eukaryotic dna is packaged into chromatin incorporating repeating nucleosomes by wrapping dna around core histones ( h2a , h2b , h3 and h4 ) . in mammalian cells ,
the n - terminal tail of histone is subject to many chemical modifications such as methylation , acetylation , ubiquitylation , phosphorylation and adp - ribosylation .
histone modifications provide accessible targets for effectors such as histone methyltransferase and acetyltransferase ( 1 ) and have different impact on chromatin structure and gene transcription , depending on the types and locations of the modifications ( 2,3 ) .
currently , methyl- , acetyl- and ubiquityl - groups among various modification types have been studied mainly by chip - based technologies . for specific loci at the n - terminal tails of arginines and lysines of histone , up to three methyl - groups can be added .
histone methylation types at different locations have been ascribed to either activating or repressive functions ( 4 ) .
for example , h3k4me3 is positively correlated with gene expression , while h3k9me3 is implicated in heterochromatin formation and gene silencing ( 5 ) . the normal pattern of histone modifications is vital for chromatin stability and transcriptional regulation ( 5 ) .
rar2 promoter is silenced by h3k27me3 enrichment specifically in prostate cancer without dna hypermethylation dependence ( 7 ) .
chip - based experiments including chip - seq , chip - chip and qchip are efficient at probing histone modifications , and have produced a large amount of histone modification data ( 8) . it is useful to have a repository of such data so that in - depth data mining can be performed .
there have been a few resources for histone or histone modifications , such as the chromatindb ( 9 ) , histone database ( 1012 ) , sysptm ( 13 ) and histonehits ( 14 ) .
although providing post - translational modification ( ptm ) information and modified residues information of histone sequences and structures , the histone database lacks large - scale profiles of histone modifications for functional evaluation .
the major difference between sysptm and hhmd is that sysptm is a curated ptm platform for online query and analysis while hhmd is a repository of large - scale histone modification data and also has built - in functions for analysis and visualization .
histonehits is a database for systematic collections of histone mutants in yeast ( 14 ) . to the best of our knowledge
, there has been no specialized database that focuses on histone modifications in mammals , which hinders further systematic and in - depth data mining .
therefore , there is need to build such a database that dedicates to the storage and analysis of experimental histone modification data .
a database of this kind would be beneficial to histone modification studies such as identification of differential histone modification regions ( d - hmrs ) for a given set of histone modifications .
cancer has been considered as a complex disease , which involves both genetic and epigenetic alteration . until
now there have been several comprehensive projects dedicated to cancer studies , including the cancer genome atlas ( tcga ) ( 15 ) and a large amount of researches studying cancer from epigenomic perspective ( 6,16,17 ) .
although dna methylation undergoes significant changes , other epigenetic changes such as histone modifications also reflect the tumorigenesis process ( 6,18 ) .
global aberrant histone modification patterns in tumorigenesis provide novel potential of molecular screening for cancer prevention , diagnosis and treatment ( 19,20 ) .
tools for histone modification data integration and analysis are still needed for cancer biomarker identification .
we developed the human histone modification database ( hhmd ) , which is available at http://bioinfo.hrbmu.edu.cn/hhmd or http://www.hhmd.org .
the latest release of hhmd provides genomic context ( hg18 ) for histone modification alignment , which can be used to make comparisons between genomic and epigenomic data .
five search options were provided for advanced searches , namely histone modification , gene i d , functional categories , chromosome location and cancer name .
it has the capability of investigating histone modifications in a genomic context by superimposing histone modification data on dna methylation , gc contents and gene information .
hhmd may be a useful resource for researchers who are interested in epigenetic regulation and computational epigenetics in human and other species .
with the in - house data and built - in tools in hhmd , it makes d - hmrs identification between cancer and control possible , and therefore benefits cancer biomarker identifications .
hhmd contains four types of data : ( i ) high - throughput histone modifications , ( ii ) medip methylation , ( iii ) curated information of aberrant histone modifications , genes and cancers and ( iv ) gc contents , refseq gene ( 21 ) and other genomic annotations .
a total of 43 histone modification types ( table 1 ) classified by histone types have been included in the current release of hhmd .
all histone modifications were collected from biological experiments , including high - throughput datasets and curated information from literature .
all the high - throughput datasets were probed from chip - based technology . among these datasets ,
81 were from chip - chip , 87 from chip - seq and 55 from qchip .
summarized chip - seq data files derived from tag - based bed files with window size of 200 bp were used to speed up hhmd .
there are 50 datasets for h4 , 11 for h2a and six for histone h2b .
regarding modification types , 122 datasets are methylation - related , 87 of acetylation - related and 1 of ubiquitylation - related .
high - throughput datasets were collected from various institutes and websites ( table 2 ) . an interface form for submission of new histone modification information
table 1.list of histone modifications for 228 high - throughput datasetshistone typehistone modificationsh2ah2ak5ac , h2ak9ac , h2azh2bh2bk120ac , h2bk12ac , h2bk20ac , h2bk5ac , h2bk5me1 , ubh2bh3h3k14ac , h3k18ac , h3k23ac , h3k27ac , h3k27me1 , h3k27me2 , h3k27me3 , h3k36ac , h3k36me1 , h3k36me3 , h3k4ac , h3k4me1 , h3k4me2 , h3k4me3 , h3k79me1 , h3k79me2 , h3k79me3 , h3k9ac , h3k9me1 , h3k9me2 , h3k9me3 , h3r2me1 , h3r2me2 , h3ach4h4k12ac , h4k16ac , h4k20me1 , h4k20me3 , h4k5ac , h4k8ac , h4k91ac , h4kac , h4r3me2 , h4acnon - specific antibodies are used.the list is classified according to histone types . 

table 2.list of institutes and websites of high - throughput datasets used in this databaseinstitute / projectwebsiteucschttp://genome.ucsc.edu / cgi - bin / hgtablesencodehttp://hgdownload.cse.ucsc.edu / goldenpath / hg18/wgencode broadchipseq / canada
s michael smith genome sciences centrehttp://www.bcgsc.ca / data / histone - modificationlaboratory of molecular immunology , national heart , lung and blood institute , nihhttp://dir.nhlbi.nih.gov / papers / lmi / epigenomes / hgtcell.aspx list of histone modifications for 228 high - throughput datasets non - specific antibodies are used .
list of institutes and websites of high - throughput datasets used in this database to investigate the relationships between histone modifications and dna methylation , hhmd integrated a panel of large - scale methylation data from medip ( 22 ) .
the methylation data comprises information from 16 tissues including gm06990 cell line data ( 23 ) .
the functional and genomic annotations of the genes in hhmd were obtained from various databases [ i.e. ncbi ( 24 ) , ucsc ( 25 ) , go ( gene ontology ) ( 26 ) , uniprot ( 14 ) , affymetrix probe i d , kegg ( 27 ) , refseq protein ( 28 ) , omim ( 29 ) , gi , unigene ( 30 ) , pir ( 31 ) and ensembl ( 32 ) ] . to elucidate the interplay of histone modifications and cancer types ,
first , we collected a panel of aberrant histone modifications in various human cancer types from the literature by manual curation .
the current version compiled 833 curated relationships involving 17 human histone modifications and 555 genes that are related to nine human cancers . among these relationships ,
the nine cancers are gastric , ovarian , colon , leukemia , prostate , lymphoid , breast , lung and pancreatic cancer .
hhmd has imbedded search functions that can be used to query human transcript ids ( 21 ) and official gene symbols .
second , hhmd includes several high throughput aberrant histone modification data such as k562 , gm06990 and hela cell lines .
hhmd is a highly cross - linked database , which facilitates data acquisition and visualization .
figure 1a presents three starting points of hismodview and five search options . to visually understand the data in hhmd , hismodview and searching tools are fully cross - linked .
search results can be downloaded from result pages of hismodview ( figure 1b ) , or reviewed offline ( figure 1c ) . in this way
for example , users can start by searching cancer name and visualize the genes in hismodview and then download the specific histone modification data in defined genomic range for further review . 

hhmd offers five advanced search options , namely search by histone modification , search by gene i d , search by functional categories , search by chromosome location and by cancer name .
meanwhile , genomic annotations such as gc contents and refseq genes and medip methylation data can also be visualized .
users can fine - tune the genomic regions by left / right shifting or zooming in / out .
users can hover on the the texts of histone modification types to gain further details .
users who are interested in genomic annotations can get details from pop - up menus .
( c ) screenshot showing the  search by histone modification result when specifying hm i d : hm-26 .
hhmd offers five advanced search options , namely search by histone modification , search by gene i d , search by functional categories , search by chromosome location and by cancer name .
meanwhile , genomic annotations such as gc contents and refseq genes and medip methylation data can also be visualized .
users can fine - tune the genomic regions by left / right shifting or zooming in / out .
users can hover on the the texts of histone modification types to gain further details .
users who are interested in genomic annotations can get details from pop - up menus .
( c ) screenshot showing the  search by histone modification result when specifying hm i d : hm-26 .
hhmd supports flexible query for various histone modifications and related genomic and functional annotations by providing five search tools . taking the histone modification search as an example
, users can specify the query options such as cytogenetic position , histone modification , technology , tissue , etc . for new users , histone modification search is the suggested option .
for example , users can enter the page  search by histone modification and input  hm-26 in the textbox labeled 
histone modification search by id: , and then proceed . the sample result page is shown in figure 1c , where a download icon and a hismodview icon are available .
functional categories search is a specific module dedicated to study the relationships between histone modification patterns and functional classifications .
users who are interested in the histone modification distribution for genes of similar functions may find this module helpful .
for example , users interested in kegg pathway : hsa00030 can select  h3k4me3 or other options from the pull - down menu  histone modification: , then type  hsa00030 in the search field of kegg i d and leave others as default . in this case ,
a report of six genes annotated with hsa00030 and 26 histone modification summaries will be returned . as an efficient visualization tool
, hismodview allows users to browse histone modifications and genomic annotations in the context of human genome ( hg18 ) .
start by chromosome location. the hismodview result page has four sections , namely histone modification , medip methylation , gc contents and refseq gene annotations .
labels and histograms of histone modifications are displayed in histone modification section for each genomic track .
for each label , the description of the tissue and histone modification type is available and can be displayed by moving the mouse over the label . to study the relationships between histone modifications and dna methylation , we have integrated a panel of methylation data from 16 tissues generated by medip into the hismodview tool . in this section ,
methylation data is represented as matrices , each element within represents one roi [ region of interest , defined by rakyan et al .
( 23 ) ] and color coding represents different methylation level ( from yellow to blue , represents from 0 to 100% methylation ) .
links to another epigenetic database for homo sapiens : methycancer ( 33 ) are available .
the description of roi , tissue type and the methylation level are also available in the popup menus .
a detailed annotation report will be brought up by clicking any of the gene structures in refseq genes section .
the gene structures such as introns and exons can be characterized for a specific gene and the range for viewing can be adjusted by centering on a specific gene .
the scripts for data analysis were written in java , which are available on hhmd website .
recent studies of histone modification co - localization suggested that the co - localized histone modifications may mark functionally important regions .
co - localized histone modifications can provide specific cubic targets for biological effectors ( 34,35 ) .
to date , co - localization of h3k4me3 ( activating ) and h3k27me3 ( repressive ) is the most studied co - localized pair of markers , which has been ascribed to the developmental control of es cells ( 36,37 ) . yet ,
identification of more co - localized histone modification pairs with functionalities is still of great interest .
we used h3k4me3 and h3k9me3 profiles ( 38,39 ) in hhmd to further study the relationships between histone modification co - localization and gene function .
h3k4me3 is a histone modification type associated with relaxed chromatin structure and active transcription , while h3k9me3 is a marker associated with heterochromatin formation , gene imprinting and repressive transcription ( 3,4 ) .
significant imbalance of co - localized h3k4me3 and h3k9me3 is also suggested to have influences on developmental processes .
we summarized the distribution of co - localization of h3k4me3 and h3k9me3 ( repressive ) from resting cd4+t cells to explore histone modification co - localization patterns in genomic context .
as shown in figure 2a  c , h3k4me3 and h3k9me3 co - localized regions ( 200 bp window size ) reveal intermediate genomic pattern which seems to be contributed by both h3k4me3 and h3k9me3 .
the observation is in good agreement with the findings that methylation of h3 lys4 and lys9 play the contrary roles in chromatin regulation ( 35,40 ) .
the genes occupied by co - localized h3k4me3 and h3k9me3 are exemplified in supplementary files , from which we find that protocadherin alpha gene cluster is marked by such co - localization . 

figure 2.the pie charts show the genomic distributions of histone modifications in co - localization study of h3k4me3 and h3k9me3 .
the pie charts show the genomic distributions of histone modifications in co - localization study of h3k4me3 and h3k9me3 .
genomic annotations such as refseq genes , gc contents and various epigenomic annotations including histone modifications and dna methylation data were compiled in hhmd .
users can access the data by searching and investigating histone modifications using hismodview and offline analysis tools .
hismodview is a visualization tool for data profiling and comparison , which makes the identification of variable histone modification regions in multiple tissues feasible .
virtual analysis tools such as d - hmrs identification and other analysis functions are to be integrated in a later release .
a standalone version of hhmd that supports complex calculation will be released . as a resource to study the potential function of histone modification markers
, hhmd could be extended with utilities for identification of cancer - related histone modification markers for candidate genes .
we will continue to investigate the relationships between histone modifications and other diseases in addition to cancer .
since histone modifications in other species are also accumulating , we will extend the research scope to build specific databases for histone modifications in other species as well .
30871394 ) ; the national high tech development project of china , the 863 program ( grant no .
2007aa02z329 ) ; the national basic research program of china , the 973 program ( grant no .
2008cb517302 ) ; the natural science foundation of heilongjiang province ( grant no . d2007 - 35 ) ; the innovation and technology special fund for researchers of harbin ( grant no .
 histone modifications play important roles in chromatin remodeling , gene transcriptional regulation , stem cell maintenance and differentiation . 
 alterations in histone modifications may be linked to human diseases especially cancer . 
 histone modifications including methylation , acetylation and ubiquitylation probed by chip - seq , chip - chip and qchip have become widely available . 
 mining and integration of histone modification data can be beneficial to novel biological discoveries . 
 there has been no comprehensive data repository that is exclusive for human histone modifications . 
 therefore , we developed a relatively comprehensive database for human histone modifications . 
 human histone modification database ( hhmd , http://bioinfo.hrbmu.edu.cn/hhmd ) focuses on the storage and integration of histone modification datasets that were obtained from laboratory experiments . 
 the latest release of hhmd incorporates 43 location - specific histone modifications in human . to facilitate data extraction , 
 flexible search options are built in hhmd . 
 it can be searched by histone modification , gene i d , functional categories , chromosome location and cancer name . 
 hhmd also includes a user - friendly visualization tool named hismodview , by which genome - wide histone modification map can be shown . 
 hismodview facilitates the acquisition and visualization of histone modifications . 
 the database also has manually curated information of histone modification dysregulation in nine human cancers . 
INTRODUCTION
DATA COLLECTION
DATABASE USAGE
DATABASE IMPLEMENTATION
DISCUSSION AND FUTURE DEVELOPMENT
SUPPLEMENTARY DATA
FUNDING