BACKGROUND
the traditional strategy for studying the role of transcription factors in gene regulation relies on either small or large-scale expression analysis following overexpression, somatic knockdown, or germ line deletion.
while these approaches permit the identification of gene networks regulated by transcription factors, little information is gathered about the chromatin coupling events that ultimately drive gene expression.
this gap in knowledge is unfortunate as transcription factors are often modular proteins armed with multiple sites for potential interactions with chromatin cofactors.
thus, knocking-out a transcription factor will, in theory, disrupt all chromatin-coupling events.
for this reason, it is important to dissect whether different transcriptional regulators use single or combinatorial coupling events to regulate distinct gene expression networks.
this knowledge is particularly important for proteins in which dysfunction contributes to disease.
impairment in each of the coupling events may yield differential effects, affecting disease penetrance as well as progression.
moreover, with the development of novel chromatin-centric pharmacology, different drugs can partially inactivate certain gene networks regulated by a transcription factor while leaving others intact.
therefore, the dissection of individual and combinatorial chromatin coupling events possesses both biological and medical relevance.
consequently, the current study focuses on klf <dig> as a model system for addressing these important questions.
klf <dig> is a well characterized human disease-causing gene that couples to several chromatin partners  <cit> .
alterations in klf <dig>  originally discovered by its role in growth regulation, causes juvenile  and neonatal  diabetes  <cit> .
klf <dig> is an inducible gene, responsive to a large variety of growth regulatory and metabolic stimuli, and functions in the nucleus to regulate gene expression by coupling to distinct chromatin partners.
upon stimuli, klf <dig> binds to promoters containing the consensus ccccgc/ccccac sequences via its three c-terminal zinc finger domains and, through well-characterized protein-protein interaction modules in its n-terminus, differentially recruits chromatin partners such as the sin3-hdac complex  <cit> , wd <dig> proteins  <cit> , and the hp1-hmt system  <cit> .
thus, through these domains, klf <dig> translates environmental signals into distinct programs of gene expression, which remain to be defined in detail.
in the current study, we have employed a combination of genome-wide chip-on-chip and gene expression profiles to reconstruct both the direct and indirect effects of klf <dig> on the regulation of different gene networks.
furthermore, we used site-directed mutants to disrupt individual chromatin coupling domains to generate genome-wide expression for the identification of the genes regulated by klf <dig> in response to binding to each of its chromatin cofactors.
our results demonstrate that certain gene programs require a single chromatin machine interaction with klf <dig> while others require interaction with multiple chromatin systems.
accordingly, we provide analyses of the distinct gene networks regulated by klf <dig> and its coupling to chromatin partners.
this type of comprehensive genome-wide analysis has never been performed for any member of the klf family.
when applied to klf <dig>  our analysis demonstrates that this protein behaves in a modular fashion with chromatin machinery to alter gene expression and impacts a variety of biological processes.
RESULTS
distinct gene expression networks are regulated by the differential coupling of klf <dig> to individual chromatin partners
recent studies support a model whereby klf <dig> functions by binding to gc-rich sites within promoters of different gene networks involved in the regulation of metabolism and cell growth  <cit> .
this data is in agreement with the biological role of this transcription factor in cancer and diabetes.
however, how binding of klf <dig> to these promoters regulates gene expression remains to be fully understood.
recent data demonstrate that the klf <dig> protein behaves as a scaffold for recruiting different chromatin cofactors via distinct structural motifs .
the sin <dig> interacting domain  between amino acids 22– <dig> enables coupling of klf <dig> to the sin3/hdac system  <cit> .
introduction of proline residues at amino acids 29– <dig> interrupts the association between klf <dig> and the sin <dig> scaffold protein, which includes interactions with hdac1/ <dig>  the region between  <dig> and  <dig> amino acids is a proline-rich domain that couples with a variety of wd <dig> proteins, including g-protein coupled receptors  <cit> .
the a347s mutation observed in mody <dig>  falls within this domain and has been demonstrated to decouple klf <dig> from novel transcription factor gβ <dig>  the zinc finger domain has been demonstrated to interact with histone acetyltransferases p <dig> and cbp  <cit> .
finally, the region between  <dig> and  <dig> dictates interaction between klf <dig> and hp1α  <cit> .
deletion of this c-terminal portion decouples hp1α from histone methyltransferases suv <dig> and g9a, leading to defects in metabolism and tumor suppression.
currently, it is unknown whether these systems work in isolation or in a cooperative manner to regulate gene expression.
to address this question, we first preformed genome-wide promoter binding analysis of wild type klf <dig>  we then limited our analysis to 5 kb upstream and downstream of the transcription start site of known gene promoters.
examination of the distance of klf11-bound peaks from each tss revealed that the majority  was within 500– <dig> base pairs of the tss .
gene ontological analysis of klf11-bound targets reveals association to a large number of metabolic processes, including many already characterized as regulated by klf <dig>  such as insulin regulation  <cit>  and akt/tor signaling  <cit>  .
next, we performed genome-wide expression profiles using mutants for these sites which have been shown to specifically uncouple klf <dig> from each of these chromatin remodeling proteins, namely eapp , a347s , and Δ <dig>  .
of the  <dig> gene targets identified by chromatin immunoprecipitation, for which corresponding gene expression data was available, 19%  display alteration in the presence of the wild type klf <dig> .
clustering analysis reveals that the expression pattern of these gene targets is disrupted in the presence of one or all of the described mutants.
thus, we conclude that klf11-mediated gene expression may be disrupted by decoupling of the transcription factor from its chromatin co-factors in a singular or combinatorial fashion.
distinct gene networks are regulated by the combinatorial coupling of klf <dig> to chromatin partners
next, we investigated which gene targets are uniquely regulated by each of these pathways.
statistical analyses of each expression profile show that, indeed, when compared with the empty vector control condition, each mutation induces changes in gene expression that are either specific to each chromatin system or similar to the wild type.
thus, comparison of the genes significantly affected by each condition  shows that 97% of transcripts that are modulated by the wild type klf <dig> are also regulated by any of the three mutations, with the a347s and Δ <dig> mutations sharing the largest number of genes with the wild type .
in fact, of the  <dig> genes modulated by the Δ <dig> mutation,  <dig> % are also modulated by the a347s mutation.
finally, the three mutants share with klf <dig> only  <dig> different genes, which likely require an intact coupling of this transcription factor to all the chromatin proteins involved in its function .
we observe that the a347s mutation shows the largest number of significantly regulated transcripts  upon the decoupling of klf <dig> from wd <dig> proteins .
by contrast, the subsets of genes uniquely regulated by the Δ <dig> or eapp mutants are much smaller,  <dig> and  <dig> genes, respectively .the Δ <dig> mutation uniquely modulates only  <dig> genes  of the  <dig> genes significantly modulated under expression of the mutant.
almost half of all Δ <dig> modulated genes  are common to the a347s mutant  and the other half are common to both a347s and wild type klf <dig> .
interestingly, the Δ <dig> and the a347s mutants have, in general, a similar direction of modulation, although with varying degrees of signal intensity.
on the other hand, the a347s and Δ <dig> modulated genes are almost always completely reverted by the eapp mutation, in which the signals are constantly proximal to empty vector values.under the same conditions, the a347s mutant, which associates to the development of human juvenile diabetes, induces changes in  <dig> genes.
<dig> % of the modulated genes  are specific to this mutation, while approximately half of the genes  are common to the Δ <dig> mutant and  <dig> % are common to both a347s and wild type klf <dig> .
finally, 7% of the genes  are common to wild type klf <dig>  and only  <dig> common to the eapp mutant.
the eapp mutation, which disrupts coupling to the sin3-hdac complex, reverses the expression of most genes induced by the wild type klf <dig> or by the other two mutations.
identification of klf11-regulated genes for which expression is independent of known chromatin coupling events
subsequently, we examined genes for which expression might be regulated by klf <dig> in a manner that does not involve the chromatin remodeling machines examined above.
a small percentage of genes are still significantly modulated uniquely by wild type klf <dig> .
ontological classification demonstrates that these  <dig> genes are associated with diabetes , cancer , or metabolism .
taken together, these data indicate that the vast majority of klf11’s biological processes are dependent on its interaction with chromatin regulators within our model system.
a repertoire of chromatin-independent function is predicted, as the transcription factor is capable of interaction with gene promoters directly.
alternatively, the regulation of these genes may involve yet unidentified chromatin co-factor.
pathway reconstruction of combinatorial klf11-regulated chromatin pathways by ontological approach
gene ontology  enrichment analysis of the genes modulated by wild type klf <dig> and each of the three mutations show various common biological processes .
indeed, the wild type shares 60%  of the biological processes with at least one of the mutants.
one of the biological processes, lipid cellular metabolic process, is enriched in wild type klf <dig> and all three mutants .
among the most highly enriched biological processes,  <dig> terms are common to wild type klf <dig> and the a347s and Δ <dig> mutations.
variations of the intensity of signal, the number of genes implicated, and the score of go enrichment for these  <dig> biological processes are shown in additional file 2: table s <dig>  interestingly, wild type klf <dig> and mutants conserve several biological processes related to anabolism or catabolism of lipids, amino acids, and glycoconjugates.
other common groups include redox processes of protein homodimerization, regulation of the activity of protein kinases, and response to the estrogen.
two of the wild type klf <dig> biological processes, the g2/m transition during dna damage checkpoint and cilium assembly, are also conserved in the a347s mutation.
there are  <dig> biological processes exclusively conserved between wild type klf <dig> and the Δ <dig> mutant.
these include cellular metabolic processes, such as aldehyde and nitrogen compound metabolism, isocitrate and 2-oxoglutarate metabolism, glycosaminoglycan catabolic processes, and fatty acid beta-oxidation.
interestingly, the a347s and Δ <dig> mutations share more than a quarter of biological processes  that are absent in wild type klf <dig> and the eapp mutant.
between these  <dig> go terms, we found enrichment of terms associated to the monitoring of the dna transcription, rna polymerase i transcription promoter regulation, and epigenetic control of gene expression, such as histone h <dig> acetylation, chromatin remodeling, protein acetylation and deacetylation, and regulation of phosphorylation.
wild type klf <dig> and eapp share only two go-terms in common: branched chain amino acid family catabolic processes and negative regulation of tor and signaling cascade.
overall,  <dig> of  <dig>  of the biological processes enriched in the wild type klf <dig> are not present any of the three mutants.
among the  <dig> most enriched biological process found only in the wild type klf <dig>  we observed several metabolic processes, such as glucose, carbohydrate, and very long-chain fatty acid metabolism, oxaloacetate metabolism, fatty acid homeostasis, and oligosaccharide metabolism.
top processes associated with wild type klf <dig> expression are shown in figure 4b and additional file 3: table s <dig>  other specific mechanisms of wild type klf <dig> lost by mutation are two mechanisms associated with gene expression  and two biological processes associated with the integrity of the dna .
finally, three important biological processes  are also lost by the mutants.
from these data, we conclude that the combinatorial association between the transcription factor and a variety of chromatin coupling systems dictates the majority of klf <dig> function, particularly in regulating gene expression and metabolism.
the high degree of overlap suggests a form of “epigenetic redundancy” to ensure the proper regulation of these gene targets and processes.
pathway reconstruction of singular klf11-regulated chromatin pathways by ontological approach
of the mutants, a347s displays the highest number of unique biological process that are not enriched in the wild type .
top processes associated with a347s mutant expression are shown in figure 4c and additional file 3: table s <dig>  interestingly, almost 30% of these biological processes are associated with the structure and maintenance of dna or telomeres , processes related to phosphorylation and dephosphorylation, deacetylation, methylation, and demethylation , and processes of transcriptional regulation by poly-ii and mrna transport .
in turn, the Δ <dig> mutant exclusively enriches  <dig> biological processes that are mediated by the wild type klf <dig> or in other mutants.
top processes associated with Δ <dig> expression are shown in figure 4c additional file 3: table s <dig>  unlike the a347s mutant, mutation Δ <dig> is particularly enriched in processes associated with metabolic and biosynthetic processes, protein export–import, and energy protein modification associated processes .
other groups of processes specifically enriched in this mutant are associated with proliferation and epithelial cell survival .
the eapp mutant did not display robust gene expression changes within the experiment.
however, utilizing a more permissive cutoff yields a list of  <dig> biological categories, although each only contains a single gene.
we find enrichment of biological processes involving nitric oxide transport, catabolic processes, and developmental morphogenesis function, among others.
top processes associated with eapp expression are shown in figure 4e and additional file 3: table s <dig>  from these data, we are able to identify singular klf <dig> chromatin coupling events dictating a number of critical processes, particularly in management of cell cycle control and dna replication, ones that inherently require a more specific and finely regulated degree of control to execute their programs under strict spatial and temporal constraints.
identification of canonical signaling pathways mediated by klf <dig> and mutants
subsequently, we wished to further assess the relatedness between the gene targets significantly modulated by klf <dig> and its mutants utilizing a semantic-based approach and the ingenuity global canonical pathways  algorithm.
the input dataset of significantly regulated gene targets was compared against the ipa canonical pathways, which are curated from published literature.
the significance of the associations between the data set and a given canonical pathway is determined by the ratio of the number of genes mapping to the pathway divided by the total number of pathway genes.
a p-value is calculated using fischer's exact test determining the probability that the association between the data set and the pathway occurs by chance alone.
gpc analysis of genes modulated by the wild type klf <dig> and the three mutants shows a variety of significantly enriched pathways .
the wild type klf <dig> shares about 71%  of its pathways with at least one of the mutants .
the most important intersection of common pathways is observed between wild type klf <dig> and mutants a347s and Δ <dig>  indeed, 90%  of these pathways are also enriched in at least one of these two mutants.
only two pathways are shared between the wild type and the eapp mutant.nevertheless, all genes implicated in these pathways are differently modulated by wild type klf <dig> and each one of the mutants.
the expression of these genes significantly regulated by wild type klf <dig> and the a347s and Δ <dig> mutants are almost always modulated in the same sense and clustered together .
in contrast, the eapp site produces a complete reversion of the signals with the levels of gene expression close to the empty vector level, as evidence by its clustering with empty vector apart from the wild type klf <dig> and other mutants.
twelve of the  <dig> significantly enriched pathways are conserved only in the wild type klf <dig>  these normal functions of the wild type klf <dig> are potentially lost by the three studied mutations.
the most enriched of these pathways includes the nucleotide excision repair pathway and the alpha-adrenergic signaling pathways.
interestingly, of all the remaining pathways, 80%  are metabolic biosynthetic pathways.mutant a347s possesses the most exclusive pathways.
indeed,  <dig> pathways are enriched in the a347s mutant compared to wild type klf <dig> and other mutants .
interestingly, over 30%  of these pathways are signaling pathways, including, egf, pdgr, p <dig>  rank and reelin signaling, and glucocorticoid receptor signaling.
furthermore, pathways involved in diabetes and pancreatic adenocarcinoma are also present.
these pathways may explain the massive number of genes differentially modulated by this mutation  and suggests that this mutant could induce new potentially unexpected and significant physiopathological changes.86% of the enriched pathways in the Δ <dig> mutant are shared with the wild type klf <dig> or the other mutants.
only four new pathways are specifically enriched by the Δ <dig> mutation .
interestingly, these pathways are implicated in the degradation of biogenic amines .
finally, the eapp mutant influences only six pathways exclusively , also primarily related to the degradation of biogenic amines.
taken together, we conclude that klf <dig> chromatin-coupled gene targets delineate into specific, well-ordered pathways, consistent with klf11’s position as a master transcriptional regulator that mediates large cascades of other transcription factors and regulators.
the effects of klf <dig> chromatin coupling are therefore not incidental to its function but a primary means by which to execute gene activation or repression.
reconstruction of downstream biological and disease networks mediated by klf <dig> and mutants
we next explored the degree of connectivity between klf <dig> and mutant-regulated genes and their pathobiological associations.
networks of significantly regulated genes were algorithmically generated based on their connectivity and assigned a score that encapsulates the relevance of the generated network to the original list of focus genes.
all edges are supported by at least one literature reference of direct physical, transcriptional, and enzymatic interactions, or from canonical information stored in the ingenuity pathways knowledge base.
a right-tailed fisher’s exact test was used to calculate the p-value for networks and a threshold of p <  <dig>  used to determine significance.
a functional analysis of a network then determined the biological functions and/or diseases that are most significant to the genes contained within the network .
of the identified processes, only three were common to wild type klf <dig> and all three mutants: cell death and survival, hereditary disorders, and molecular transport mechanisms.
wild type klf <dig> possessed the most unique networks  and included a number of characterized biological and disease networks, including cellular growth and proliferation  <cit> , endocrine disorders, reproductive system development and function  <cit> , and biliary hyperplasia, among others.
the a347s mutant uniquely associates to  <dig> networks, including ones linked to auditory disease, cellular compromise, and organismal development.
the Δ <dig> mutant uniquely associated to  <dig> networks.
among the Δ <dig> mediated networks are significant associations to gastrointestinal disease, connective tissue development, and immune presentation and response.
together, these data recapitulate the repertoire of known klf11-mediated functions and uncover a number of new potential functional and disease associations for future studies.
identification of upstream regulators of klf11-chromatin coupled pathways
finally, we wished to identify the upstream transcriptional regulators using ingenuity’s upstream regulator  analysis.
the ur analytic is based on prior knowledge of the expected effects of transcriptional regulators and their target genes stored in the ingenuity public knowledge base.
the algorithm examines how many known targets of each transcriptional regulator are present in the list of significantly regulated targets and compares direction of expression to that expected from previously published data.
for each potential regulator, an overlap p-value using fisher’s exact test and an activation z-score was computed, the latter of which determines the activation state.
a threshold p-value of < <dig>  and activation score of +/-  <dig> was considered significant for our purposes.
the end result was the identification of upstream regulators of klf <dig> or its mutants that permit the generation of plausible signaling cascades mediated by these upstream regulators through klf <dig> chromatin coupling.
the results of the ur analysis are presented in additional file 6: table s <dig>  wild type klf <dig> displayed two significant upstream inhibitory regulators, pparg and hnf1a, both of which have previously been implicated in the regulation of the transcription factor or as a co-regulator   <cit> .
the a347s mutant, consistent with the gene level, pathway, and network analyses, is the most disruptive, possessing  <dig> activating upstream regulators and  <dig> inhibitory upstream regulators.
interestingly, ezh <dig>  a histone methyltransferases, is identified as potential upstream activator.
previous reports have identified an antagonistic relationship between klf <dig> and the functions of ezh2/polycomb repressive complex  <cit> .
this data hints to other important chromatin couplings to klf <dig> that remain to be completely elucidated.
figure 7b shows the genes affected in the presence of the mutant when  <dig> is inhibited by srebf <dig> or jun and atf <dig>  for the  <dig> mutant,  <dig> upstream activators were identified and  <dig> upstream inhibitors, including srebf <dig> and hif1a .
although the eapp did not display any significant upstream regulators as defined by our full criteria, a number of other klf proteins  were identified as upstream regulators by p-value alone, suggesting that inter-association of klf proteins is also critical to klf <dig> function.
in addition, we find that klf <dig> is identified as affected downstream of both the  <dig> and a347s mutants through multiple klf <dig> upstream regulators.
together, these data suggest that transregulation among klf proteins is critical to their function and reveal the potential for a chromatin-dependent interplay between family members, permitting additional specificity in the regulation of distinct nodes within their targeted gene expression networks.
although a number of the regulators are common to the wild type klf <dig> and the a347s and Δ <dig> mutants, indicating that mutation does not necessarily impair upstream regulation or co-regulation, the activation or inhibition of these proteins regulates a distinct subset of endpoint genes.
the absence of wild type klf <dig> targets and acquisition of new, inappropriate targets in the presence of mutation places klf <dig> chromatin coupling events at the center of the transcription factor’s function or dysfunction.
in summary, here we have identified the precise interplay between klf <dig> and three of its chromatin coupling systems, namely sin3/hdac, hp1/hmts, and wd <dig> proteins, on a genome-wide scale.
the functional repertoire of klf <dig> appears to be dictated by both combinatorial effects between these systems and singular chromatin coupling events.
redundancy, particularly in critical biological processes such as metabolism and organismal development, occurs between systems to ensure preservation of critical functions.
on the other hand, singular chromatin coupling events exist to ensure precision in processes requiring strict spatial and temporal parameters to avoid inappropriate signaling.
discussion
until recently, the relationship between chromatin and transcription factors appeared largely passive.
however, the evidence that overexpression of four transcription factors—oct <dig>  sox <dig>  m-myc, and klf4—is sufficient to reprogram fully differentiated fibroblasts into pluripotent es-like cells suggests an active role for transcription factors in the epigenetic regulation of chromatin state  <cit> .
klf proteins, in particular, have demonstrated a multitude of feed-forward effects on epigenetic regulation of gene expression.
here, we propose klf <dig> as a model for understanding the critical role of sequence-specific factors in epigenetic regulation of a multitude of pathobiological processes.
klf proteins deliver epigenetic information to gene promoters through three primary mechanisms:  sensing and translating environmental stimuli into a program of gene expression,  sequence-specific targeting of chromatin remodeling complexes to gene promoters, and  transactivation of other transcription factors to assist in the regulation of large networks of interdependent genes.
transient regulation of gene expression occurs through association with histone acetyltransferases  and histone deacetyltransferases , while long-term gene silencing is enacted by interactions with histone methyltransferases  and dna methyltransferases .
no single classification scheme may accurately characterize the function of klf <dig> in its entirety.
however, the classification of family members by virtue of its co-factors provides a framework to understand the functional differences in the manner in which it engages chromatin to activate or repress transcription in a dynamic and reversible manner.
the association between klf <dig> and chromatin coupling is well established.
initial biochemical characterization of klf <dig> revealed that its n-terminus possesses a domain that adopts an α-helical conformation and that mutations within this domain significantly disrupted its binding to sin <dig> and the subsequent recruitment of hdacs  <cit> .
subsequently, klf <dig> was found to associate with hp1α  <cit> , one of the epigenetic “gatekeepers” of gene silencing through its extreme c-terminus  <cit> .
hp <dig> proteins repress gene expression by binding to h3k9me marks and interacting to h3k <dig> histone methyltransferases, such as g9a or suv39h <dig>  which methylate this same residue on adjacent nucleosomes  <cit> .
deletion of the c-terminus leads to deregulation of tumor suppression functions mediated by klf <dig>  finally, klf <dig> has also been demonstrated to associate with wd <dig>  wwi, wwii, and sh3-domain containing proteins through a proline-rich domain  <cit> .
interaction of klf <dig> and wd <dig> protein gβ <dig>  for example, is disrupted in the presence of the a347s mutation, the variant associated with development of maturity onset diabetes of the young  <dig> .
the effect of decoupling of klf <dig> to chromatin co-factors, however, has only been studied on small subsets of genes related to the system of focus.
the investigation presented here is the first to attempt to dissect the roles of klf <dig> and chromatin coupling on a genome-wide level to ascertain the combinatorial or unique effects of each type of previously identified interacting chromatin system.
utilizing a single cell type with overexpression of wild type klf <dig> and the three previously described mutants, a347s, Δ <dig>  and eapp, we were able to examine the interplay between coupling of the transcription factor to the wd <dig>  hp1/hmt, and sin3/hdac systems, respectively.
by introducing saturating amounts of each mutant into pancreatic epithelial cells, we were able to tease apart the relative and overlapping contributions of each chromatin coupling mechanism to the regulation of pathobiological gene networks.
our results reveal that in the pancreatic epithelial cell utilized as our model, a large number of the affected genes, networks, and signaling cascades are modulated by the coupling of klf <dig> to its variety of chromatin systems.
the number of genes identified as regulated independently of these systems represented <1% of the total genes mediated in the presence of wild type or mutant proteins.
it is important to underscore that the study reported here is unique as it uses genome-wide approaches to dissect the function of several different chromatin-based pathways in the transcriptional function of a single klf protein.
the klf family of transcription factors consists of  <dig> members  <cit> .
sequence identity at the carboxyl terminus among klf family members is greater than >65%, suggesting the regulation of similar types of gene promoters and the potential for synergistic or antagonistic regulation by family members.
despite the high degree of sequence identity in their dna-binding ability, the functional activities of klf members differ widely due to the high degree of variability within the n-terminal domain of each protein, the region responsible for interaction with chromatin machinery.
although the highly conserved dna-binding domain denotes their familial origins, the ultimate functional identity of each klf protein rests in its their ability to couple to the molecular machinery that regulate gene expression, in particular to bind writers, readers, and erasers of the histone code.
although some family members bind to the same chromatin partners as klf <dig>  <cit> , the degree to which the gene networks and biological processes identified here may be generalized to other family members remains to be elucidated.
however, it is tempting to speculate that the most closely related proteins klf <dig>  klf <dig>  klf <dig>  klf <dig>  klf <dig>  and klf <dig> being extensively studied in our laboratory because of their participation in metabolic processes, may at least regulate some overlapping gene networks.
these proteins contain distinct motifs which couple to similar and also distinct chromatin remodeling complexes.
in fact, it has been shown that these proteins couple to the same chromatin regulators in organisms ranging from flies to human.
notably, disruption of the klf <dig> orthologous fly gene cabut, leads to metabolic problems  <cit> .
in human, alterations in klf <dig> causes neonatal and juvenile diabetes while its closely related gene, klf <dig> causes obesity and adult type ii diabetes  <cit> .
interestingly, this function of klf <dig> appears to be influenced by a cross regulation with klf <dig>  this is interesting since our microarray data shows that, klf <dig> is significantly downregulated by wild type klf <dig> and klf <dig> is upregulated by the  <dig> and a347s mutants.
therefore, this data suggests that this subgroup of highly related subfamily of proteins, which appears to have evolved from the fly ancestor cabut, share similar chromatin coupling mechanisms, similar general function in metabolism, and may, to some extent, share targeted gene networks however, they still have some degree of biochemical differences as well as phenotypic outcomes.
on the other hand, the rest of the klf proteins, which are structurally and functionally less related to klf <dig> but may still share some chromatin partners, are predicted to have fewer overlapping co-regulated gene networks.
in this regard, we are optimistic that the type of investigations reported here will fuel the interest of both basic biologists and translational scientists who are keen on better understanding the function of this extended family of transcription factor proteins as it relates to biochemistry, genetics, epigenetics, general biology and the pathobiology of human diseases.
CONCLUSIONS
more than fifteen years ago, our research team hypothesized that the discovery and study of klf proteins and their chromatin cofactors would assist in unraveling complex human diseases.
in the intervening years, our understanding of the interplay between klf proteins and epigenetic machinery in transient and long-term gene regulation has grown exponentially.
with knowledge inferred from the predictive power of rationally derived computational models, we propose a new paradigm for klf regulation of gene networks through the translation of input from cellular milieu into epigenetic information to affect changes in chromatin structure.
klf proteins currently provide the best model for understanding the interactions between mechanisms of “hard inheritance”  and “soft inheritance”  in underscoring the phenotypic variability observed in complex disease mechanisms.
perhaps most importantly, these new pathways open up infinite possibilities for targeting new molecules and processes in therapeutic intervention and disease management.

 the traditional strategy for studying the role of transcription factors in gene regulation relies on either small or large-scale expression analysis following overexpression, somatic knockdown, or germ line deletion. while these approaches permit the identification of gene networks regulated by transcription factors, little information is gathered about the chromatin coupling events that ultimately drive gene expression. this gap in knowledge is unfortunate as transcription factors are often modular proteins armed with multiple sites for potential interactions with chromatin cofactors. thus, knocking-out a transcription factor will, in theory, disrupt all chromatin-coupling events. for this reason, it is important to dissect whether different transcriptional regulators use single or combinatorial coupling events to regulate distinct gene expression networks. this knowledge is particularly important for proteins in which dysfunction contributes to disease. impairment in each of the coupling events may yield differential effects, affecting disease penetrance as well as progression. moreover, with the development of novel chromatin-centric pharmacology, different drugs can partially inactivate certain gene networks regulated by a transcription factor while leaving others intact. therefore, the dissection of individual and combinatorial chromatin coupling events possesses both biological and medical relevance. consequently, the current study focuses on klf as a model system for addressing these important questions. klf is a well characterized human disease-causing gene that couples to several chromatin partners. alterations in klf originally discovered by its role in growth regulation, causes juvenile and neonatal diabetes. klf is an inducible gene, responsive to a large variety of growth regulatory and metabolic stimuli, and functions in the nucleus to regulate gene expression by coupling to distinct chromatin partners. upon stimuli, klf binds to promoters containing the consensus ccccgc/ccccac sequences via its three c-terminal zinc finger domains and, through well-characterized protein-protein interaction modules in its n-terminus, differentially recruits chromatin partners such as the sin3-hdac complex, wd proteins, and the hp1-hmt system. thus, through these domains, klf translates environmental signals into distinct programs of gene expression, which remain to be defined in detail. in the current study, we have employed a combination of genome-wide chip-on-chip and gene expression profiles to reconstruct both the direct and indirect effects of klf on the regulation of different gene networks. furthermore, we used site-directed mutants to disrupt individual chromatin coupling domains to generate genome-wide expression for the identification of the genes regulated by klf in response to binding to each of its chromatin cofactors. our results demonstrate that certain gene programs require a single chromatin machine interaction with klf while others require interaction with multiple chromatin systems. accordingly, we provide analyses of the distinct gene networks regulated by klf and its coupling to chromatin partners. this type of comprehensive genome-wide analysis has never been performed for any member of the klf family. when applied to klf our analysis demonstrates that this protein behaves in a modular fashion with chromatin machinery to alter gene expression and impacts a variety of biological processes.  distinct gene expression networks are regulated by the differential coupling of klf to individual chromatin partners recent studies support a model whereby klf functions by binding to gc-rich sites within promoters of different gene networks involved in the regulation of metabolism and cell growth. this data is in agreement with the biological role of this transcription factor in cancer and diabetes. however, how binding of klf to these promoters regulates gene expression remains to be fully understood. recent data demonstrate that the klf protein behaves as a scaffold for recruiting different chromatin cofactors via distinct structural motifs. the sin interacting domain between amino acids 22– enables coupling of klf to the sin3/hdac system. introduction of proline residues at amino acids 29– interrupts the association between klf and the sin scaffold protein, which includes interactions with hdac1/ the region between and amino acids is a proline-rich domain that couples with a variety of wd proteins, including g-protein coupled receptors. the a347s mutation observed in mody falls within this domain and has been demonstrated to decouple klf from novel transcription factor gβ the zinc finger domain has been demonstrated to interact with histone acetyltransferases p and cbp. finally, the region between and dictates interaction between klf and hp1α. deletion of this c-terminal portion decouples hp1α from histone methyltransferases suv and g9a, leading to defects in metabolism and tumor suppression. currently, it is unknown whether these systems work in isolation or in a cooperative manner to regulate gene expression. to address this question, we first preformed genome-wide promoter binding analysis of wild type klf we then limited our analysis to 5 kb upstream and downstream of the transcription start site of known gene promoters. examination of the distance of klf11-bound peaks from each tss revealed that the majority was within 500– base pairs of the tss. gene ontological analysis of klf11-bound targets reveals association to a large number of metabolic processes, including many already characterized as regulated by klf such as insulin regulation and akt/tor signaling. next, we performed genome-wide expression profiles using mutants for these sites which have been shown to specifically uncouple klf from each of these chromatin remodeling proteins, namely eapp, a347s, and Δ. of the gene targets identified by chromatin immunoprecipitation, for which corresponding gene expression data was available, 19% display alteration in the presence of the wild type klf. clustering analysis reveals that the expression pattern of these gene targets is disrupted in the presence of one or all of the described mutants. thus, we conclude that klf11-mediated gene expression may be disrupted by decoupling of the transcription factor from its chromatin co-factors in a singular or combinatorial fashion. distinct gene networks are regulated by the combinatorial coupling of klf to chromatin partners next, we investigated which gene targets are uniquely regulated by each of these pathways. statistical analyses of each expression profile show that, indeed, when compared with the empty vector control condition, each mutation induces changes in gene expression that are either specific to each chromatin system or similar to the wild type. thus, comparison of the genes significantly affected by each condition shows that 97% of transcripts that are modulated by the wild type klf are also regulated by any of the three mutations, with the a347s and Δ mutations sharing the largest number of genes with the wild type. in fact, of the genes modulated by the Δ mutation, % are also modulated by the a347s mutation. finally, the three mutants share with klf only different genes, which likely require an intact coupling of this transcription factor to all the chromatin proteins involved in its function. we observe that the a347s mutation shows the largest number of significantly regulated transcripts upon the decoupling of klf from wd proteins. by contrast, the subsets of genes uniquely regulated by the Δ or eapp mutants are much smaller, and genes, respectively.the Δ mutation uniquely modulates only genes of the genes significantly modulated under expression of the mutant. almost half of all Δ modulated genes are common to the a347s mutant and the other half are common to both a347s and wild type klf. interestingly, the Δ and the a347s mutants have, in general, a similar direction of modulation, although with varying degrees of signal intensity. on the other hand, the a347s and Δ modulated genes are almost always completely reverted by the eapp mutation, in which the signals are constantly proximal to empty vector values.under the same conditions, the a347s mutant, which associates to the development of human juvenile diabetes, induces changes in genes. % of the modulated genes are specific to this mutation, while approximately half of the genes are common to the Δ mutant and % are common to both a347s and wild type klf. finally, 7% of the genes are common to wild type klf and only common to the eapp mutant. the eapp mutation, which disrupts coupling to the sin3-hdac complex, reverses the expression of most genes induced by the wild type klf or by the other two mutations. identification of klf11-regulated genes for which expression is independent of known chromatin coupling events subsequently, we examined genes for which expression might be regulated by klf in a manner that does not involve the chromatin remodeling machines examined above. a small percentage of genes are still significantly modulated uniquely by wild type klf. ontological classification demonstrates that these genes are associated with diabetes, cancer, or metabolism. taken together, these data indicate that the vast majority of klf11’s biological processes are dependent on its interaction with chromatin regulators within our model system. a repertoire of chromatin-independent function is predicted, as the transcription factor is capable of interaction with gene promoters directly. alternatively, the regulation of these genes may involve yet unidentified chromatin co-factor. pathway reconstruction of combinatorial klf11-regulated chromatin pathways by ontological approach gene ontology enrichment analysis of the genes modulated by wild type klf and each of the three mutations show various common biological processes. indeed, the wild type shares 60% of the biological processes with at least one of the mutants. one of the biological processes, lipid cellular metabolic process, is enriched in wild type klf and all three mutants. among the most highly enriched biological processes, terms are common to wild type klf and the a347s and Δ mutations. variations of the intensity of signal, the number of genes implicated, and the score of go enrichment for these biological processes are shown in additional file 2: table s interestingly, wild type klf and mutants conserve several biological processes related to anabolism or catabolism of lipids, amino acids, and glycoconjugates. other common groups include redox processes of protein homodimerization, regulation of the activity of protein kinases, and response to the estrogen. two of the wild type klf biological processes, the g2/m transition during dna damage checkpoint and cilium assembly, are also conserved in the a347s mutation. there are biological processes exclusively conserved between wild type klf and the Δ mutant. these include cellular metabolic processes, such as aldehyde and nitrogen compound metabolism, isocitrate and 2-oxoglutarate metabolism, glycosaminoglycan catabolic processes, and fatty acid beta-oxidation. interestingly, the a347s and Δ mutations share more than a quarter of biological processes that are absent in wild type klf and the eapp mutant. between these go terms, we found enrichment of terms associated to the monitoring of the dna transcription, rna polymerase i transcription promoter regulation, and epigenetic control of gene expression, such as histone h acetylation, chromatin remodeling, protein acetylation and deacetylation, and regulation of phosphorylation. wild type klf and eapp share only two go-terms in common: branched chain amino acid family catabolic processes and negative regulation of tor and signaling cascade. overall, of of the biological processes enriched in the wild type klf are not present any of the three mutants. among the most enriched biological process found only in the wild type klf we observed several metabolic processes, such as glucose, carbohydrate, and very long-chain fatty acid metabolism, oxaloacetate metabolism, fatty acid homeostasis, and oligosaccharide metabolism. top processes associated with wild type klf expression are shown in figure 4b and additional file 3: table s other specific mechanisms of wild type klf lost by mutation are two mechanisms associated with gene expression and two biological processes associated with the integrity of the dna. finally, three important biological processes are also lost by the mutants. from these data, we conclude that the combinatorial association between the transcription factor and a variety of chromatin coupling systems dictates the majority of klf function, particularly in regulating gene expression and metabolism. the high degree of overlap suggests a form of “epigenetic redundancy” to ensure the proper regulation of these gene targets and processes. pathway reconstruction of singular klf11-regulated chromatin pathways by ontological approach of the mutants, a347s displays the highest number of unique biological process that are not enriched in the wild type. top processes associated with a347s mutant expression are shown in figure 4c and additional file 3: table s interestingly, almost 30% of these biological processes are associated with the structure and maintenance of dna or telomeres, processes related to phosphorylation and dephosphorylation, deacetylation, methylation, and demethylation, and processes of transcriptional regulation by poly-ii and mrna transport. in turn, the Δ mutant exclusively enriches biological processes that are mediated by the wild type klf or in other mutants. top processes associated with Δ expression are shown in figure 4c additional file 3: table s unlike the a347s mutant, mutation Δ is particularly enriched in processes associated with metabolic and biosynthetic processes, protein export–import, and energy protein modification associated processes. other groups of processes specifically enriched in this mutant are associated with proliferation and epithelial cell survival. the eapp mutant did not display robust gene expression changes within the experiment. however, utilizing a more permissive cutoff yields a list of biological categories, although each only contains a single gene. we find enrichment of biological processes involving nitric oxide transport, catabolic processes, and developmental morphogenesis function, among others. top processes associated with eapp expression are shown in figure 4e and additional file 3: table s from these data, we are able to identify singular klf chromatin coupling events dictating a number of critical processes, particularly in management of cell cycle control and dna replication, ones that inherently require a more specific and finely regulated degree of control to execute their programs under strict spatial and temporal constraints. identification of canonical signaling pathways mediated by klf and mutants subsequently, we wished to further assess the relatedness between the gene targets significantly modulated by klf and its mutants utilizing a semantic-based approach and the ingenuity global canonical pathways algorithm. the input dataset of significantly regulated gene targets was compared against the ipa canonical pathways, which are curated from published literature. the significance of the associations between the data set and a given canonical pathway is determined by the ratio of the number of genes mapping to the pathway divided by the total number of pathway genes. a p-value is calculated using fischer's exact test determining the probability that the association between the data set and the pathway occurs by chance alone. gpc analysis of genes modulated by the wild type klf and the three mutants shows a variety of significantly enriched pathways. the wild type klf shares about 71% of its pathways with at least one of the mutants. the most important intersection of common pathways is observed between wild type klf and mutants a347s and Δ indeed, 90% of these pathways are also enriched in at least one of these two mutants. only two pathways are shared between the wild type and the eapp mutant.nevertheless, all genes implicated in these pathways are differently modulated by wild type klf and each one of the mutants. the expression of these genes significantly regulated by wild type klf and the a347s and Δ mutants are almost always modulated in the same sense and clustered together. in contrast, the eapp site produces a complete reversion of the signals with the levels of gene expression close to the empty vector level, as evidence by its clustering with empty vector apart from the wild type klf and other mutants. twelve of the significantly enriched pathways are conserved only in the wild type klf these normal functions of the wild type klf are potentially lost by the three studied mutations. the most enriched of these pathways includes the nucleotide excision repair pathway and the alpha-adrenergic signaling pathways. interestingly, of all the remaining pathways, 80% are metabolic biosynthetic pathways.mutant a347s possesses the most exclusive pathways. indeed, pathways are enriched in the a347s mutant compared to wild type klf and other mutants. interestingly, over 30% of these pathways are signaling pathways, including, egf, pdgr, p rank and reelin signaling, and glucocorticoid receptor signaling. furthermore, pathways involved in diabetes and pancreatic adenocarcinoma are also present. these pathways may explain the massive number of genes differentially modulated by this mutation and suggests that this mutant could induce new potentially unexpected and significant physiopathological changes.86% of the enriched pathways in the Δ mutant are shared with the wild type klf or the other mutants. only four new pathways are specifically enriched by the Δ mutation. interestingly, these pathways are implicated in the degradation of biogenic amines. finally, the eapp mutant influences only six pathways exclusively, also primarily related to the degradation of biogenic amines. taken together, we conclude that klf chromatin-coupled gene targets delineate into specific, well-ordered pathways, consistent with klf11’s position as a master transcriptional regulator that mediates large cascades of other transcription factors and regulators. the effects of klf chromatin coupling are therefore not incidental to its function but a primary means by which to execute gene activation or repression. reconstruction of downstream biological and disease networks mediated by klf and mutants we next explored the degree of connectivity between klf and mutant-regulated genes and their pathobiological associations. networks of significantly regulated genes were algorithmically generated based on their connectivity and assigned a score that encapsulates the relevance of the generated network to the original list of focus genes. all edges are supported by at least one literature reference of direct physical, transcriptional, and enzymatic interactions, or from canonical information stored in the ingenuity pathways knowledge base. a right-tailed fisher’s exact test was used to calculate the p-value for networks and a threshold of p <  used to determine significance. a functional analysis of a network then determined the biological functions and/or diseases that are most significant to the genes contained within the network. of the identified processes, only three were common to wild type klf and all three mutants: cell death and survival, hereditary disorders, and molecular transport mechanisms. wild type klf possessed the most unique networks and included a number of characterized biological and disease networks, including cellular growth and proliferation, endocrine disorders, reproductive system development and function, and biliary hyperplasia, among others. the a347s mutant uniquely associates to networks, including ones linked to auditory disease, cellular compromise, and organismal development. the Δ mutant uniquely associated to networks. among the Δ mediated networks are significant associations to gastrointestinal disease, connective tissue development, and immune presentation and response. together, these data recapitulate the repertoire of known klf11-mediated functions and uncover a number of new potential functional and disease associations for future studies. identification of upstream regulators of klf11-chromatin coupled pathways finally, we wished to identify the upstream transcriptional regulators using ingenuity’s upstream regulator analysis. the ur analytic is based on prior knowledge of the expected effects of transcriptional regulators and their target genes stored in the ingenuity public knowledge base. the algorithm examines how many known targets of each transcriptional regulator are present in the list of significantly regulated targets and compares direction of expression to that expected from previously published data. for each potential regulator, an overlap p-value using fisher’s exact test and an activation z-score was computed, the latter of which determines the activation state. a threshold p-value of < and activation score of +/- was considered significant for our purposes. the end result was the identification of upstream regulators of klf or its mutants that permit the generation of plausible signaling cascades mediated by these upstream regulators through klf chromatin coupling. the results of the ur analysis are presented in additional file 6: table s wild type klf displayed two significant upstream inhibitory regulators, pparg and hnf1a, both of which have previously been implicated in the regulation of the transcription factor or as a co-regulator. the a347s mutant, consistent with the gene level, pathway, and network analyses, is the most disruptive, possessing activating upstream regulators and inhibitory upstream regulators. interestingly, ezh a histone methyltransferases, is identified as potential upstream activator. previous reports have identified an antagonistic relationship between klf and the functions of ezh2/polycomb repressive complex. this data hints to other important chromatin couplings to klf that remain to be completely elucidated. figure 7b shows the genes affected in the presence of the mutant when is inhibited by srebf or jun and atf for the mutant, upstream activators were identified and upstream inhibitors, including srebf and hif1a. although the eapp did not display any significant upstream regulators as defined by our full criteria, a number of other klf proteins were identified as upstream regulators by p-value alone, suggesting that inter-association of klf proteins is also critical to klf function. in addition, we find that klf is identified as affected downstream of both the and a347s mutants through multiple klf upstream regulators. together, these data suggest that transregulation among klf proteins is critical to their function and reveal the potential for a chromatin-dependent interplay between family members, permitting additional specificity in the regulation of distinct nodes within their targeted gene expression networks. although a number of the regulators are common to the wild type klf and the a347s and Δ mutants, indicating that mutation does not necessarily impair upstream regulation or co-regulation, the activation or inhibition of these proteins regulates a distinct subset of endpoint genes. the absence of wild type klf targets and acquisition of new, inappropriate targets in the presence of mutation places klf chromatin coupling events at the center of the transcription factor’s function or dysfunction. in summary, here we have identified the precise interplay between klf and three of its chromatin coupling systems, namely sin3/hdac, hp1/hmts, and wd proteins, on a genome-wide scale. the functional repertoire of klf appears to be dictated by both combinatorial effects between these systems and singular chromatin coupling events. redundancy, particularly in critical biological processes such as metabolism and organismal development, occurs between systems to ensure preservation of critical functions. on the other hand, singular chromatin coupling events exist to ensure precision in processes requiring strict spatial and temporal parameters to avoid inappropriate signaling. discussion until recently, the relationship between chromatin and transcription factors appeared largely passive. however, the evidence that overexpression of four transcription factors—oct sox m-myc, and klf4—is sufficient to reprogram fully differentiated fibroblasts into pluripotent es-like cells suggests an active role for transcription factors in the epigenetic regulation of chromatin state. klf proteins, in particular, have demonstrated a multitude of feed-forward effects on epigenetic regulation of gene expression. here, we propose klf as a model for understanding the critical role of sequence-specific factors in epigenetic regulation of a multitude of pathobiological processes. klf proteins deliver epigenetic information to gene promoters through three primary mechanisms: sensing and translating environmental stimuli into a program of gene expression, sequence-specific targeting of chromatin remodeling complexes to gene promoters, and transactivation of other transcription factors to assist in the regulation of large networks of interdependent genes. transient regulation of gene expression occurs through association with histone acetyltransferases and histone deacetyltransferases, while long-term gene silencing is enacted by interactions with histone methyltransferases and dna methyltransferases. no single classification scheme may accurately characterize the function of klf in its entirety. however, the classification of family members by virtue of its co-factors provides a framework to understand the functional differences in the manner in which it engages chromatin to activate or repress transcription in a dynamic and reversible manner. the association between klf and chromatin coupling is well established. initial biochemical characterization of klf revealed that its n-terminus possesses a domain that adopts an α-helical conformation and that mutations within this domain significantly disrupted its binding to sin and the subsequent recruitment of hdacs. subsequently, klf was found to associate with hp1α, one of the epigenetic “gatekeepers” of gene silencing through its extreme c-terminus. hp proteins repress gene expression by binding to h3k9me marks and interacting to h3k histone methyltransferases, such as g9a or suv39h which methylate this same residue on adjacent nucleosomes. deletion of the c-terminus leads to deregulation of tumor suppression functions mediated by klf finally, klf has also been demonstrated to associate with wd wwi, wwii, and sh3-domain containing proteins through a proline-rich domain. interaction of klf and wd protein gβ for example, is disrupted in the presence of the a347s mutation, the variant associated with development of maturity onset diabetes of the young. the effect of decoupling of klf to chromatin co-factors, however, has only been studied on small subsets of genes related to the system of focus. the investigation presented here is the first to attempt to dissect the roles of klf and chromatin coupling on a genome-wide level to ascertain the combinatorial or unique effects of each type of previously identified interacting chromatin system. utilizing a single cell type with overexpression of wild type klf and the three previously described mutants, a347s, Δ and eapp, we were able to examine the interplay between coupling of the transcription factor to the wd hp1/hmt, and sin3/hdac systems, respectively. by introducing saturating amounts of each mutant into pancreatic epithelial cells, we were able to tease apart the relative and overlapping contributions of each chromatin coupling mechanism to the regulation of pathobiological gene networks. our results reveal that in the pancreatic epithelial cell utilized as our model, a large number of the affected genes, networks, and signaling cascades are modulated by the coupling of klf to its variety of chromatin systems. the number of genes identified as regulated independently of these systems represented <1% of the total genes mediated in the presence of wild type or mutant proteins. it is important to underscore that the study reported here is unique as it uses genome-wide approaches to dissect the function of several different chromatin-based pathways in the transcriptional function of a single klf protein. the klf family of transcription factors consists of members. sequence identity at the carboxyl terminus among klf family members is greater than >65%, suggesting the regulation of similar types of gene promoters and the potential for synergistic or antagonistic regulation by family members. despite the high degree of sequence identity in their dna-binding ability, the functional activities of klf members differ widely due to the high degree of variability within the n-terminal domain of each protein, the region responsible for interaction with chromatin machinery. although the highly conserved dna-binding domain denotes their familial origins, the ultimate functional identity of each klf protein rests in its their ability to couple to the molecular machinery that regulate gene expression, in particular to bind writers, readers, and erasers of the histone code. although some family members bind to the same chromatin partners as klf, the degree to which the gene networks and biological processes identified here may be generalized to other family members remains to be elucidated. however, it is tempting to speculate that the most closely related proteins klf klf klf klf klf and klf being extensively studied in our laboratory because of their participation in metabolic processes, may at least regulate some overlapping gene networks. these proteins contain distinct motifs which couple to similar and also distinct chromatin remodeling complexes. in fact, it has been shown that these proteins couple to the same chromatin regulators in organisms ranging from flies to human. notably, disruption of the klf orthologous fly gene cabut, leads to metabolic problems. in human, alterations in klf causes neonatal and juvenile diabetes while its closely related gene, klf causes obesity and adult type ii diabetes. interestingly, this function of klf appears to be influenced by a cross regulation with klf this is interesting since our microarray data shows that, klf is significantly downregulated by wild type klf and klf is upregulated by the and a347s mutants. therefore, this data suggests that this subgroup of highly related subfamily of proteins, which appears to have evolved from the fly ancestor cabut, share similar chromatin coupling mechanisms, similar general function in metabolism, and may, to some extent, share targeted gene networks however, they still have some degree of biochemical differences as well as phenotypic outcomes. on the other hand, the rest of the klf proteins, which are structurally and functionally less related to klf but may still share some chromatin partners, are predicted to have fewer overlapping co-regulated gene networks. in this regard, we are optimistic that the type of investigations reported here will fuel the interest of both basic biologists and translational scientists who are keen on better understanding the function of this extended family of transcription factor proteins as it relates to biochemistry, genetics, epigenetics, general biology and the pathobiology of human diseases.  more than fifteen years ago, our research team hypothesized that the discovery and study of klf proteins and their chromatin cofactors would assist in unraveling complex human diseases. in the intervening years, our understanding of the interplay between klf proteins and epigenetic machinery in transient and long-term gene regulation has grown exponentially. with knowledge inferred from the predictive power of rationally derived computational models, we propose a new paradigm for klf regulation of gene networks through the translation of input from cellular milieu into epigenetic information to affect changes in chromatin structure. klf proteins currently provide the best model for understanding the interactions between mechanisms of “hard inheritance” and “soft inheritance” in underscoring the phenotypic variability observed in complex disease mechanisms. perhaps most importantly, these new pathways open up infinite possibilities for targeting new molecules and processes in therapeutic intervention and disease management. 
in the current study, we use genome-wide analyses of chromatin immunoprecipitation and affymetrix-based expression profiling to gain insight into how klf a human transcription factor involved in tumor suppression and metabolic diseases, works by coupling to three co-factor groups: the sin3-histone deacetylase system, wd40-domain containing proteins, and the hp1-histone methyltransferase system. this study, the first of its type for any klf protein, reveals that interactions with multiple chromatin systems are required for the full gene regulatory function of these proteins. our results reveal that klf regulates distinct gene networks involved in metabolism and growth by using single or combinatorial coupling events. however, scant information is available on either the mechanisms or functional impact of the coupling of klf proteins to chromatin remodeling machines, a deterministic step in transcriptional regulation. krüppel-like factors are a group of master regulators of gene expression conserved from flies to human. krüppel-like factortranscription factorgene expression profilinggene networksmetabolismcellular growthproliferationsignaling pathways
 krüppel-like factors are a group of master regulators of gene expression conserved from flies to human. however, scant information is available on either the mechanisms or functional impact of the coupling of klf proteins to chromatin remodeling machines, a deterministic step in transcriptional regulation. results and discussion in the current study, we use genome-wide analyses of chromatin immunoprecipitation and affymetrix-based expression profiling to gain insight into how klf a human transcription factor involved in tumor suppression and metabolic diseases, works by coupling to three co-factor groups: the sin3-histone deacetylase system, wd40-domain containing proteins, and the hp1-histone methyltransferase system. our results reveal that klf regulates distinct gene networks involved in metabolism and growth by using single or combinatorial coupling events. conclusion this study, the first of its type for any klf protein, reveals that interactions with multiple chromatin systems are required for the full gene regulatory function of these proteins. krüppel-like factortranscription factorgene expression profilinggene networksmetabolismcellular growthproliferationsignaling pathways  in the current study, we use genome-wide analyses of chromatin immunoprecipitation and affymetrix-based expression profiling to gain insight into how klf a human transcription factor involved in tumor suppression and metabolic diseases, works by coupling to three co-factor groups: the sin3-histone deacetylase system, wd40-domain containing proteins, and the hp1-histone methyltransferase system. our results reveal that klf regulates distinct gene networks involved in metabolism and growth by using single or combinatorial coupling events.  this study, the first of its type for any klf protein, reveals that interactions with multiple chromatin systems are required for the full gene regulatory function of these proteins. krüppel-like factortranscription factorgene expression profilinggene networksmetabolismcellular growthproliferationsignaling pathways 