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

Abstract

Eukaryotic chromatin is composed of DNA and protein components-core histones-that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins.

Full text

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Database Updates
The Histone Database: an integrated
resource for histones and histone
fold-containing proteins
Leonardo Marin ˜o-Ramı ´rez1,*, Kevin M. Levine1, Mario Morales2, Suiyuan Zhang3,
R. Travis Moreland3, Andreas D. Baxevanis3and David Landsman1
1Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health,
8600 Rockville Pike, MSC 6075, Bethesda, MD 20894-6075, USA,2Polytechnic Institute of New York University, Six MetroTech Center, Brooklyn,
NY 11201, USA and3Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Building 50,
Room 5222, Bethesda, MD 20892-8002
*Corresponding author: Tel: +1 301 402 3708; Fax: +1 301 480 2288; Email: marino@ncbi.nlm.nih.gov
Submitted 6 July 2011; Revised 30 September 2011; Accepted 3 October 2011
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Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA
into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleo-
somes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifica-
tions and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene
expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of
histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a
substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins
available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone
sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones
(H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current informa-
tion on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of
chromatin structure and function focused on histones and histone fold-containing proteins.
Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/
histones/.
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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 con-
sists 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 his-
tone 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 modi-
fications, in addition to histone variants present during de-
velopment, contribute to epigenetic mechanisms that
signal transcriptional activation, repression and recombin-
ation events. Histone proteins and their variants have an
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? The Author(s) 2011. Published by Oxford University Press on behalf of the National Institutes of Health.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
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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 regula-
tory regions (8), and other variants are involved in the tran-
sition 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 a-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 con-
served, 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 inter-
nucleosomal interactions (16). In addition, H1 variants have
been shown to be involved in the regulation of develop-
mental genes (17). The overall structural state of chromatin
controls DNA replication, recombination and gene expres-
sion, 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
forother fungi,plants,insects
Therefore, different species have developed unique gene
regulatory mechanisms for core histone genes that con-
verge in the same gene expression phenotype, high expres-
sion 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 spe-
cialized roles in DNA repair, gene silencing, gene expression
and centromere function (5, 6). Interestingly, the centro-
mere H3 variant appears to form tetrameric nucleosomes
that induce positive supercoils, and these specialized
and mammals (19).
‘centromeric nucleosomes’ have been proposed as the epi-
genetic 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 loca-
lized 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 con-
tains a histone fold and is localized in the cytoplasm. We
hypothesize that histone folds in cytoplasm-localized pro-
teins 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 his-
tone fold-containing proteins (23). The information pre-
sented in this Database includes a list of published
three-dimensional structures for histones and histone
fold-containing proteins, as well as manually curated mul-
tiple sequence alignments for each histone family.
Database and software
Data tables
The Histone Sequence Database, which has been developed
and expanded significantly since its last release (23), has
three tables stored in a relational database schema using
Oracle 10g (Figure 1). The HISTONES table stores informa-
tion about the histone category, its accession, the sequence
string, the submitting database, as well as NCBI’s taxonomic
information on the sequence. The ORGANISM table con-
tains detailed taxonomic information for the sequences
contained in theHistone
STRUCTURES table stores information on the experimental-
ly determined structures of proteins contained in the data-
base, including the method of determination (i.e. X-ray
crystallography or NMR spectroscopy).
Sequence Database. The
Software
The Histone Sequence Database uses Common Gateway
Interfaces (CGIs) written in the Perl programming language
that communicate with the relational database software.
The connectivity between the CGIs and the Oracle 10g
Relational Database Management Software was imple-
mented using Perl’s Database Interface (DBI) and the
Oracle database driver for the DBI module (DBD::Oracle),
available through the Comprehensive Perl Archive Network
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(CPAN; http://search.cpan.org/). The use of object-oriented
design methodologies and Perl modules that are both open
source and developed in-house allows for flexibility and
scalability. The Web pages displaying data, such as the sum-
mary of contents, non-redundant sets, and search pages are
dynamically generated using CGI. Comments concerning
the Web front-end are welcomed and encouraged.
Data sources and histone protein identification
The protein databases searched for the update and cur-
ation of the Histone Sequence Database were the NCBI
non-redundant (nr) database (18 November 2010); nr in-
cludes sequences of all non-redundant GenBank CDS trans-
lations (24), as well as the sequences of RefSeq proteins,
sequences of structures represented in the Protein Data
Bank (PDB) (25), and sequences from UniProtKB/Swiss-Prot
(26), the Protein Information Resource (PIR) (27), and the
Protein Research Foundation (PRF) (http://www.prf.or.jp/
index-e.html). The collection of histones was extended
and revised, using the HMMER3 software package (28).
We constructed hidden Markov models (HMMs) for each
of the four core histones and the linker histone H1 from
the alignments generated in the last release of the Histone
Database. Additional HMMs were generated for archaeal
histones(29)and bacterial proteinsthat containa
histone-likefold (30); only the protein entries that have a
complete domain hit with an E<0.01 are collected for fur-
ther analysis. For each histone family, multiple sequence
alignments were generated using MUSCLE (31). The align-
ments that are manually curated to include proteins with
complete folds are also available in PDF format and are
color-coded to allow easy identification of amino acid vari-
ants. The Histone Database uses a color scheme designed to
highlight the specific amino acid differences that a particu-
lar group of sequences may have inside the core or linker
histone alignments by coloring amino acids with similar
physicochemical properties differently. A summary table
of the number of sequences found grouped by family and
species represented in the database is provided (Table 1).
Identification of histone fold-containing proteins
Histone fold-containing proteins were identified using a
different search strategy. We used the sequences from
each of the four core histone MUSCLE alignments (H2A,
H2B, H3 and H4) as seeds for PSI-BLAST (32) searches.
The PSI-BLAST searches were run to convergence with an
E-value inclusion threshold of 0.01; the core histone seeds
were excluded from the final list of histone fold-containing
proteins. Additionally, related structures were identified
using NCBI’s VAST-related structures searches (33, 34), in
an effort to identify more distant histone fold-containing
proteins that could not be identified through PSI-BLAST
searches. Using this strategy, we were able to identify a
total of 2180 histone fold-containing proteins.
Results and Discussion
The computational approach presented here has identified
proteins throughout a wider evolutionary spread of gen-
omes. Currently, the Histone Database contains entries that
represent a total of 7356 unique NCBI taxonomic identi-
fiers, which correspond to approximately the same number
of organisms. The sequences of core histones, linker his-
tones and archeal histones are available in FASTA format.
Figure 1. Histone Database data model. The Histone Database
stores selected manually curated information from GenBank
records. The information stored as part of each record in-
cludes the GenBank unique identifier (GI), accession number,
definition line, sequence string, histone class, database source,
NCBI taxonomic identifier and organism name. The database
front-end is written in Perl, the data is stored in an Oracle 10g
relational database, and data is retrieved using Perl DBI and
DBD libraries.
Table 1. Histone Database content
Core histone
profile
Number
of unique
sequences
Increase
since last
update (%) (23)
Number
of unique
taxonomic
identifiers
H1/H5591138.3156
H2A1016214.6 331
H2B 755161.2308
H32287476.17096
H4 341181.8 344
Archaeal182–89
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Figure 2. Histone Database query and results. The Histone Database main page displays a search engine that allows users to find
histone sequences from a large variety of organisms. Additionally, users have the possibility of exploring other features to access
complete collections of Histone Protein Sequences, Multiple Sequence Alignments, The Human Histone Gene Complement,
Non-Histone Proteins Containing the Histone fold Motif and Histone Structures. The upper panel shown (A) presents the criteria
used for the query, which requires the sequence to contain a fragment with amino acids PRK from the angulate sea urchin
(Parechinus angulosus) histone H2B. The search results presented in the lower panel (B) include two protein sequences that meet
the criteria specified by the query.
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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 se-
quence for which three-dimensional structure data is avail-
able is linked to the corresponding entry in both PDB and
the Molecular Modeling Database (MMDB) (35).
The Histone Sequence Database has been expanded sig-
nificantly 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
Figure 3. Histone-like folds in A. aeolicus and M. kandleri. Protein Aq_328 from the hyperthermophilic bacterium A. aeolicus
(PDB:1R4V) and archaeal histone from M. kandleri (PDB:1F1E) have two histone like folds. These are colored as dark blue and
dark green (for 1R4V) and light blue and light green (for 1F1E). The electrostatic surface potential ranges from +2 kTe?1(blue) to
?2 kTe?1(red). (A) and (D) the front and back views, respectively, of the electrostatic surface potential of Protein Aq_328. (B) and
(E) superimposed structures of protein Aq_328 and the archaeal histone from M. kandleri. (C) and (F) the front and back views,
respectively, of the electrostatic surface potential of the archaeal histone from M. kandleri. The figures were generated with
PyMOL (49) and the APBS plug-in for PyMOL (50).
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