The Histone Database: an integrated resource for histones and histone fold-containing proteins

Introduction

Histones play central roles in both chromatin organization and gene regulation, as they constitute the fundamental protein units of the nucleosome (1). The nucleosome consists of DNA wrapped around an octameric core histone complex, composed of a central H3–H4 tetramer and two adjacent H2A–H2B dimers; the nucleosome is commonly identified as the first order of compaction of eukaryotic chromatin (2). Core histone genes also display conserved expression patterns that show periodic expression across the eukaryotic cell cycle, with a pronounced peak during S-phase (3). This allows for histone proteins to be produced at the same time DNA is being synthesized. Thus, the histone proteins can be readily assembled into nucleosomes and then compacted into chromatin.

Core histones are highly conserved across eukaryotes in terms of sequence and structure. Despite overall sequence conservation, extensive histone tail post-translational modifications, in addition to histone variants present during development, contribute to epigenetic mechanisms that signal transcriptional activation, repression and recombination events. Histone proteins and their variants have an essential function in gene regulation (4–6). Nucleosomes are disassembled at transcriptionally active promoters via histone post-translational modifications (7), specific histone variants are known to mark active promoters and regulatory regions (8), and other variants are involved in the transition between transcriptionally active or silent chromatin (9). In recent years, much progress has been made toward genome-wide profiling of chromatin modifications (10), where histones play critical roles in defining the overall structure and function of chromatin and, by extension, in gene regulation.

The histone fold is a common structural motif shared by each of the four core histones, which mediates interactions between the individual core histones. The histone fold is structurally composed of three α-helices connected by two loops, and this overall architecture allows for heterodimeric interactions between core histones (11). Interestingly, even though each individual histone protein family is highly conserved, the histone fold is not conserved at the sequence level but, rather, at the structural level (12). Higher-resolution crystal structure of the nucleosome core particle has demonstrated detailed structures of the histone folds in each of the histones (13, 14). The DNA wrapped around each nucleosome is held in place by linker histones (called H1, or H5 in avian species). The linker histones, which do not contain the histone fold motif and have a different evolutionary origin from the core histones (15), are critical to chromatin higher-order compaction and facilitate internucleosomal interactions (16). In addition, H1 variants have been shown to be involved in the regulation of developmental genes (17). The overall structural state of chromatin controls DNA replication, recombination and gene expression, with histones playing critical roles during these processes (18).

Interestingly, despite the conservation of core histone gene expression patterns, the regulatory machinery that controls core histone gene expression has changed greatly among eukaryotic evolutionary lineages. Specifically, the identity of the core histone gene cis-regulatory sequence motifs and the protein factors that bind these motifs are distinct for the yeast Saccharomyces cerevisiae, as well as for other fungi, plants, insects and mammals (19). Therefore, different species have developed unique gene regulatory mechanisms for core histone genes that converge in the same gene expression phenotype, high expression levels specifically during S phase, concomitant with DNA replication.

Although the core histones are among the most slowly evolving eukaryotic proteins, members of the histone H2A and H3 families have diversified extensively, assuming specialized roles in DNA repair, gene silencing, gene expression and centromere function (5, 6). Interestingly, the centromere H3 variant appears to form tetrameric nucleosomes that induce positive supercoils, and these specialized ‘centromeric nucleosomes’ have been proposed as the epigenetic inheritance mechanism for centromeres (20).

The histone fold motif—common to all core histones—has also been found in a variety of non-histone proteins. The large majority of these non-histone proteins are localized in the nucleus and their functions are related to DNA metabolism; they include nuclear factor Y (NF-Y) and the TFIIB transcription factors (12). A few histone fold-containing proteins localized in the cytoplasm include the Ras activator Son of Sevenless (SOS) (21): SOS1 is localized primarily in the nucleus and SOS2 localized in the cytoplasm (22). Huntingtin interacting protein M (CXorf27) also contains a histone fold and is localized in the cytoplasm. We hypothesize that histone folds in cytoplasm-localized proteins are used to mediate protein–protein interactions.

Given the central role of histones and related proteins in a wide variety of critical cellular functions, we feel the need to continue to provide a centralized, curated source of important information on these proteins to the biomedical community. To this end, the Histone Sequence Database represents an organized collection of all histones and histone fold-containing proteins (23). The information presented in this Database includes a list of published three-dimensional structures for histones and histone fold-containing proteins, as well as manually curated multiple sequence alignments for each histone family.

Database and software

Results and Discussion

The computational approach presented here has identified proteins throughout a wider evolutionary spread of genomes. Currently, the Histone Database contains entries that represent a total of 7356 unique NCBI taxonomic identifiers, which correspond to approximately the same number of organisms. The sequences of core histones, linker histones and archeal histones are available in FASTA format. They are also available as a series of multiple sequence alignments, one for each class of proteins. A number of search engines can be used to query the database in several different ways: by protein family, organism, keyword or based on a sequence pattern (Figure 2). Each histone sequence for which three-dimensional structure data is available is linked to the corresponding entry in both PDB and the Molecular Modeling Database (MMDB) (35).

The Histone Sequence Database has been expanded significantly since its last update (23) (Table 1). However, the expansion is not proportional for each of the core histones. The H3 sequences, which contain a large number of variants with specialized roles in chromosome segregation and transcription, show an increase over 400% since the last database update. Similarly, the H2A core histone sequences that include variants with specialized functions in DNA repair and transcription regulation show an increase over 200% since the last update. In contrast, we observe a more modest growth in sequence numbers for the relatively invariant H4 and H2B core histones.

The Histone Sequence Database now includes archaeal histone sequences. The current update contains 182 sequences from 89 archaeal organisms, which includes members of all classified archaeal phyla (i.e. euryarchaeota, crenarchaeota, nanoarchaeota, korarchaeota and the newly proposed phylum thaumarchaeota). The presence of histone folds in all classified archaeal phyla indicates that the histone fold originated before the archaeal and eukaryotic lineage divergence (29). Most of the archaeal histones have a single histone fold domain; however, there are a number of sequences that contain two histone folds, with the C-terminal histone fold sharing higher sequence similarity with archaeal histones with a single histone fold. Archaeal histones containing two histone folds have been proposed as intermediates between archaeal and eukaryotic histones (36, 37), where both core histones H3 and H4 would have originated at the same time, followed by a second event that gave rise to core histones H2A and H2B. In the current release of the Histone Sequence Database, archaeal histones with two histone folds are confined to two distinct branches: Halobacteriaceae and the hyperthermophilic methanogen Methanopyrus kandleri. Although archaeal histones containing two histone folds have been previously identified in these lineages, it is not clear how these histones could also contribute to pack DNA in extreme high temperature or high salinity environments.

Structural comparisons confirmed the presence of the histone fold in the extreme bacterial thermophile Aquifex aeolicus (30). Additionally, the RIKEN Structural Genomics/Proteomics Initiative (RSGI) (38) has solved two Thermus thermophilus structures for a protein that also contain the histone fold (PDB:1WWI and PDB:1WWS). The histone fold was also found in diverse types of bacteria, including aquificales, ε-proteobacteria, thermaceae, actinobacteria and nostocaceae. This suggests that the histone fold appeared in bacteria by lateral gene transfer (29, 39). Interestingly, the structure from T. thermophilus (PDB:1WWS), predicted to be a dimer, is strikingly similar to the H3–H4 tetramer. However, an analysis of the electrostatic surface potential for protein Aq_328 from the hyperthermophilic bacterium A. aeolicus (PDB:1R4V) and archaeal histone from M. kandleri (PDB:1F1E) (Figure 3) reveals the DNA binding surface in the archaeal histone (Figure 3F) but shows no conservation of any of the DNA-binding residues present in both archaeal and eukaryotic histones (Figures 3A and 3D) (29). Therefore, it is possible that histone fold-like bacterial proteins have functions unrelated to DNA binding. However, it is likely that the histone-like fold is used as a dimerization domain in these species.

Conclusions

Researchers studying chromatin structure and function have traditionally relied on the Histone Sequence Database to explore the taxonomic breadth of histones and their variants (40–43). Others have focused on epigenetics and transcriptional regulation and use the database to discover newly reported core histones and histone-fold-containing proteins (44–48). The Histone Database continues to be a comprehensive bioinformatic resource that organizes and stores histone sequences and groups them into families (that now includes archaeal histones), maintains a collection of histone fold-containing sequences, and provides information on three-dimensional structures available in PDB. In the future, we will enhance our histone fold identification pipeline with state-of-the-art sequence- and structure-based methods to continue to identify new members of this biologically critical family of proteins. We also plan to integrate functional information from other publicly available Web resources.

Funding

Funding for open access charge: The Intramural Research Programs of the National Center for Biotechnology Information, National Library of Medicine and the National Human Genome Research Institute, both at the National Institutes of Health.

Conflict of interest. None declared.