BACKGROUND
dna methylation is a well-studied component of epigenetics that, in the mammalian system, involves the 5' covalent modification of cytosine nucleotides by a methyl group.
in humans, cytosine methylation almost always occurs in the context of a cg di-nucleotide, except in undifferentiated cells where methylation was recently identified in cytosines that do not precede guanines   <cit> .
regions of high cg density, termed "cpg islands" are usually un-methylated and found mainly in the 5' promoter ends of genes.
however high resolution maps of genome-wide methylation now show that cytosine methylation occurs throughout the genome, particularly in bodies of highly expressed genes  <cit> , and up to  <dig> % of cytosines in the human genome are methylated  <cit> .
although the functional difference between cg and non-cg methylation requires further investigation, it is clear that dna methylation itself significantly regulates gene expression and affects cellular processes in disease and development  <cit> .
for example, genome-wide methylation is altered during aging  <cit>  and malignant transformation  <cit> , and recent evidence supports the notion that methylation can be modulated by diet and environment  <cit> .
moreover evidence of rapid and dynamic dna methylation/de-methylation in vivo  <cit>  challenges the conventional view that dna methylation is a stable or permanent epigenetic mark.
mechanisms to explain aberrant de novo methylation in these contexts include  targeted recruitment of dna methyl-transferases by cis-acting factors such as g9a or ezh <dig>  <cit> , or  loss of boundaries or "protective" transcription factors leading to the spread of dna methylation into affected regions in the genome  <cit> .
indeed several non-redundant sequences matching the consensus motifs for transcription factors such as sp <dig> have been identified at sites that are resistant to de novo methylation in cancer  <cit> .
de novo methylated cpg islands in cancer however were characterized by the lack of sequence motif combinations and the absence of activating tf binding  <cit> .
conversely, the classical mechanism by which dna methylation regulates transcription is through altered accessibility of transcription factor complexes to their cognate dna binding sites  <cit> .
this mechanism is supported by many locus-specific examples  <cit>  but one that links the mechanism to environmental influences is the rodent model of maternal grooming  <cit> .
"highly groomed" neonates developed hypo-methylation in the first exon of the glucocorticoid receptor gene which in turn permits binding of the transcription factor ngfi-a to this dna regulatory region and up-regulates glucocorticoid receptor expression  <cit> .
in contrast, "lesser groomed" neonates developed methylation in the same dna regulatory sequence with corresponding inhibition of ngfi-a binding and down-regulation of glucocorticoid receptor expression.
conserved consensus motifs have been predicted for transcription factor binding across the human genome, and empirical transcription factor binding sites  have been determined biologically using the genome-wide technique which couples chromatin immunoprecipitation and high throughput sequencing .
we have previously examined the genome-wide methylome of human hearts  <cit>  and sperm  <cit> .
we therefore set out to analyze the methylation state of tfbs in these methylation maps.
RESULTS
conserved transcription factor consensus motifs  are hyper-methylated
we analyzed genome-wide dna methylation profile in  <dig> normal adult human hearts  and human sperm  by employing the technique of medip-seq  <cit> .
analysis of our medip-seq datasets was performed using the bayesian deconvolution algorithm called batman  <cit> .
using batman we assigned methylation scores across the genome for hearts and sperm.
because we hypothesized that the interaction between transcription factor complexes and their cognate dna binding sites is modulated and influenced by methylation of cytosines within the dna sequence  <cit> , we examined methylation profiles at genome-wide sites of transcription factor binding.
first, we made use of a computational dataset of transcription factor motifs where locations of motifs were determined based on a score which met the threshold for its conserved binding matrix in the alignment for all  <dig> species: human, rat and mouse  http://www.gene-regulation.com.
score and threshold were computed with the transfac matrix database , created by biobase, and are currently found on the ucsc genome web browser .
we observed a profile of increased average methylation, centred on the predicted tfbs at  <dig> , <dig> locations where the consensus motifs for  <dig> transcription factor families were conserved .
a similar methylation profile was found for the same  <dig> , <dig> genomic locations in sperm cells .
in contrast, a separate control analysis performed with a set of random genomic locations  showed no modulation in the methylation profile across random genomic locations in both hearts and sperm .
when examined in further detail for each individual tf, the large majority of tf showed a hyper-methylation profile in both hearts and sperm  but a smaller proportion showed a variation of hypo-methylation pattern in both tissues, an opposite pattern in either, or a neutral methylation pattern.
although the latter analysis implicates the possibility that binding of sub-groups of transcription factors are variably affected by dna methylation pattern, and differences between hearts and sperm may exist for specific sets of tf, the aggregate hyper-methylation profile in the former analysis suggests that conserved consensus tf motifs are mainly hyper-methylated.
empirical tfbs with conserved consensus motifs are hypo-methylated
empirical or bona fide genome-wide sites of transcription factor binding are now determined by chip-seq  <cit>  and the encode consortium http://www.genome.gov/ <dig> has now performed chip-seq for at least  <dig> different tf.
we therefore examined predicted tfbs  for these  <dig> tf .
at the locations of predicted tfbs for this subset of  <dig> tf , a hyper-methylation profile  was again found in both hearts and sperm.
1yale/ucd/harvard
2hudsonalpha institute
3yale/ucd/harvard/duke unc/ut, genome institute of singapore
4yale/ucd/harvard, gis
5lister et al, nature 2009
6gis
next we compared predicted tfbs  to empirical tfbs  for the same  <dig> tf.
this revealed that only  <dig>  locations were both predicted tfbs and empirical tfbs ; i.e.
<dig> %  of the empirical tfbs were predicted by motif and conservation, and  <dig> %  of the predicted tfbs were biologically proven tbfs as determined by chip-seq.
in contrast to the aggregate hyper-methylation profile at all predicted tfbs  and at the subset of  <dig> tfbs , predicted tfbs that were biologically proven tfbs  showed an aggregate profile of hypo-methylation in both hearts and sperm .
table  <dig> shows the detailed methylation profile for each tf in "set 4".
all tf were associated with either a hypo-methylation or neutral profile in hearts whereas  <dig> out of  <dig> tf showed a hyper-methylation profile in sperm.
the latter detailed analysis may reflect specific differences in empirical tf binding between a post-mitotic organ and germ cell.
empirical tfbs with conserved consensus motif are more likely to reside in cpg islands than predicted tfbs
cpg islands  are cg-rich genomic regions often located at the 5' promoter region of genes.
since cpg islands are largely hypo-methylated  <cit>  and the interaction between transcription factor complexes and dna may be regulated by cgi/promoter methylation, we asked what proportion of our sets of genomic locations corresponded to cgi.
only 7% of the subset of  <dig> predicted tfbs  resided in cgi, whereas 40% of locations of empirical tfbs containing the expected conserved consensus motif  were in cgi.
we therefore divided the predicted tfbs  into cgi and non-cgi, and examined the methylation profile for each subset.
as expected, predicted tfbs in cgi were hypo-methylated , whereas predicted tfbs outside of cgis were hyper-methylated .
this hyper-methylation profile again suggests that where conserved consensus motifs for tf binding exist outside of cgi, promiscuous or disorderly tf binding may be controlled by dna hyper-methylation.
similarly, although empirical tfbs containing the expected conserved consensus motif in cgi  were hypo-methylated , non-cgi of this dataset showed a hyper-methylation profile in sperm  and a neutral methylation profile in hearts .
discussion
interaction between dna and proteins or protein complexes can be modulated by dna methylation.
indeed there are examples of dna methylation-dependent binding for transcription factors such as ctcf  <cit>  and ngfi-a  <cit> .
promiscuous or disorderly transcription factor binding may therefore be controlled by dna methylation at potential binding sites throughout the genome where there are conserved consensus motifs.
here our global analysis largely supports this hypothesis for tf in general.
although the possibility remains that we are only sampling methylation profiles at these sites of conserved consensus motifs at a single time point, and previous or subsequent tf binding may occur as a result of dynamic changes in dna methylation, this is the first genome-wide study to associate conserved consensus motifs  with dna hyper-methylation.
we found similar aggregate methylation profiles for the various sets of tfbs in parallel analyses using methylation maps from both hearts and sperm.
analysis of all conserved consensus motifs throughout the genome and the subset of those that reside outside cgi showed an aggregate profile of hyper-methylation, but detailed analysis of individual tf suggests that there may be subsets of tf that behave differently.
these may indeed represent specific tf whose combinatorial function is to establish or maintain the un-methylated dna state  <cit> .
indeed our finding of sp <dig> and nrf <dig> in this latter group corresponds to previous reports  <cit>  proposing this function for these two tf.
we have further found that only a very small number of predicted tfbs containing conserved consensus motif are biologically proven tbfs .
conversely, only a very small subset of empirical tfbs has the expected conserved consensus motif.
most importantly, we found that while conserved consensus motifs without biologically proven tf binding have a hyper-methylated profile, sites of biologically proven tfbs have the opposite hypo-methylation profile.
although the scales of methylation scores  in our analysis are generally narrow , these scores represent composite/aggregate scores at over  <dig> m locations in the genome and confidence intervals as indicated on the graphs do not show overlap from peaks to troughs, reflecting the significance of altered methylation patterns in these regions.
moreover these methylation scores are not representative of "whole-genome" methylation but only of the local regions that are being analyzed in each graph , peak-to-trough scores therefore differ between analyses.
interestingly, we also found that only a very small proportion of the sites of conserved consensus motif without biologically proven tf binding were within cgi s; whereas a larger proportion of sites of biologically proven tf binding were within cgis .
the lack of methylation modulation in sites of biologically proven tf binding outside of cgis  serves as a negative control for the other profiles of methylation differences that we have detected, but may also indicate that at these sites of empirical tf binding, a neutral methylation profile allows potential tf binding.
alternatively, potential tf binding at these sites may not be regulated by dna methylation.
most importantly and in contrast to that, predicted consensus tfbs that are non-cgis maintain a significant hyper-methylation pattern .
CONCLUSIONS
our data provides genome-wide evidence that the majority of conserved consensus motifs in the human genome are hyper-methylated, whereas biologically proven tfbs with conserved consensus motifs are hypo-methylated.
this implicates a role for dna methylation in preventing promiscuous or disorderly tf binding, at least for the majority of tf.

 dna methylation is a well-studied component of epigenetics that, in the mammalian system, involves the 5' covalent modification of cytosine nucleotides by a methyl group. in humans, cytosine methylation almost always occurs in the context of a cg di-nucleotide, except in undifferentiated cells where methylation was recently identified in cytosines that do not precede guanines. regions of high cg density, termed "cpg islands" are usually un-methylated and found mainly in the 5' promoter ends of genes. however high resolution maps of genome-wide methylation now show that cytosine methylation occurs throughout the genome, particularly in bodies of highly expressed genes, and up to % of cytosines in the human genome are methylated. although the functional difference between cg and non-cg methylation requires further investigation, it is clear that dna methylation itself significantly regulates gene expression and affects cellular processes in disease and development. for example, genome-wide methylation is altered during aging and malignant transformation, and recent evidence supports the notion that methylation can be modulated by diet and environment. moreover evidence of rapid and dynamic dna methylation/de-methylation in vivo challenges the conventional view that dna methylation is a stable or permanent epigenetic mark. mechanisms to explain aberrant de novo methylation in these contexts include targeted recruitment of dna methyl-transferases by cis-acting factors such as g9a or ezh, or loss of boundaries or "protective" transcription factors leading to the spread of dna methylation into affected regions in the genome. indeed several non-redundant sequences matching the consensus motifs for transcription factors such as sp have been identified at sites that are resistant to de novo methylation in cancer. de novo methylated cpg islands in cancer however were characterized by the lack of sequence motif combinations and the absence of activating tf binding. conversely, the classical mechanism by which dna methylation regulates transcription is through altered accessibility of transcription factor complexes to their cognate dna binding sites. this mechanism is supported by many locus-specific examples but one that links the mechanism to environmental influences is the rodent model of maternal grooming. "highly groomed" neonates developed hypo-methylation in the first exon of the glucocorticoid receptor gene which in turn permits binding of the transcription factor ngfi-a to this dna regulatory region and up-regulates glucocorticoid receptor expression. in contrast, "lesser groomed" neonates developed methylation in the same dna regulatory sequence with corresponding inhibition of ngfi-a binding and down-regulation of glucocorticoid receptor expression. conserved consensus motifs have been predicted for transcription factor binding across the human genome, and empirical transcription factor binding sites have been determined biologically using the genome-wide technique which couples chromatin immunoprecipitation and high throughput sequencing. we have previously examined the genome-wide methylome of human hearts and sperm. we therefore set out to analyze the methylation state of tfbs in these methylation maps.  conserved transcription factor consensus motifs are hyper-methylated we analyzed genome-wide dna methylation profile in normal adult human hearts and human sperm by employing the technique of medip-seq. analysis of our medip-seq datasets was performed using the bayesian deconvolution algorithm called batman. using batman we assigned methylation scores across the genome for hearts and sperm. because we hypothesized that the interaction between transcription factor complexes and their cognate dna binding sites is modulated and influenced by methylation of cytosines within the dna sequence, we examined methylation profiles at genome-wide sites of transcription factor binding. first, we made use of a computational dataset of transcription factor motifs where locations of motifs were determined based on a score which met the threshold for its conserved binding matrix in the alignment for all species: human, rat and mouse http://www.gene-regulation.com. score and threshold were computed with the transfac matrix database, created by biobase, and are currently found on the ucsc genome web browser. we observed a profile of increased average methylation, centred on the predicted tfbs at, locations where the consensus motifs for transcription factor families were conserved. a similar methylation profile was found for the same, genomic locations in sperm cells. in contrast, a separate control analysis performed with a set of random genomic locations showed no modulation in the methylation profile across random genomic locations in both hearts and sperm. when examined in further detail for each individual tf, the large majority of tf showed a hyper-methylation profile in both hearts and sperm but a smaller proportion showed a variation of hypo-methylation pattern in both tissues, an opposite pattern in either, or a neutral methylation pattern. although the latter analysis implicates the possibility that binding of sub-groups of transcription factors are variably affected by dna methylation pattern, and differences between hearts and sperm may exist for specific sets of tf, the aggregate hyper-methylation profile in the former analysis suggests that conserved consensus tf motifs are mainly hyper-methylated. empirical tfbs with conserved consensus motifs are hypo-methylated empirical or bona fide genome-wide sites of transcription factor binding are now determined by chip-seq and the encode consortium http://www.genome.gov/ has now performed chip-seq for at least different tf. we therefore examined predicted tfbs for these tf. at the locations of predicted tfbs for this subset of tf, a hyper-methylation profile was again found in both hearts and sperm. 1yale/ucd/harvard 2hudsonalpha institute 3yale/ucd/harvard/duke unc/ut, genome institute of singapore 4yale/ucd/harvard, gis 5lister et al, nature 2009 6gis next we compared predicted tfbs to empirical tfbs for the same tf. this revealed that only locations were both predicted tfbs and empirical tfbs; i.e. % of the empirical tfbs were predicted by motif and conservation, and % of the predicted tfbs were biologically proven tbfs as determined by chip-seq. in contrast to the aggregate hyper-methylation profile at all predicted tfbs and at the subset of tfbs, predicted tfbs that were biologically proven tfbs showed an aggregate profile of hypo-methylation in both hearts and sperm. table shows the detailed methylation profile for each tf in "set 4". all tf were associated with either a hypo-methylation or neutral profile in hearts whereas out of tf showed a hyper-methylation profile in sperm. the latter detailed analysis may reflect specific differences in empirical tf binding between a post-mitotic organ and germ cell. empirical tfbs with conserved consensus motif are more likely to reside in cpg islands than predicted tfbs cpg islands are cg-rich genomic regions often located at the 5' promoter region of genes. since cpg islands are largely hypo-methylated and the interaction between transcription factor complexes and dna may be regulated by cgi/promoter methylation, we asked what proportion of our sets of genomic locations corresponded to cgi. only 7% of the subset of predicted tfbs resided in cgi, whereas 40% of locations of empirical tfbs containing the expected conserved consensus motif were in cgi. we therefore divided the predicted tfbs into cgi and non-cgi, and examined the methylation profile for each subset. as expected, predicted tfbs in cgi were hypo-methylated, whereas predicted tfbs outside of cgis were hyper-methylated. this hyper-methylation profile again suggests that where conserved consensus motifs for tf binding exist outside of cgi, promiscuous or disorderly tf binding may be controlled by dna hyper-methylation. similarly, although empirical tfbs containing the expected conserved consensus motif in cgi were hypo-methylated, non-cgi of this dataset showed a hyper-methylation profile in sperm and a neutral methylation profile in hearts. discussion interaction between dna and proteins or protein complexes can be modulated by dna methylation. indeed there are examples of dna methylation-dependent binding for transcription factors such as ctcf and ngfi-a. promiscuous or disorderly transcription factor binding may therefore be controlled by dna methylation at potential binding sites throughout the genome where there are conserved consensus motifs. here our global analysis largely supports this hypothesis for tf in general. although the possibility remains that we are only sampling methylation profiles at these sites of conserved consensus motifs at a single time point, and previous or subsequent tf binding may occur as a result of dynamic changes in dna methylation, this is the first genome-wide study to associate conserved consensus motifs with dna hyper-methylation. we found similar aggregate methylation profiles for the various sets of tfbs in parallel analyses using methylation maps from both hearts and sperm. analysis of all conserved consensus motifs throughout the genome and the subset of those that reside outside cgi showed an aggregate profile of hyper-methylation, but detailed analysis of individual tf suggests that there may be subsets of tf that behave differently. these may indeed represent specific tf whose combinatorial function is to establish or maintain the un-methylated dna state. indeed our finding of sp and nrf in this latter group corresponds to previous reports proposing this function for these two tf. we have further found that only a very small number of predicted tfbs containing conserved consensus motif are biologically proven tbfs. conversely, only a very small subset of empirical tfbs has the expected conserved consensus motif. most importantly, we found that while conserved consensus motifs without biologically proven tf binding have a hyper-methylated profile, sites of biologically proven tfbs have the opposite hypo-methylation profile. although the scales of methylation scores in our analysis are generally narrow, these scores represent composite/aggregate scores at over m locations in the genome and confidence intervals as indicated on the graphs do not show overlap from peaks to troughs, reflecting the significance of altered methylation patterns in these regions. moreover these methylation scores are not representative of "whole-genome" methylation but only of the local regions that are being analyzed in each graph, peak-to-trough scores therefore differ between analyses. interestingly, we also found that only a very small proportion of the sites of conserved consensus motif without biologically proven tf binding were within cgi s; whereas a larger proportion of sites of biologically proven tf binding were within cgis. the lack of methylation modulation in sites of biologically proven tf binding outside of cgis serves as a negative control for the other profiles of methylation differences that we have detected, but may also indicate that at these sites of empirical tf binding, a neutral methylation profile allows potential tf binding. alternatively, potential tf binding at these sites may not be regulated by dna methylation. most importantly and in contrast to that, predicted consensus tfbs that are non-cgis maintain a significant hyper-methylation pattern.  our data provides genome-wide evidence that the majority of conserved consensus motifs in the human genome are hyper-methylated, whereas biologically proven tfbs with conserved consensus motifs are hypo-methylated. this implicates a role for dna methylation in preventing promiscuous or disorderly tf binding, at least for the majority of tf. 
conserved consensus motifs exist across the human genome but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing not to be biological transcription factor binding sites. we hypothesize that dna methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. dna methylation can regulate gene expression by modulating the interaction between dna and proteins or protein complexes. using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside cpg islands have an aggregate profile of hyper-methylation. 40% of empirical tfbs with conserved consensus motifs resided in cpg islands whereas only 7% of all conserved consensus motifs were in cpg islands. in contrast, empirical tfbs with conserved consensus motifs have a profile of hypo-methylation. finally we further identified a minority subset of tf whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these tf may be responsible for establishing or maintaining an un-methylated dna state, or whose binding is not regulated by dna methylation. our analysis supports the hypothesis that at least for a subset of tf, empirical binding to conserved consensus motifs genome-wide may be controlled by dna methylation.
 dna methylation can regulate gene expression by modulating the interaction between dna and proteins or protein complexes. conserved consensus motifs exist across the human genome but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing not to be biological transcription factor binding sites. we hypothesize that dna methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding.  using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside cpg islands have an aggregate profile of hyper-methylation. in contrast, empirical tfbs with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical tfbs with conserved consensus motifs resided in cpg islands whereas only 7% of all conserved consensus motifs were in cpg islands. finally we further identified a minority subset of tf whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these tf may be responsible for establishing or maintaining an un-methylated dna state, or whose binding is not regulated by dna methylation.  our analysis supports the hypothesis that at least for a subset of tf, empirical binding to conserved consensus motifs genome-wide may be controlled by dna methylation. 