Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays

Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers, which are directed towards modified histone tails. The arrays contained 384 peptides from eight different regions of the N-terminal tails of histones, viz. H3 1–19, 7–26, 16–35 and 26–45, H4 1–19 and 11–30, H2A 1–19 and H2B 1–19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profiles of the tested antibodies. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition, some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed toward the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies.Key words: histone modification, histone methylation, histone acetylation, histone phosphorylation, chromatin, antibody, specificity, ChIP     

Physical methods used to study core histone tail structures and interactions in solution.

The core histone tail domains are key regulatory elements in chromatin. The tails are essential for folding oligonucleosomal arrays into both secondary and tertiary structures, and post-translational modifications within these domains can directly alter DNA accessibility. Unfortunately, there is little understanding of the structures and interactions of the core histone tail domains or how post-translational modifications within the tails may alter these interactions. Here we review NMR, thermal denaturation, cross-linking, and other selected solution methods used to define the general structures and binding behavior of the tail domains in various chromatin environments. All of these methods indicate that the tail domains bind primarily electrostatically to sites within chromatin. The data also indicate that the tails adopt specific structures when bound to DNA and that tail structures and interactions are plastic, depending on the specific chromatin environment. In addition, post-translational modifications, such as acetylation, can directly alter histone tail structures and interactions.     

The CW domain, a new histone recognition module in chromatin proteins

Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.     

The evolutionary history of histone H3 suggests a deep eukaryotic root of chromatin modifying mechanisms

Background  

The phenotype of an organism is an outcome of both its genotype, encoding the primary sequence of proteins, and the developmental orchestration of gene expression. The substrate of gene expression in eukaryotes is the chromatin, whose fundamental units are nucleosomes composed of DNA wrapped around each two of the core histone types H2A, H2B, H3 and H4. Key regulatory steps involved in the determination of chromatin conformations are posttranslational modifications (PTM) at histone tails as well as the assembly of histone variants into nucleosomal arrays. Although the mechanistic background is fragmentary understood, it appears that the chromatin signature of metazoan cell types is inheritable over generations. Even less understood is the conservation of epigenetic mechanisms among eukaryotes and their origins.     

Histone tails cooperate to control the breathing of genomic nucleosomes

Genomic DNA is packaged in chromatin, a dynamic fiber variable in size and compaction. In chromatin, repeating nucleosome units wrap 145–147 DNA basepairs around histone proteins. Genetic and epigenetic regulation of genes relies on structural transitions in chromatin which are driven by intra- and inter-nucleosome dynamics and modulated by chemical modifications of the unstructured terminal tails of histones. Here we demonstrate how the interplay between histone H3 and H2A tails control ample nucleosome breathing motions. We monitored large openings of two genomic nucleosomes, and only moderate breathing of an engineered nucleosome in atomistic molecular simulations amounting to 24 μs. Transitions between open and closed nucleosome conformations were mediated by the displacement and changes in compaction of the two histone tails. These motions involved changes in the DNA interaction profiles of clusters of epigenetic regulatory aminoacids in the tails. Removing the histone tails resulted in a large increase of the amplitude of nucleosome breathing but did not change the sequence dependent pattern of the motions. Histone tail modulated nucleosome breathing is a key mechanism of chromatin dynamics with important implications for epigenetic regulation.     

The relationship between histone H3 phosphorylation and acetylation throughout the mammalian cell cycle.

During interphase, histone amino-terminal tails play important roles in regulating the extent of DNA compaction. Post-translational modifications of the histone tails are intimately associated with regulating chromatin structure: phosphorylation of histone H3 is associated with proper chromosome condensation and dynamics during mitosis, while multiple H2B, H3, and H4 tail acetylations destabilize the chromatin fiber and are sufficient to decondense chromatin fibers in vitro. In this study, we investigate the spatio-temporal dynamics of specific histone H3 phosphorylations and acetylations to better understand the interplay of these post-translational modifications throughout the cell cycle. Using a panel of antibodies that individually, or in combination, recognize phosphorylated serines 10 and 28 and acetylated lysines 9 and 14, we define a series of changes associated with histone H3 that occur as cells progress through the cell cycle. Our results establish that mitosis appears to be a period of the cell cycle when many modifications are highly dynamic. Furthermore, they suggest that the upstream histone acetyltransferases/deacetylases and kinase/phosphatases are temporally regulated to alter their function globally during specific cell cycle time points.     

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.     

The effect of epigenetic modifications on the secondary structures and possible binding positions of the N-terminal tail of histone H3 in the nucleosome: a computational study

The roles of histone tails as substrates for reversible chemical modifications and dynamic cognate surfaces for the binding of regulatory proteins are well established. Despite these crucial roles, experimentally derived knowledge of the structure and possible binding sites of histone tails in chromatin is limited. In this study, we utilized molecular dynamics of isolated histone H3 N-terminal peptides to investigate its structure as a function of post-translational modifications that are known to be associated with defined chromatin states. We observed a structural preference for α-helices in isoforms associated with an inactive chromatin state, while isoforms associated with active chromatin states lacked α-helical content. The physicochemical effect of the post-translational modifications was highlighted by the interaction of arginine side-chains with the phosphorylated serine residues in the inactive isoform. We also showed that the isoforms exhibit different tail lengths, and, using molecular docking of the first 15 N-terminal residues of an H3 isoform, identified potential binding sites between the superhelical gyres on the octamer surface, close to the site of DNA entry/exit in the nucleosome. We discuss the possible functional role of the binding of the H3 tail within the nucleosome on both nucleosome and chromatin structure and stability.     

Chromatin fiber folding: requirement for the histone H4 N-terminal tail

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.     

Histone tail network and modulation in a nucleosome

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A/H2B heterodimers and one histone (H3/H4)₂ tetramer, wrapped around by ∼146-bp core DNA and linker DNA. Flexible histone tails sticking out from the core undergo posttranslational modifications that are responsible for various epigenetic functions. Recently, the functional dynamics of histone tails and their modulation within the nucleosome and nucleosomal complexes have been investigated by integrating NMR, molecular dynamics simulations, and cryo-electron microscopy approaches. In particular, recent NMR studies have revealed correlations in the structures of histone N-terminal tails between H2A and H2B, as well as between H3 and H4 depending on linker DNA, suggesting that histone tail networks exist even within the nucleosome.     

Switching on Chromatin: Mechanistic Role of Histone H4-K16 Acetylation

DNA in eukaryotic organisms does not exist free in cells, but instead is present as chromatin, a complex assembly of DNA, histone proteins, and chromatin-associated proteins. Chromatin exhibits a complex hierarchy of structures, but in its simplest form it is composed of long linear arrays of nucleosomes. Nucleosomes contain 147 base pairs of DNA wrapped around a histone octamer, consisting of two copies each of histones H2A, H2B, H3 and H4, where 15-38 amino terminal residues of each histone protein extends past the DNA gyres to form histone “tails” 1. Chromatin provides a versatile regulatory platform for nearly all cellular processes that involve DNA, and improper chromatin regulation results in a wide range of diseases, including various cancers and congenital defects. One major way that chromatin regulates DNA utilization is through a wide range of post-translational modification of histones, including serine and threonine phosphorylation, lysine acetylation, methylation, ubiquitination, and sumoylation, and arginine methylation 2. Histone H4 K16 acetylation is a modification that occurs on the H4 histone tail and is one of the most frequent of the known histone modifications. We have demonstrated that this mark both disrupts formation of higher-order chromatin structure and changes the functional interaction of chromatin-associated proteins 3. Our results suggest a dual mechanism by which H4 K16 acetylation can ultimately facilitate genomic functions.     

Modifications of human histone H3 variants during mitosis

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.     

Structural role of RKS motifs in chromatin interactions: a molecular dynamics study of HP1 bound to a variably modified histone tail

The current understanding of epigenetic signaling assigns a central role to post-translational modifications that occur in the histone tails. In this context, it has been proposed that methylation of K9 and phosphorylation of S10 in the tail of histone H3 represent a binary switch that controls its reversible association to heterochromatin protein 1 (HP1). To test this hypothesis, we performed a comprehensive molecular dynamics study in which we analyzed a crystallographically defined complex that involves the HP1 chromodomain and an H3 tail peptide. Microsecond-long simulations show that the binding of the trimethylated K9 H3 peptide in the aromatic cage of HP1 is only slightly affected by S10 phosphorylation, because the modified K9 and S10 do not interact directly with one another. Instead, the phosphate group of S10 seems to form a persistent intramolecular salt bridge with R8, an interaction that can provoke a major structural change and alter the hydrogen-bonding regime in the H3-HP1 complex. These observations suggest that interactions between adjacent methyl-lysine and phosphoserine side chains do not by themselves provide a binary switch in the H3-HP1 system, but arginine-phosphoserine interactions, which occur in both histones and nonhistone proteins in the context of a conserved RKS motif, are likely to serve a key regulatory function.