BACKGROUND
twist <dig> is a highly conserved basic-helix-loop-helix  transcription factor, which regulates cell migration, proliferation, and differentiation in progenitor populations, such as embryonic neural crest cells, cranial mesoderm, limb buds, and endocardial cushions
.
in addition to its role in developmental processes, human twist <dig> is highly expressed in diseased heart valves and in metastatic tumor cells
.
twist <dig> expression is normally silenced after embryonic development, but expression is reactivated during disease, including cancer.
twist <dig> controls gene expression through binding e-box  consensus sites as a homodimer or heterodimer with other bhlh transcription factors
<cit> .
however, a comparison of twist <dig> direct transcriptional targets in developing and diseased tissues has yet to be reported.
this study focuses on identifying and comparing twist <dig> genomic occupancy in developing ecc, limb buds, and a cancer cell line.
in multiple structures in the developing embryo, including the heart, limb buds, cranial sutures, skeletal elements, and neural crest derivatives, twist <dig> regulates cell migration and proliferation of progenitor populations
<cit> .
in the developing heart, twist <dig> expression is robust in the mesenchymal cells of the ecc and is subsequently down-regulated during remodeling and differentiation of the valve leaflets
<cit> .
in the ecc, twist <dig> promotes cell migration and proliferation of mesenchymal cells.
direct transcriptional targets of twist <dig> include genes involved in cell migration  and proliferation
<cit> .
in the developing limbs, twist <dig> is broadly expressed in the limb bud mesenchyme and strictly regulated genetic dosage of twist <dig> is required for limb bud outgrowth and patterning
<cit> .
although extensive genetic studies have been performed demonstrating an essential role for twist <dig> in limb patterning and morphogenesis, the direct transcriptional targets underlying these functions are only recently being elucidated
<cit> .
in addition, it is not known if twist <dig> regulates the same downstream targets in distinct progenitor populations or if there is tissue-specificity to twist <dig> regulatory mechanisms in the developing embryo.
while twist <dig> is largely absent from adult differentiated tissues, it is expressed in diseased heart valves and highly metastatic cancers such as breast, pancreatic, gastric, prostate, and malignant peripheral nerve sheath tumors
<cit> .
human mpnst cells can harbor nf <dig> and p <dig> mutations, and murine nf <dig> and p <dig> mutations can cause similar nerve-associated sarcomas, peripheral nerve sheath tumors
.
sirna-mediated knockdown of twist <dig> abrogates the migratory activity of human mpnst cells in vitro
<cit> .
twist <dig> also is expressed in invasive carcinoma and promotes endothelial-to-mesenchymal transformation  and metastasis of tumor cells through regulation of genes involved in differentiation, adhesion, and proliferation
<cit> .
it has not previously been determined if the downstream targets of twist <dig> related to regulation of cell migration or proliferation in embryonic structures and cancer cells are shared or are cell-type specific.
to investigate the tissue-specificity of twist <dig> genomic occupancy, we performed chromatin immunoprecipitation  with anti-twist <dig> followed by nextgen sequencing  in mouse embryonic day  <dig>  eccs, e <dig>  forelimb buds, and cultured pnst cells.
the specificity of twist <dig> binding was confirmed by chip qpcr in each tissue.
potential twist <dig> cofactors and intersecting signaling pathways also were identified.
RESULTS
analysis of twist <dig> genomic occupancy in eccs, limb buds, and pnst cells
twist <dig> genomic occupancy was assessed by chip with anti-twist <dig> in mouse e <dig>  eccs, e <dig>  limb buds, and pnst cells followed by deep sequencing .
next gen sequencing was used to generate  <dig> , <dig> reads for e <dig>  eccs,  <dig> , <dig> reads for e <dig>  limb buds, and  <dig> , <dig> reads for pnst cells.
resulting sequences were aligned to the mouse genome , and peaks were discovered using macs analysis.
a p-value cut-off of  <dig> e- <dig> was used to identify  <dig>  peaks for e <dig>  eccs,  <dig>  peaks for e <dig>  limb buds, and  <dig>  peaks for pnst cells  relative to comparable reads for fvb/n mouse genomic dna as a control.
in order to confirm enrichment of e-box binding sites in each data set, de-novo motif enrichment was performed using pscanchip and meme-chip
<cit> .
e-box containing motifs were significantly enriched in all datasets when analyzed by pscanchip and meme-chip .
peaks were filtered based on distance from the nearest transcriptional start site  with a threshold of  <dig> kilobases  resulting in  <dig>  peaks for e <dig>  eccs,  <dig>  peaks for e <dig>  limb buds, and  <dig>  peaks for pnst cells .
in order to compare twist <dig> genomic occupancy with genes expressed in each tissue, genes associated with binding peaks in each tissue were compared to respective affymetrix microarray gene expression data for e <dig>  eccs, e <dig>  limb buds , and pnst cells
<cit> .
the identities of genes associated with binding peaks within  <dig> kb of tss, identified by chip-seq were compared to the total expressed genes as detected by microarray for each tissue individually .
these comparisons were used to generate lists of twist <dig> binding regions corresponding to expressed genes in each tissue.
for e <dig>  eccs,  <dig>  twist <dig> binding peaks corresponding to  <dig> expressed genes were identified .
twist <dig> occupancy was detected for  <dig>  peaks corresponding to  <dig> expressed genes in e <dig>  limb buds, and  <dig> peaks corresponding to  <dig> expressed genes were detected for pnst cells .
the list of genes for pnst cells is likely an underestimate of twist <dig> genomic occupancy because only genes that are expressed in both human mpnst and mouse pnst cell lines were included
<cit> .
these chip-seq data indicate that twist <dig> occupies genomic regions associated with large numbers of genes expressed in eccs, limb buds, and pnst cells.figure  <dig>
twist <dig> genomic occupancy in endocardial cushions, limb buds, and peripheral nerve sheath tumor cells.
a. twist <dig> binding regions were identified by chip-seq followed by macs analysis in e <dig>  eccs, e <dig>  limb buds, and pnst cells compared to a control strain matched whole genome dataset.
the total number of peaks, the number of peaks within  <dig> kilobases  of a transcriptional start site , and the number of peaks associated with expressed genes, as determined by microarray gene expression analysis of the tissue of interest, are shown.
b. the total number of shared peaks detected by twist <dig> chip-seq of ecc-limb, limb-pnst, ecc-pnst and ecc-limb-pnst groups is shown.
the number of overlapping peaks within  <dig> kb of a tss for expressed genes also was determined.
tissue-specific and shared gene targets of twist <dig> in eccs, limb buds, and pnst cells corresponding to binding regions within  <dig> kb of tss were analyzed
<cit> .
binding region overlap determination was performed before and after peaks were compared to gene expression profiles .
these analyses produced datasets containing binding regions that are unique to a specific tissue type or shared among multiple tissue types.
groups of binding regions were classified as ecc only, limb only, pnst only, ecc-limb, ecc-pnst, limb-pnst, or ecc-limb-pnst.
e <dig>  eccs and e <dig>  limb buds share the most binding regions, with  <dig>  shared peaks in the ecc-limb group before comparison to mrna expression data, and  <dig> ecc-limb binding regions associated with expressed genes after comparison with mrna expression.
similarly the limb-pnst group contains  <dig> shared binding regions, before the expression filter, with only  <dig> shared binding regions corresponding to expressed genes.
the ecc-pnst group contains  <dig> total shared binding regions, with  <dig> shared binding regions corresponding to expressed genes.
surprisingly, only  <dig> binding regions associated with expressed genes were occupied by twist <dig> in all three cell types , indicating a high degree of tissue-specificity in twist <dig> genomic occupancy.
genomic sequences of the shared binding regions associated with expressed genes were analyzed for presence of an e-box consensus site on the sense or antisense strand , which would indicate a potential twist <dig> binding site.
of the  <dig> ecc-limb shared binding regions associated with expressed genes,  <dig> contain an e-box consensus site, corresponding to  <dig> genes.
in addition, e-box consensus sites are present in 7/ <dig> ecc-pnst shared binding regions, 6/ <dig> of the limb-pnst binding regions, and 5/ <dig> of the ecc-limb-pnst shared binding regions.
collectively, these chip-seq experiments demonstrate extensive twist <dig> binding to genomic sequences and indicate that twist <dig> bound predominantly in a tissue-specific manner, with much smaller numbers of shared binding regions detected among eccs, limb buds, and pnst cells.
in order to examine twist1-dna binding distribution across the genome and determine if twist <dig> is bound to specific genomic regions relative to gene organization, location analysis was performed.
twist <dig> binding regions located within  <dig> kb of a tss for genes expressed in each tissue were subjected to genomic location and peak distribution analysis using genomatix regionminer .
twist <dig> binding regions associated with expressed genes were analyzed in tissue-specific gene groups , and also in groups of binding regions shared between tissue types .
distribution of the tissue-specific binding regions and ecc-limb shared peaks is largely within intergenic and intronic regions.
however, ecc-pnst and limb-pnst shared binding regions are more evenly distributed in genomic regions.
this lack of specificity in peak distribution relative to gene organization is consistent with other transcription factors, including twist <dig>  in other cell types
<cit> .
these data suggest that twist <dig> occupies genomic sequences associated with expressed genes located in intergenic and intragenic genomic regions and does not bind exclusively to promoter regions.
functional classification of genes associated with twist <dig> binding peaks
the major biological functions of genes with twist <dig> binding regions identified within  <dig> kb of tss, that also are expressed in eccs, limb buds, or pnst cells, were assessed using gene ontology  analysis .
the four go categories containing the greatest numbers of genes in each tissue were chosen for further analysis.
in eccs, the four predominant go groups were cell-cell signaling, cell adhesion, cell morphogenesis, and neurogenesis .
the top four go categories of twist <dig> occupied genes in limb buds were kinase activity, cell migration, neurogenesis, and cytoskeletal proteins .
for pnst cells, the four go groups with the most genes were cell adhesion, skeletal system development, cell recognition, and ventricular septum morphogenesis .
cell adhesion-cell migration was among the most predominant go categories in each tissue type, indicating a conserved role for twist <dig> in cell adhesion-migration in multiple cellular contexts .
however, the specific genes corresponding to twist <dig> binding regions in each of these go categories were unique to each tissue type, suggesting cell-type specificity of twist <dig> genomic occupancy of gene targets associated with cell adhesion and migration.figure  <dig>
gene ontology analysis of the genes associated with twist <dig> binding regions in eccs, limb buds, and pnst cells.
a, b, c. toppgene was utilized to perform go analysis of the genes associated with twist <dig> chip-seq peaks that are within  <dig> kb of a transcriptional start site  of expressed genes in e <dig>  eccs , e <dig>  limb buds , or pnst cells .
the four most predominant go categories and the total number of genes in each category are represented.
the biological functions of ecc-limb shared binding regions associated with expressed genes were further examined resulting in  <dig> genes associated with the  <dig> binding regions containing an e-box consensus site .
again, adhesion-migration was the most predominant go category in genes associated with ecc-limb shared binding regions containing e-box consensus sites associated with expressed genes.
additional go categories include mrna metabolic process, microtubule-based process, and organ morphogenesis.
thus, the expressed gene identities associated with twist <dig> shared binding regions in eccs and limb buds suggest conserved functions for twist <dig> in regulation of cell adhesion-migration during development in different tissues.
expressed genes associated with shared binding regions containing e-box binding sequences related to cell adhesion and migration also were identified for ecc-pnst, limb-psnt and ecc-limb-pnst groups .
the overlapping peaks in the ecc-limb-pnst group correspond to six shared binding regions associated with  <dig> genes: cd <dig> antigen , carbohydrate sulfotransferase  <dig> , lps-induced tn factor , receptor tyrosine kinase-like orphan receptor  <dig> , and spermatogenesis associated  <dig> .
these five genes relate to the biological processes of cell adhesion-migration, cell differentiation, signal transduction, and cartilage development .
these biological processes are consistent with known twist <dig> functions during embryogenesis and in cancer cells.figure  <dig>
shared twist <dig> binding regions detected by chip-seq in ecc, limb buds, and pnst cells.
a. twist <dig> chip-seq peaks identified in e <dig>  eccs, e <dig>  limb buds, and pnst cells were filtered based on a cut off of  <dig> kb from the nearest tss and gene expression as determined by microarray in each cell type.
the peaks associated with expressed genes from each tissue type were analyzed for shared peaks and are represented in the venn diagram.
b. a total of  <dig> twist <dig> binding regions were detected in both ecc and limb bud, and  <dig> contain e-box consensus sequences.
the four predominant go categories for the  <dig> genes associated with shared ecc-limb e-box-containing binding regions are shown.
c. gene names and descriptions of the four go categories containing the most genes for ecc-limb e-box containing binding regions from b are shown.
d. gene names and descriptions are shown for shared binding regions detected in ecc-pnst, limb-pnst, and ecc-limb-pnst groups.
validation of twist <dig> genomic occupancy in ecc, limb buds and pnst cells
to further examine tissue-specificity of twist <dig> binding, the top three e-box containing binding regions associated with expressed genes, as determined by the highest fold-enrichment in each cell type, were chosen for further analysis.
chip with anti-twist <dig> was performed in e <dig>  eccs, e <dig>  limb buds, and pnst cells followed by quantification using qpcr relative to normal rabbit igg control.
ecc tissue-specific binding regions chosen for further analysis were associated with the genes leukocyte cell derived chemotaxin  <dig>  shroom family member  <dig> , and bromodomain and wd repeat domain containing  <dig>  twist <dig> chip followed by qpcr demonstrated binding of twist <dig> to ecc-specific regions in eccs, but not in limb buds or pnst cells .
likewise, the limb bud-specific binding regions were associated with the genes son of sevenless homolog  <dig>  transmembrane protein  <dig> , and abhydrolase domain containing  <dig> , and were bound by twist <dig> in limb buds, but not in eccs or pnst cells .
finally, pnst cell-specific binding regions associated with the genes cd <dig> antigen , calcium/calmodulin-dependent serine protein kinase , and transforming growth factor, beta  <dig>  were bound by twist <dig> in pnst cells, but not in ecc or limb buds .
these data further support the tissue-specificity of twist <dig> binding to genomic sequences detected by chip-seq in ecc, limb buds, and pnst cells.figure  <dig>
validation of twist <dig> chip-seq peak specificity ecc, limb, or pnst cells.
chip was performed with anti-twist <dig> in e <dig>  eccs , e <dig>  limb buds , and pnst cells  followed by sequence-specific qpcr to examine relative binding of twist <dig> to the three binding regions with the highest fold enrichment, from macs analysis, associated with expressed genes in each tissue.
in e <dig>  eccs, twist <dig> occupies binding regions associated with leukocyte cell derived chemotaxin 1
, shroom family member  <dig> , and bromodomain and wd repeat domain containing 3
.
in e <dig>  limb buds, twist <dig> occupies binding regions associated with son of sevenless homolog 2
, transmembrane protein  <dig> , and abhydrolase domain containing  <dig> .
in pnst cells, twist <dig> occupies binding regions associated with cd <dig> antigen  and calcium/calmodulin-dependent serine protein kinase , and transforming growth factor, beta  <dig> .
chip quantification was performed by qpcr, and fold-enrichment was calculated versus an anti-rabbit igg control for each tissue.
all analyses were performed in biological triplicate.
* indicates p <  <dig>  as determined by student’s t-test comparing igg and twist <dig> chip qpcr ΔΔct values.
in order to examine twist <dig> occupancy at shared sequences associated with genes expressed in ecc-limb or ecc-limb-pnst groups, chip was performed and enrichment at overlapping binding regions was quantified by sequence-specific qpcr.
ecc-limb overlapping binding regions containing e-box binding sites associated with expressed genes from the four major go categories  were sorted according to proximity to tss for greater probability of identifying proximal regulatory sequences, and validation was performed on the two binding peaks nearest the tss.
intronic binding regions associated with midline  <dig>  and cdc <dig> binding protein kinase alpha  were bound by twist <dig> in both eccs and limb buds .
of the six binding regions shared among all three-tissue types associated with expressed genes, five binding regions contain e-box consensus sites and four were analyzed by chip-qpcr for twist <dig> occupancy.
twist <dig> occupancy at shared binding regions associated with chst <dig>  litaf, ror <dig> , and spata <dig> was confirmed in eccs, limb buds, and pnst cells, but binding to the shared cd <dig> site was not detected .
thus, twist <dig> binding was detected at small group of shared binding regions in ecc, limb bud, and pnst cells, which may represent common targets of twist <dig> in multiple cell types.figure  <dig>
twist <dig> and candidate cofactors occupy binding regions shared among ecc, limb bud, and pnst cells.
a. binding of twist <dig> to regions associated with the genes midline  <dig>  and cdc <dig> binding protein kinase alpha  detected in e <dig>  eccs and e <dig>  limb chip-seq was validated by chip with anti-twist <dig> in e <dig>  eccs and e <dig>  limb buds followed by sequence-specific qpcr.
b. twist <dig> binding regions containing an e-box sequence, as detected by chip-seq in e <dig>  eccs, e <dig>  limb buds, and pnst, are associated with receptor tyrosine kinase-like orphan receptor  <dig> , carbohydrate sulfotransferase  <dig> , spermatogenesis associated  <dig>  and lps-induced tn factor .
twist <dig> chip followed by qpcr indicates that twist <dig> binds each of these sequences in e <dig>  eccs, e <dig>  limb buds, and pnst cells.
c. genomatix regionminer was utilized to identify rxr as a potential cofactor with twist <dig> in e <dig>  limb bud.
twist <dig> binding regions containing an e-box within  <dig> bp of an rxr binding consensus associated with ror2-a and wnt5b genes involved in limb development were chosen for validation.
chip was performed with anti-rxr in e <dig>  limb buds followed qpcr amplification of twist <dig> binding region sequences.
d. rreb <dig> consensus binding sites were identified within  <dig> base pairs of an e-box in twist <dig> binding peak sequences of chst <dig>  litaf, ror2-a, and spata <dig> detected in ecc, limb bud and pnst cells.
chip was performed with anti-rreb <dig> followed by qpcr amplification of twist <dig> binding region sequences in eccs, limb buds, and pnst cells.
e. motif of rxr consensus site.
f. motif of rreb consensus site.
fold-enrichment was calculated relative to normal rabbit igg control.
all analyses were performed in biological triplicate.
* indicates p <  <dig>  as determined by student’s t-test comparing igg and twist <dig>  igg and rxr, or igg and rreb <dig> chip qpcr ΔΔct values.
identification of potential twist <dig> cofactors in eccs, limb buds, and pnst cells
the presence of tissue-specific twist <dig> cofactors was examined as a potential mechanism underlying specificity of target occupancy.
twist <dig> binding regions associated with expressed genes for each tissue type  were analyzed for transcriptional modules containing a transcription factor-binding sequence within +/-  <dig> base pairs of the e-box consensus site using genomatix regionminer .
in ecc, cp2-erythrocyte factor related to drosophila elf <dig>  sites and heat shock factor sites are located in proximity to e-boxes in approximately 10% of the twist <dig> occupied sequences.
retinoid x receptor  sites  are present in 43% of the limb-only twist <dig> occupied sites and also in 44% of the ecc-limb shared twist <dig> occupied sites.
interestingly, binding sites for ras-related element binding protein  sites  are present in 4/ <dig> e-box-containing peaks in ecc-limb-pnst shared twist <dig> binding regions.
to validate binding occupancy of twist <dig> and candidate cofactors to twist <dig> binding regions identified by chip-seq, sequence-specific chip was performed with antibodies directed against rxr and rreb1
<cit> .
these analyses confirm that twist <dig> and rxr can occupy twist <dig> binding regions associated with wnt5b and ror <dig>  in limb buds .
rreb <dig> binding to shared twist <dig> binding region sequences associated with spata <dig>  ror <dig> , litaf, and chst <dig> was detected in pnst cells, and rreb <dig> bound to spata <dig> and litaf twist <dig> binding regions in limb buds .
however, rreb <dig> occupancy at shared binding regions was not detected in eccs.
together these data provide evidence that rxr can also occupy twist <dig> binding regions in limb buds, and that rreb <dig> can occupy twist <dig> binding regions in both limb buds and pnst cells, but not ecc.
therefore, rxr is a candidate cofactor for twist <dig> in limb buds, and rreb <dig> is a candidate cofactor for twist <dig> in limb buds and pnst cells.
twist <dig> dna occupancy is associated with genes in the wnt signaling pathway
to examine pathways corresponding to genes associated with twist <dig> binding regions, toppgene suite and genemania were utilized to integrate information on protein-protein interactions, gene regulation, coexpression, and functional studies, as well as to determine pathway association
<cit> .
these analyses demonstrated that genes associated with wnt signaling pathway components and interacting proteins are prominent in binding regions within  <dig> kb of a tss associated with expressed genes from ecc-limb shared twist <dig> binding peaks.
in eccs, limb buds, and pnst cells, twist <dig> occupied a binding peak  associated with the wnt receptor ror <dig> .
twist <dig> binding to peak sequences associated with wnt pathway genes was confirmed by chip-qpcr in e <dig>  eccs and e <dig>  limb buds.
in eccs, twist <dig> bound sequences associated with a second region associated with ror <dig> , as well as sequences associated with homeodomain interacting protein kinase  <dig> , secreted frizzled-related protein  <dig> , frizzled homolog  <dig> , and frizzled homolog  <dig>  .
in limb buds, twist <dig> binding to peak sequences associated with wingless-related mmtv integration site 5b , lymphoid enhancer binding factor  <dig> , nemo like kinase , casein kinase  <dig>  epsilon , disabled  <dig> interacting protein , mutated in colorectal cancers , and frizzled homolog  <dig>  were confirmed .
thus twist <dig> binding was detected at genomic sequences associated with wnt pathway genes and wnt interacting genes in developing eccs and limb buds.figure  <dig>
twist <dig> binding region sequences are associated with genes in the wnt signaling pathway.
a. wnt pathway genes associated with twist <dig> e-box-containing binding regions detected by chip-seq in e <dig>  eccs include receptor tyrosine kinase-like orphan receptor  <dig> , homeodomain interacting protein kinase  <dig> , secreted frizzled-related protein  <dig> , frizzled homolog  <dig> , and frizzled homolog  <dig> .
twist <dig> binding to these sequences was confirmed by chip with anti-twist <dig> in e <dig>  eccs followed by quantification by qpcr.
b. wnt pathway genes associated with twist <dig> e-box containing binding regions detected by chip-seq in e <dig>  limb buds include wnt5b, lymphoid enhancer binding factor  <dig> , nemo like kinase , casein kinase  <dig>  epsilon , disabled  <dig> interacting protein , mutated in colorectal cancers , and frizzled homolog  <dig> .
twist <dig> binding to these sequences was confirmed by chip with anti-twist <dig> in e <dig>  limb bud followed by quantification by qpcr.
all chip experiments were quantified with qpcr, and fold-enrichment was calculated relative to normal rabbit igg control.
all analyses were performed in biological triplicate.
* indicates p <  <dig>  as determined by student’s t-test comparing igg and twist <dig> chip qpcr ΔΔct values.
discussion
tissue-specificity of twist <dig> binding to distinct subsets of gene sequences was observed by chip-seq and then confirmed by chip and sequence-specific qpcr in ecc, limb buds, and pnst cells.
the tissue-specific binding regions occupied by twist <dig> are consistent with known functions of twist <dig> in each cell type, although the individual genes are distinct.
twist <dig> binds to dna sequences associated with genes that regulate migration and other biological functions in each tissue, but few shared binding regions were detected between samples.
among the six sequences occupied by twist <dig> in all three tissues are genes involved in cell adhesion-migration that are also bound by the ras-related element binding protein .
moreover, twist <dig> binding regions are associated with several wnt pathway genes and pathway components.
these data suggest that twist <dig> binds target genomic sequences predominantly in a tissue-specific manner and provide initial evidence that twist <dig> occupies sequences of genes acting in the wnt signaling pathway.
however, the mechanisms that dictate the tissue-specificity of twist <dig> binding to distinct transcriptional targets in different cell types remain to be determined.
analysis of twist <dig> and its downstream target genes in a variety of cell types have revealed roles in cell proliferation, adhesion/migration, and ecm protein expression.
in the ecc mesenchymal cells of the developing heart valves, alterations in twist <dig> expression affect cell migration and expression of tbx <dig>  cadherin- <dig>  and periostin
<cit> .
additionally, twist <dig> binding to genomic regions associated with genes that regulate cell proliferation and migration, including tbx <dig>  cdh <dig>  rab39b, sema3c, and gadd45a, have been identified
<cit> .
likewise, expression analysis of avc of twist1-null embryos relative to wt mice revealed reduced expression of cell migration and ecm genes
<cit> .
interestingly, ror <dig> expression is decreased in twist1-null ecc and also was found to contain a twist <dig> binding region in this study
<cit> .
in synovial sarcoma cells, twist <dig> direct downstream targets are involved in maintenance of mesenchymal differentiation
<cit> , and twist <dig> targets promote cell migration and proliferation in thyroid cancer cells
<cit> .
interestingly, in the current study only one binding region, ror <dig>  overlaps with these  <dig> other twist <dig> chip-seq studies.
these studies further support the ability of twist <dig> to regulate different networks of downstream genes depending on the specific cellular context.
however, predominant roles for twist <dig> downstream target genes in cell migration and adhesion are highly reproducible.
potential twist <dig> cofactors were identified based on the presence of transcription factor binding sites in twist <dig> binding regions and were confirmed by chip-qpcr in the tissues of interest.
the retinoic acid  receptor rxr was identified as a potential twist <dig> cofactor in limb buds.
both ra signaling and twist <dig> have been implicated in limb bud patterning, and the binding of both factors to the same genomic target sequence supports rxr as a candidate cofactor for twist1
<cit> .
in skeletal muscle, the bhlh transcription factor myod physically interacts with rxr to promote cell-type specific gene regulation, suggesting the possibility of a related or intersecting regulatory mechanism with twist1
<cit> .
rreb <dig> binding sites are present in proximity to twist <dig> occupied sites detected in ecc, limb buds, and pnst cells.
rreb <dig> is expressed in developing tissues and cancer where it acts downstream of ras and mapk signaling twist <dig> is required for k-ras mediated progression of lung tumors, supporting a role for rreb <dig> in this regulatory interaction
<cit> .
in addition, rreb <dig> has been shown to interact and bind with bhlh factor neurod to enhance transcriptional activity
<cit> .
thus twist <dig> and rreb <dig> may act together in transcriptional regulation of genes activated by ras-mapk signaling in multiple tissue types.
together, these studies have uncovered candidate cofactors, previously demonstrated to interact with related bhlh factors, which may contribute to the cell-type specificity of twist <dig> genomic occupancy and target gene regulation.
increasing evidence implicates the wnt signaling pathway as a key pathway active in developing heart valves and limb buds and also in disease states, such as cancer
<cit> .
twist <dig> genomic occupancy was observed in association with genes in the wnt pathway in ecc and limb buds.
in eccs, wnt/β-catenin signaling is required for emt of the endothelial cells, and for proliferation of mesenchymal cells
<cit> .
in twist1-null avc and oft, genes associated with the wnt signaling pathway are significantly differentially regulated relative to control embryos
<cit> .
likewise, conditional deletion of the twist <dig> target gene tbx <dig> in endothelial cells leads to reduced expression of wnt pathway genes sfrp <dig> and fzd <dig> in eccs
<cit> .
the current study identified genomic sequences associated with sfrp <dig> and fzd <dig> occupied by twist <dig> in eccs, supporting an intersecting regulatory network for twist <dig>  tbx <dig>  and wnt signaling in heart valve development.
in developing limb buds, wnt signaling is critical for limb bud outgrowth, while wnt and ra signaling are required for proximal-distal limb patterning
.
here, we identify wnt5b associated sequences bound by twist <dig> and rxr in limb buds.
in limb bud cartilage progenitors, ra and wnt signaling pathways intersect, and wnt5b promotes cell migration and inhibition of cell adhesion mediated by cadherins, which are often regulated by twist1
<cit> .
additionally, mutations in wnt/β-catenin signaling pathway genes have been identified in patients with neurofibromatosis type  <dig>  and in vitro experiments in mpnst cells demonstrate that wnt signaling promotes cell proliferation
<cit> .
furthermore, wnt signaling through ror <dig> is linked with cell invasion and emt in human osteosarcoma cells, and the same binding region, ror2-b, was recently identified as a twist <dig> target in both e <dig>  avc of the heart and developing limb buds
<cit> .
the identification of twist <dig> binding regions in proximity to multiple genes associated with wnt signaling suggests that twist <dig> may regulate components of the wnt signaling pathway in eccs, limb buds, and pnst cells.
CONCLUSIONS
this study examined twist <dig> genomic occupancy in two murine developmental tissues  and a murine cancer cell line .
interestingly, little overlap between binding regions was observed in eccs, limb buds, and pnst cells, although the predominant functions of twist <dig> occupied genes, including cell adhesion and migration are similar in the three tissues.
the specificity of twist <dig> binding was confirmed for tissue-specific targets, while twist <dig> binding in multiple tissues was detected for shared target genes, including four genes that represent core targets of twist <dig> in multiple cell types.
the molecular basis for the tissue-specificity of twist <dig> binding remains to be determined.
interestingly rreb <dig> binding sites are located in proximity to twist <dig> sites in the occupied regions detected in multiple tissues providing initial evidence for a core regulatory interaction.
in addition, we provide evidence that twist <dig> interacts with genes in the wnt signaling pathway in eccs, limb buds, and pnst cells.
a limitation of our analysis is the focus on twist <dig> genomic occupancy of expressed genes, which is indicative of twist <dig> acting as a transcriptional activator, not repressor.
however, the identification of tissue-specific targets and dna binding mechanisms of genes potentially activated by twist <dig> will aid in our understanding of its role in transcriptional hierarchies active in development and disease.

 twist is a highly conserved basic-helix-loop-helix transcription factor, which regulates cell migration, proliferation, and differentiation in progenitor populations, such as embryonic neural crest cells, cranial mesoderm, limb buds, and endocardial cushions. in addition to its role in developmental processes, human twist is highly expressed in diseased heart valves and in metastatic tumor cells. twist expression is normally silenced after embryonic development, but expression is reactivated during disease, including cancer. twist controls gene expression through binding e-box consensus sites as a homodimer or heterodimer with other bhlh transcription factors. however, a comparison of twist direct transcriptional targets in developing and diseased tissues has yet to be reported. this study focuses on identifying and comparing twist genomic occupancy in developing ecc, limb buds, and a cancer cell line. in multiple structures in the developing embryo, including the heart, limb buds, cranial sutures, skeletal elements, and neural crest derivatives, twist regulates cell migration and proliferation of progenitor populations. in the developing heart, twist expression is robust in the mesenchymal cells of the ecc and is subsequently down-regulated during remodeling and differentiation of the valve leaflets. in the ecc, twist promotes cell migration and proliferation of mesenchymal cells. direct transcriptional targets of twist include genes involved in cell migration and proliferation. in the developing limbs, twist is broadly expressed in the limb bud mesenchyme and strictly regulated genetic dosage of twist is required for limb bud outgrowth and patterning. although extensive genetic studies have been performed demonstrating an essential role for twist in limb patterning and morphogenesis, the direct transcriptional targets underlying these functions are only recently being elucidated. in addition, it is not known if twist regulates the same downstream targets in distinct progenitor populations or if there is tissue-specificity to twist regulatory mechanisms in the developing embryo. while twist is largely absent from adult differentiated tissues, it is expressed in diseased heart valves and highly metastatic cancers such as breast, pancreatic, gastric, prostate, and malignant peripheral nerve sheath tumors. human mpnst cells can harbor nf and p mutations, and murine nf and p mutations can cause similar nerve-associated sarcomas, peripheral nerve sheath tumors. sirna-mediated knockdown of twist abrogates the migratory activity of human mpnst cells in vitro. twist also is expressed in invasive carcinoma and promotes endothelial-to-mesenchymal transformation and metastasis of tumor cells through regulation of genes involved in differentiation, adhesion, and proliferation. it has not previously been determined if the downstream targets of twist related to regulation of cell migration or proliferation in embryonic structures and cancer cells are shared or are cell-type specific. to investigate the tissue-specificity of twist genomic occupancy, we performed chromatin immunoprecipitation with anti-twist followed by nextgen sequencing in mouse embryonic day eccs, e forelimb buds, and cultured pnst cells. the specificity of twist binding was confirmed by chip qpcr in each tissue. potential twist cofactors and intersecting signaling pathways also were identified.  analysis of twist genomic occupancy in eccs, limb buds, and pnst cells twist genomic occupancy was assessed by chip with anti-twist in mouse e eccs, e limb buds, and pnst cells followed by deep sequencing. next gen sequencing was used to generate, reads for e eccs,, reads for e limb buds, and, reads for pnst cells. resulting sequences were aligned to the mouse genome, and peaks were discovered using macs analysis. a p-value cut-off of e- was used to identify peaks for e eccs, peaks for e limb buds, and peaks for pnst cells relative to comparable reads for fvb/n mouse genomic dna as a control. in order to confirm enrichment of e-box binding sites in each data set, de-novo motif enrichment was performed using pscanchip and meme-chip. e-box containing motifs were significantly enriched in all datasets when analyzed by pscanchip and meme-chip. peaks were filtered based on distance from the nearest transcriptional start site with a threshold of kilobases resulting in peaks for e eccs, peaks for e limb buds, and peaks for pnst cells. in order to compare twist genomic occupancy with genes expressed in each tissue, genes associated with binding peaks in each tissue were compared to respective affymetrix microarray gene expression data for e eccs, e limb buds, and pnst cells. the identities of genes associated with binding peaks within kb of tss, identified by chip-seq were compared to the total expressed genes as detected by microarray for each tissue individually. these comparisons were used to generate lists of twist binding regions corresponding to expressed genes in each tissue. for e eccs, twist binding peaks corresponding to expressed genes were identified. twist occupancy was detected for peaks corresponding to expressed genes in e limb buds, and peaks corresponding to expressed genes were detected for pnst cells. the list of genes for pnst cells is likely an underestimate of twist genomic occupancy because only genes that are expressed in both human mpnst and mouse pnst cell lines were included. these chip-seq data indicate that twist occupies genomic regions associated with large numbers of genes expressed in eccs, limb buds, and pnst cells.figure twist genomic occupancy in endocardial cushions, limb buds, and peripheral nerve sheath tumor cells. a. twist binding regions were identified by chip-seq followed by macs analysis in e eccs, e limb buds, and pnst cells compared to a control strain matched whole genome dataset. the total number of peaks, the number of peaks within kilobases of a transcriptional start site, and the number of peaks associated with expressed genes, as determined by microarray gene expression analysis of the tissue of interest, are shown. b. the total number of shared peaks detected by twist chip-seq of ecc-limb, limb-pnst, ecc-pnst and ecc-limb-pnst groups is shown. the number of overlapping peaks within kb of a tss for expressed genes also was determined. tissue-specific and shared gene targets of twist in eccs, limb buds, and pnst cells corresponding to binding regions within kb of tss were analyzed. binding region overlap determination was performed before and after peaks were compared to gene expression profiles. these analyses produced datasets containing binding regions that are unique to a specific tissue type or shared among multiple tissue types. groups of binding regions were classified as ecc only, limb only, pnst only, ecc-limb, ecc-pnst, limb-pnst, or ecc-limb-pnst. e eccs and e limb buds share the most binding regions, with shared peaks in the ecc-limb group before comparison to mrna expression data, and ecc-limb binding regions associated with expressed genes after comparison with mrna expression. similarly the limb-pnst group contains shared binding regions, before the expression filter, with only shared binding regions corresponding to expressed genes. the ecc-pnst group contains total shared binding regions, with shared binding regions corresponding to expressed genes. surprisingly, only binding regions associated with expressed genes were occupied by twist in all three cell types, indicating a high degree of tissue-specificity in twist genomic occupancy. genomic sequences of the shared binding regions associated with expressed genes were analyzed for presence of an e-box consensus site on the sense or antisense strand, which would indicate a potential twist binding site. of the ecc-limb shared binding regions associated with expressed genes, contain an e-box consensus site, corresponding to genes. in addition, e-box consensus sites are present in 7/ ecc-pnst shared binding regions, 6/ of the limb-pnst binding regions, and 5/ of the ecc-limb-pnst shared binding regions. collectively, these chip-seq experiments demonstrate extensive twist binding to genomic sequences and indicate that twist bound predominantly in a tissue-specific manner, with much smaller numbers of shared binding regions detected among eccs, limb buds, and pnst cells. in order to examine twist1-dna binding distribution across the genome and determine if twist is bound to specific genomic regions relative to gene organization, location analysis was performed. twist binding regions located within kb of a tss for genes expressed in each tissue were subjected to genomic location and peak distribution analysis using genomatix regionminer. twist binding regions associated with expressed genes were analyzed in tissue-specific gene groups, and also in groups of binding regions shared between tissue types. distribution of the tissue-specific binding regions and ecc-limb shared peaks is largely within intergenic and intronic regions. however, ecc-pnst and limb-pnst shared binding regions are more evenly distributed in genomic regions. this lack of specificity in peak distribution relative to gene organization is consistent with other transcription factors, including twist in other cell types. these data suggest that twist occupies genomic sequences associated with expressed genes located in intergenic and intragenic genomic regions and does not bind exclusively to promoter regions. functional classification of genes associated with twist binding peaks the major biological functions of genes with twist binding regions identified within kb of tss, that also are expressed in eccs, limb buds, or pnst cells, were assessed using gene ontology analysis. the four go categories containing the greatest numbers of genes in each tissue were chosen for further analysis. in eccs, the four predominant go groups were cell-cell signaling, cell adhesion, cell morphogenesis, and neurogenesis. the top four go categories of twist occupied genes in limb buds were kinase activity, cell migration, neurogenesis, and cytoskeletal proteins. for pnst cells, the four go groups with the most genes were cell adhesion, skeletal system development, cell recognition, and ventricular septum morphogenesis. cell adhesion-cell migration was among the most predominant go categories in each tissue type, indicating a conserved role for twist in cell adhesion-migration in multiple cellular contexts. however, the specific genes corresponding to twist binding regions in each of these go categories were unique to each tissue type, suggesting cell-type specificity of twist genomic occupancy of gene targets associated with cell adhesion and migration.figure gene ontology analysis of the genes associated with twist binding regions in eccs, limb buds, and pnst cells. a, b, c. toppgene was utilized to perform go analysis of the genes associated with twist chip-seq peaks that are within kb of a transcriptional start site of expressed genes in e eccs, e limb buds, or pnst cells. the four most predominant go categories and the total number of genes in each category are represented. the biological functions of ecc-limb shared binding regions associated with expressed genes were further examined resulting in genes associated with the binding regions containing an e-box consensus site. again, adhesion-migration was the most predominant go category in genes associated with ecc-limb shared binding regions containing e-box consensus sites associated with expressed genes. additional go categories include mrna metabolic process, microtubule-based process, and organ morphogenesis. thus, the expressed gene identities associated with twist shared binding regions in eccs and limb buds suggest conserved functions for twist in regulation of cell adhesion-migration during development in different tissues. expressed genes associated with shared binding regions containing e-box binding sequences related to cell adhesion and migration also were identified for ecc-pnst, limb-psnt and ecc-limb-pnst groups. the overlapping peaks in the ecc-limb-pnst group correspond to six shared binding regions associated with genes: cd antigen, carbohydrate sulfotransferase, lps-induced tn factor, receptor tyrosine kinase-like orphan receptor, and spermatogenesis associated. these five genes relate to the biological processes of cell adhesion-migration, cell differentiation, signal transduction, and cartilage development. these biological processes are consistent with known twist functions during embryogenesis and in cancer cells.figure shared twist binding regions detected by chip-seq in ecc, limb buds, and pnst cells. a. twist chip-seq peaks identified in e eccs, e limb buds, and pnst cells were filtered based on a cut off of kb from the nearest tss and gene expression as determined by microarray in each cell type. the peaks associated with expressed genes from each tissue type were analyzed for shared peaks and are represented in the venn diagram. b. a total of twist binding regions were detected in both ecc and limb bud, and contain e-box consensus sequences. the four predominant go categories for the genes associated with shared ecc-limb e-box-containing binding regions are shown. c. gene names and descriptions of the four go categories containing the most genes for ecc-limb e-box containing binding regions from b are shown. d. gene names and descriptions are shown for shared binding regions detected in ecc-pnst, limb-pnst, and ecc-limb-pnst groups. validation of twist genomic occupancy in ecc, limb buds and pnst cells to further examine tissue-specificity of twist binding, the top three e-box containing binding regions associated with expressed genes, as determined by the highest fold-enrichment in each cell type, were chosen for further analysis. chip with anti-twist was performed in e eccs, e limb buds, and pnst cells followed by quantification using qpcr relative to normal rabbit igg control. ecc tissue-specific binding regions chosen for further analysis were associated with the genes leukocyte cell derived chemotaxin shroom family member, and bromodomain and wd repeat domain containing twist chip followed by qpcr demonstrated binding of twist to ecc-specific regions in eccs, but not in limb buds or pnst cells. likewise, the limb bud-specific binding regions were associated with the genes son of sevenless homolog transmembrane protein, and abhydrolase domain containing, and were bound by twist in limb buds, but not in eccs or pnst cells. finally, pnst cell-specific binding regions associated with the genes cd antigen, calcium/calmodulin-dependent serine protein kinase, and transforming growth factor, beta were bound by twist in pnst cells, but not in ecc or limb buds. these data further support the tissue-specificity of twist binding to genomic sequences detected by chip-seq in ecc, limb buds, and pnst cells.figure validation of twist chip-seq peak specificity ecc, limb, or pnst cells. chip was performed with anti-twist in e eccs, e limb buds, and pnst cells followed by sequence-specific qpcr to examine relative binding of twist to the three binding regions with the highest fold enrichment, from macs analysis, associated with expressed genes in each tissue. in e eccs, twist occupies binding regions associated with leukocyte cell derived chemotaxin 1, shroom family member, and bromodomain and wd repeat domain containing 3. in e limb buds, twist occupies binding regions associated with son of sevenless homolog 2, transmembrane protein, and abhydrolase domain containing. in pnst cells, twist occupies binding regions associated with cd antigen and calcium/calmodulin-dependent serine protein kinase, and transforming growth factor, beta. chip quantification was performed by qpcr, and fold-enrichment was calculated versus an anti-rabbit igg control for each tissue. all analyses were performed in biological triplicate. * indicates p <  as determined by student’s t-test comparing igg and twist chip qpcr ΔΔct values. in order to examine twist occupancy at shared sequences associated with genes expressed in ecc-limb or ecc-limb-pnst groups, chip was performed and enrichment at overlapping binding regions was quantified by sequence-specific qpcr. ecc-limb overlapping binding regions containing e-box binding sites associated with expressed genes from the four major go categories were sorted according to proximity to tss for greater probability of identifying proximal regulatory sequences, and validation was performed on the two binding peaks nearest the tss. intronic binding regions associated with midline and cdc binding protein kinase alpha were bound by twist in both eccs and limb buds. of the six binding regions shared among all three-tissue types associated with expressed genes, five binding regions contain e-box consensus sites and four were analyzed by chip-qpcr for twist occupancy. twist occupancy at shared binding regions associated with chst litaf, ror, and spata was confirmed in eccs, limb buds, and pnst cells, but binding to the shared cd site was not detected. thus, twist binding was detected at small group of shared binding regions in ecc, limb bud, and pnst cells, which may represent common targets of twist in multiple cell types.figure twist and candidate cofactors occupy binding regions shared among ecc, limb bud, and pnst cells. a. binding of twist to regions associated with the genes midline and cdc binding protein kinase alpha detected in e eccs and e limb chip-seq was validated by chip with anti-twist in e eccs and e limb buds followed by sequence-specific qpcr. b. twist binding regions containing an e-box sequence, as detected by chip-seq in e eccs, e limb buds, and pnst, are associated with receptor tyrosine kinase-like orphan receptor, carbohydrate sulfotransferase, spermatogenesis associated and lps-induced tn factor. twist chip followed by qpcr indicates that twist binds each of these sequences in e eccs, e limb buds, and pnst cells. c. genomatix regionminer was utilized to identify rxr as a potential cofactor with twist in e limb bud. twist binding regions containing an e-box within bp of an rxr binding consensus associated with ror2-a and wnt5b genes involved in limb development were chosen for validation. chip was performed with anti-rxr in e limb buds followed qpcr amplification of twist binding region sequences. d. rreb consensus binding sites were identified within base pairs of an e-box in twist binding peak sequences of chst litaf, ror2-a, and spata detected in ecc, limb bud and pnst cells. chip was performed with anti-rreb followed by qpcr amplification of twist binding region sequences in eccs, limb buds, and pnst cells. e. motif of rxr consensus site. f. motif of rreb consensus site. fold-enrichment was calculated relative to normal rabbit igg control. all analyses were performed in biological triplicate. * indicates p <  as determined by student’s t-test comparing igg and twist igg and rxr, or igg and rreb chip qpcr ΔΔct values. identification of potential twist cofactors in eccs, limb buds, and pnst cells the presence of tissue-specific twist cofactors was examined as a potential mechanism underlying specificity of target occupancy. twist binding regions associated with expressed genes for each tissue type were analyzed for transcriptional modules containing a transcription factor-binding sequence within +/- base pairs of the e-box consensus site using genomatix regionminer. in ecc, cp2-erythrocyte factor related to drosophila elf sites and heat shock factor sites are located in proximity to e-boxes in approximately 10% of the twist occupied sequences. retinoid x receptor sites are present in 43% of the limb-only twist occupied sites and also in 44% of the ecc-limb shared twist occupied sites. interestingly, binding sites for ras-related element binding protein sites are present in 4/ e-box-containing peaks in ecc-limb-pnst shared twist binding regions. to validate binding occupancy of twist and candidate cofactors to twist binding regions identified by chip-seq, sequence-specific chip was performed with antibodies directed against rxr and rreb1. these analyses confirm that twist and rxr can occupy twist binding regions associated with wnt5b and ror in limb buds. rreb binding to shared twist binding region sequences associated with spata ror, litaf, and chst was detected in pnst cells, and rreb bound to spata and litaf twist binding regions in limb buds. however, rreb occupancy at shared binding regions was not detected in eccs. together these data provide evidence that rxr can also occupy twist binding regions in limb buds, and that rreb can occupy twist binding regions in both limb buds and pnst cells, but not ecc. therefore, rxr is a candidate cofactor for twist in limb buds, and rreb is a candidate cofactor for twist in limb buds and pnst cells. twist dna occupancy is associated with genes in the wnt signaling pathway to examine pathways corresponding to genes associated with twist binding regions, toppgene suite and genemania were utilized to integrate information on protein-protein interactions, gene regulation, coexpression, and functional studies, as well as to determine pathway association. these analyses demonstrated that genes associated with wnt signaling pathway components and interacting proteins are prominent in binding regions within kb of a tss associated with expressed genes from ecc-limb shared twist binding peaks. in eccs, limb buds, and pnst cells, twist occupied a binding peak associated with the wnt receptor ror. twist binding to peak sequences associated with wnt pathway genes was confirmed by chip-qpcr in e eccs and e limb buds. in eccs, twist bound sequences associated with a second region associated with ror, as well as sequences associated with homeodomain interacting protein kinase, secreted frizzled-related protein, frizzled homolog, and frizzled homolog. in limb buds, twist binding to peak sequences associated with wingless-related mmtv integration site 5b, lymphoid enhancer binding factor, nemo like kinase, casein kinase epsilon, disabled interacting protein, mutated in colorectal cancers, and frizzled homolog were confirmed. thus twist binding was detected at genomic sequences associated with wnt pathway genes and wnt interacting genes in developing eccs and limb buds.figure twist binding region sequences are associated with genes in the wnt signaling pathway. a. wnt pathway genes associated with twist e-box-containing binding regions detected by chip-seq in e eccs include receptor tyrosine kinase-like orphan receptor, homeodomain interacting protein kinase, secreted frizzled-related protein, frizzled homolog, and frizzled homolog. twist binding to these sequences was confirmed by chip with anti-twist in e eccs followed by quantification by qpcr. b. wnt pathway genes associated with twist e-box containing binding regions detected by chip-seq in e limb buds include wnt5b, lymphoid enhancer binding factor, nemo like kinase, casein kinase epsilon, disabled interacting protein, mutated in colorectal cancers, and frizzled homolog. twist binding to these sequences was confirmed by chip with anti-twist in e limb bud followed by quantification by qpcr. all chip experiments were quantified with qpcr, and fold-enrichment was calculated relative to normal rabbit igg control. all analyses were performed in biological triplicate. * indicates p <  as determined by student’s t-test comparing igg and twist chip qpcr ΔΔct values. discussion tissue-specificity of twist binding to distinct subsets of gene sequences was observed by chip-seq and then confirmed by chip and sequence-specific qpcr in ecc, limb buds, and pnst cells. the tissue-specific binding regions occupied by twist are consistent with known functions of twist in each cell type, although the individual genes are distinct. twist binds to dna sequences associated with genes that regulate migration and other biological functions in each tissue, but few shared binding regions were detected between samples. among the six sequences occupied by twist in all three tissues are genes involved in cell adhesion-migration that are also bound by the ras-related element binding protein. moreover, twist binding regions are associated with several wnt pathway genes and pathway components. these data suggest that twist binds target genomic sequences predominantly in a tissue-specific manner and provide initial evidence that twist occupies sequences of genes acting in the wnt signaling pathway. however, the mechanisms that dictate the tissue-specificity of twist binding to distinct transcriptional targets in different cell types remain to be determined. analysis of twist and its downstream target genes in a variety of cell types have revealed roles in cell proliferation, adhesion/migration, and ecm protein expression. in the ecc mesenchymal cells of the developing heart valves, alterations in twist expression affect cell migration and expression of tbx cadherin- and periostin. additionally, twist binding to genomic regions associated with genes that regulate cell proliferation and migration, including tbx cdh rab39b, sema3c, and gadd45a, have been identified. likewise, expression analysis of avc of twist1-null embryos relative to wt mice revealed reduced expression of cell migration and ecm genes. interestingly, ror expression is decreased in twist1-null ecc and also was found to contain a twist binding region in this study. in synovial sarcoma cells, twist direct downstream targets are involved in maintenance of mesenchymal differentiation, and twist targets promote cell migration and proliferation in thyroid cancer cells. interestingly, in the current study only one binding region, ror overlaps with these other twist chip-seq studies. these studies further support the ability of twist to regulate different networks of downstream genes depending on the specific cellular context. however, predominant roles for twist downstream target genes in cell migration and adhesion are highly reproducible. potential twist cofactors were identified based on the presence of transcription factor binding sites in twist binding regions and were confirmed by chip-qpcr in the tissues of interest. the retinoic acid receptor rxr was identified as a potential twist cofactor in limb buds. both ra signaling and twist have been implicated in limb bud patterning, and the binding of both factors to the same genomic target sequence supports rxr as a candidate cofactor for twist1. in skeletal muscle, the bhlh transcription factor myod physically interacts with rxr to promote cell-type specific gene regulation, suggesting the possibility of a related or intersecting regulatory mechanism with twist1. rreb binding sites are present in proximity to twist occupied sites detected in ecc, limb buds, and pnst cells. rreb is expressed in developing tissues and cancer where it acts downstream of ras and mapk signaling twist is required for k-ras mediated progression of lung tumors, supporting a role for rreb in this regulatory interaction. in addition, rreb has been shown to interact and bind with bhlh factor neurod to enhance transcriptional activity. thus twist and rreb may act together in transcriptional regulation of genes activated by ras-mapk signaling in multiple tissue types. together, these studies have uncovered candidate cofactors, previously demonstrated to interact with related bhlh factors, which may contribute to the cell-type specificity of twist genomic occupancy and target gene regulation. increasing evidence implicates the wnt signaling pathway as a key pathway active in developing heart valves and limb buds and also in disease states, such as cancer. twist genomic occupancy was observed in association with genes in the wnt pathway in ecc and limb buds. in eccs, wnt/β-catenin signaling is required for emt of the endothelial cells, and for proliferation of mesenchymal cells. in twist1-null avc and oft, genes associated with the wnt signaling pathway are significantly differentially regulated relative to control embryos. likewise, conditional deletion of the twist target gene tbx in endothelial cells leads to reduced expression of wnt pathway genes sfrp and fzd in eccs. the current study identified genomic sequences associated with sfrp and fzd occupied by twist in eccs, supporting an intersecting regulatory network for twist tbx and wnt signaling in heart valve development. in developing limb buds, wnt signaling is critical for limb bud outgrowth, while wnt and ra signaling are required for proximal-distal limb patterning. here, we identify wnt5b associated sequences bound by twist and rxr in limb buds. in limb bud cartilage progenitors, ra and wnt signaling pathways intersect, and wnt5b promotes cell migration and inhibition of cell adhesion mediated by cadherins, which are often regulated by twist1. additionally, mutations in wnt/β-catenin signaling pathway genes have been identified in patients with neurofibromatosis type and in vitro experiments in mpnst cells demonstrate that wnt signaling promotes cell proliferation. furthermore, wnt signaling through ror is linked with cell invasion and emt in human osteosarcoma cells, and the same binding region, ror2-b, was recently identified as a twist target in both e avc of the heart and developing limb buds. the identification of twist binding regions in proximity to multiple genes associated with wnt signaling suggests that twist may regulate components of the wnt signaling pathway in eccs, limb buds, and pnst cells.  this study examined twist genomic occupancy in two murine developmental tissues and a murine cancer cell line. interestingly, little overlap between binding regions was observed in eccs, limb buds, and pnst cells, although the predominant functions of twist occupied genes, including cell adhesion and migration are similar in the three tissues. the specificity of twist binding was confirmed for tissue-specific targets, while twist binding in multiple tissues was detected for shared target genes, including four genes that represent core targets of twist in multiple cell types. the molecular basis for the tissue-specificity of twist binding remains to be determined. interestingly rreb binding sites are located in proximity to twist sites in the occupied regions detected in multiple tissues providing initial evidence for a core regulatory interaction. in addition, we provide evidence that twist interacts with genes in the wnt signaling pathway in eccs, limb buds, and pnst cells. a limitation of our analysis is the focus on twist genomic occupancy of expressed genes, which is indicative of twist acting as a transcriptional activator, not repressor. however, the identification of tissue-specific targets and dna binding mechanisms of genes potentially activated by twist will aid in our understanding of its role in transcriptional hierarchies active in development and disease. 
consistent with known twist functions during development and in cancer cells, twist1-dna binding regions associated with genes related to cell migration and adhesion were detected in all three tissues. thus, while twist has similar functions in eccs, limb buds, and pnst cells, the specific genomic sequences occupied by twist were different depending on cellular context. subgroups of shared genes, also predominantly related to cell adhesion and migration, were identified in pairwise comparisons of ecc, limb buds and pnst cells. together, these data indicate that twist interacts with genes that regulate adhesion and migration in different tissues, potentially through distinct sets of target genes. in addition, there is a small subset of genes occupied by twist in all three tissues that may represent a core group of twist target genes in multiple cell types. the basic helix-loop-helix transcription factor twist has well-documented roles in progenitor populations of the developing embryo, including endocardial cushions and limb buds, and also in cancer. twist1endocardial cushionslimb budsperipheral neuron sheath tumor cellschipchip-seqissue-copyright-statement© the author 2014 the tissue-specificity of twist genomic occupancy was examined in mouse eccs, limb buds, and peripheral nerve sheath tumor cells using chromatin immunoprecipitation followed by sequencing analysis. twist genomic occupancy was detected for six binding regions in all tissue types, and twist1-binding sequences associated with chst litaf, ror and spata also bound the potential twist cofactor rreb pathway analysis of the genes associated with twist binding suggests that twist may regulate genes associated with the wnt signaling pathway in eccs and limb buds. whether twist regulates the same transcriptional targets in different tissue types is largely unknown. however, the vast majority of twist binding regions were specific to individual tissue types.
 the basic helix-loop-helix transcription factor twist has well-documented roles in progenitor populations of the developing embryo, including endocardial cushions and limb buds, and also in cancer. whether twist regulates the same transcriptional targets in different tissue types is largely unknown.  the tissue-specificity of twist genomic occupancy was examined in mouse eccs, limb buds, and peripheral nerve sheath tumor cells using chromatin immunoprecipitation followed by sequencing analysis. consistent with known twist functions during development and in cancer cells, twist1-dna binding regions associated with genes related to cell migration and adhesion were detected in all three tissues. however, the vast majority of twist binding regions were specific to individual tissue types. thus, while twist has similar functions in eccs, limb buds, and pnst cells, the specific genomic sequences occupied by twist were different depending on cellular context. subgroups of shared genes, also predominantly related to cell adhesion and migration, were identified in pairwise comparisons of ecc, limb buds and pnst cells. twist genomic occupancy was detected for six binding regions in all tissue types, and twist1-binding sequences associated with chst litaf, ror and spata also bound the potential twist cofactor rreb pathway analysis of the genes associated with twist binding suggests that twist may regulate genes associated with the wnt signaling pathway in eccs and limb buds.  together, these data indicate that twist interacts with genes that regulate adhesion and migration in different tissues, potentially through distinct sets of target genes. in addition, there is a small subset of genes occupied by twist in all three tissues that may represent a core group of twist target genes in multiple cell types. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords twist1endocardial cushionslimb budsperipheral neuron sheath tumor cellschipchip-seqissue-copyright-statement© the author 2014 