of the second modification, (ii) the dihedral angle values of K9 residue are similarly altered majorly by the tri-methylation of K4 residue, (iii) different combinations of modifications (tri-methylation of K4 and K9, and acetylation of K14) have different influences on phi and psi values of K14 residue. Finally, we discuss the consequences of these results on the binding modes and specificity of the histone modifying enzymes such as DIM-5, GCN5, and JMJD2A.     

Polycomb group proteins: remembering how to catch chromatin during replication

Polycomb group (PcG) proteins maintain the expression state of PcG‐responsive genes during development of multicellular organisms. Recent observations suggest that “the H3K27me3 modification” acts to maintain Polycomb repressive complex (PRC) 2, the enzyme that creates this modification, on replicating chromatin. This could in turn promote propagation of H3K27me3 on newly replicated daughter chromatin, and promote recruitment of PRC1. Other work suggests that PRC1‐class complexes can be maintained on replicating chromatin, at least in vitro, independently of H3K27me3. Thus, histone modifications and PcG proteins themselves may both be maintained through replication.