BACKGROUND
regulatory transcription factors  are encoded by approximately 10% of the human genome  <cit> .
the search for an accurate and complete list of target genes for thousands of tfs and the elucidation of their complex interactions at promoter sites, particularly in embryonic stem  cells, has gained increasing interest.
however, only a small fraction of the in vivo target genes and relatively few tf-tf interactions have been elucidated  <cit> .
chromatin immunoprecipitation  and its derivatives  have been widely used for the investigation of tf-dna interactions  <cit> .
high-throughput approaches, such as chip-chip and chip-sage, are necessary for genome-wide analysis and the systematic identification of new dna-binding sequences.
real-time  pcr remains extensively used for validation of genome-wide data and for analysis of chip results in general.
high-throughput approaches are time-consuming, expensive, labor-intensive, involve multiple steps that facilitate error introduction, and require complex statistical analysis  <cit> .
therefore, advances in this field will greatly benefit from the development and use of faster and straightforward chip assay and analysis methodologies.
here, we present data obtained with a simplified, basic chip assay and analysis protocol that allowed the rapid identification of known target genes for sox <dig>  nanog, oct3/ <dig>  sox <dig>  klf <dig>  runx <dig>  olig <dig>  smad2/ <dig>  bmi- <dig>  and c-myc in the human es cell line bg01v.
we used rtpcr to initially validate the protocol/antibodies and densitometric analysis of pcr results with the imagej software as a more practical, less expansive, less time-consuming readout alternative.
in addition, we developed a novel, non-disruptive, highly sensitive sequential chip protocol for the identification of promoter co-occupancy, based on our simplified basic chip protocol.
the data obtained with this sequential chip protocol are consistent with data previously obtained with more labor-intensive, expensive, time-consuming chip-chip platforms.
furthermore, sequential chip analysis led to the identification of two tf complexes in bg01v es cells: sox2/nanog/oct3/4/c-myc and runx2/bmi-1/smad2/ <dig> complexes.
these two tf complexes associate with two different sets of target genes.
the runx2/bmi-1/smad2/ <dig> complex is associated predominantly with genes not expressed in undifferentiated bg01v cells, consistent with the reported role of those tfs as transcriptional repressors.
these simplified basic chip and novel sequential chip protocols were successfully tested with a variety of antibodies with bg01v es cells, generated a number of novel observations for future studies and might be useful for high-throughput chip-based assays.
RESULTS
development of an improved basic chip protocol
we developed a simplified, basic chip protocol  and test its usefulness with antibodies against tfs expressed in the human es cell line bg01v.
these antibodies included those against sox <dig>  nanog, oct3/ <dig>  sox <dig>  runx <dig>  olig <dig>  smad2/ <dig>  klf <dig>  bmi- <dig>  and c-myc.
this basic chip assay is characterized by the combination of simplicity , speed  and sensitivity .
recently described, commonly used protocols  <cit>  normally take longer time or lack one or more of those characteristics.
chip assays were performed with previously characterized antibodies  <cit>  and known target genes and initially analyzed by rtpcr.
we also analyzed the pcr results by densitometry using the imagej software, reducing time and resources for defining pcr parameters and, therefore, significantly decreasing experimental costs.
target genes included fgf <dig>  lefty, nanog, vegf, bcl <dig>  gli <dig>  e-cadherin, oct3/ <dig>  c-myc, hesx <dig>  zfp <dig>  and suz <dig> , lama <dig> , b2r , hoxc <dig> , c-myc and gli <dig> , p <dig>  and vegf and bax .
all primer sets have been validated previously   <cit> .
normal igg and input dna  were used as negative controls.
* all others are human genes.
the undifferentiated human es cell line bg01v, used throughout this study, shows nanog expression and nuclear localization , as expected  <cit> .
sox <dig> and oct3/ <dig> expression and nuclear localization was also verified .
fig.
2b shows fold enrichment relative to input dna, as determined by rtpcr analysis, of three known sox2/oct3/4/nanog target genes   <cit>  from sox2/oct3/4/nanog chip assays.
fig.
2c depicts pcr results using  <dig> candidate targets and fig.
2d shows fold enrichment, as determined by densitometric analysis, of the pcr results , of the three abovementioned known targets  and additional known sox2/oct3/4/nanog targets   <cit> .
figs.
2c and 2d also suggest hoxc <dig>  pten and bax as novel putative sox2/oct3/4/nanog targets in undifferentiated bg01v cells .
these results indicate that the described chip assay and analysis protocol are suitable for chip screening.
moreover, the results also suggest that the densitometric quantification of pcr results using the imagej software can be used as an alternative chip readout, reducing experimental time and resources.
to further confirm the usefulness of the protocol and test its applicability, chip assays were performed with additional es cell tfs and their known target genes.
expression and nuclear localization of all these tfs were verified .
sox <dig> plays critical roles in development regulation, stem cell function and is required for endoderm specification and maintenance in the embryo  <cit> .
as expected  <cit>  the mouse lama <dig> promoter was detected by rtpcr and densitometric analysis  from sox <dig> chip assays using endoderm differentiated mouse d <dig> es cells.
runx <dig>  linked to bone development and maintenance  <cit> , regulates vegf and bax  <cit> , which were detected by rtpcr and densitometric analysis  from runx <dig> chip assays using undifferentiated bg01v cells.
klf <dig>  implicated in transcriptional repression in smooth muscle cells, kidney development, stem cell self-renewal and pluripotency  <cit> , regulates oct3/ <dig>  nanog, and b2r  <cit> , which were detected by rtpcr and densitometric analysis  from klf <dig> chip assays using undifferentiated bg01v cells.
bmi- <dig>  implicated in cell proliferation and stem cell self-renewal  <cit> , binds to the hoxc <dig> promoter  <cit> , which was detected by rtpcr and densitometric analysis  of bmi- <dig> chip assays using undifferentiated bg01v cells.
smad2/ <dig>  involved in epithelial-to-mesenchymal transition, neuronal differentiation, and chondrogenesis  <cit> , have been shown to regulate c-myc and gli <dig>  <cit> , which were also detected by rtpcr and densitometric analysis of smad2/ <dig> chip assays using undifferentiated bg01v cells .
olig <dig>  implicated in neuron, astrocyte and oligodendrocyte differentiation, binds to the p <dig> promoter  <cit> , which was detected by rtpcr and densitometric analysis  of olig <dig> chip assays using undifferentiated bg01v cells.
as an additional negative control, an antibody against pdx <dig>  which is involved in pancreatic organogenesis  <cit> , was also used in chip assays.
as expected  <cit>  pdx <dig> was not detected by immunofluorescence in undifferentiated bg01v cells .
consistently, a know pdx <dig> target gene, insulin  <cit> , could not be detected above background by rtpcr in pdx <dig> chip assays performed with undifferentiated bg01v cells .
in addition, our basic chip protocol was directly compared to a previously described  <cit>  commonly used chip protocol, using three of the above mentioned antibodies and their target genes.
fig.
4b shows that our basic chip protocol leads to higher fold enrichment, compared to igg controls, than the fold enrichment obtained with a previously described  <cit>  commonly used chip protocol.
finally, to add experimental evidence for the applicability of the densitometric analysis of chip results using the imagej software, olig <dig> chip assays were performed with serially diluted template and primers.
fig.
4c shows that imagej measurements of p <dig> promoter signals from these olig <dig> chip assays are within a logarithmic range.
taken together, these results confirm that our simplified, basic chip assay can be used with a variety of antibodies and that the densitometric quantification of pcr results using the imagej software can be used as an alternative chip readout.
sox2/oct3/4/nanog/c-myc complex co-occupies several promoters in bg01v cells
we then sought to examine tf-tf interactions at promoter sites  using a newly developed sequential chip assay , which is based on the simplified basic chip assay.
chip-chip studies have identified numerous promoters co-occupied by the sox2/oct3/4/nanog complex  <cit> .
to validate the effectiveness of the sequential chip assay we selected a number of sox2/oct3/4/nanog known targets and subjected these targets to our new protocol.
the data obtained with the sequential chip assay in undifferentiated bg01v cells is consistent with previously reported chip-chip data  <cit> .
first, these three tfs  have been reported to co-occupy the oct3/ <dig>  nanog, hesx <dig> and vegf promoters  <cit> , and consistent with that, any combination of those tfs antibodies in the sequential chip assay led to the detection of those promoters .
second, only sox <dig> and oct3/ <dig> co-occupy the suz <dig> promoter  <cit> , and consistent with that, only immunoprecipitation with the sox <dig> antibody followed by the oct3/ <dig> antibody lead to the detection of the suz <dig> promoter , whereas other antibody combinations did not.
a sox <dig> regular chip assay was used as a positive control .
sox2/oct3/4nanog and c-myc have been found to share a large number of target genes in chip-chip studies  <cit> .
we then investigated if c-myc co-occupies promoters with the sox2/oct3/4/nanog complex.
indeed, immunoprecipitation with the sox <dig> antibody followed by the c-myc antibody led to the detection of a number of promoters in undifferentiated bg01v cells .
the same promoters were detected if the nanog antibody preceded the c-myc antibody  or if the oct3/ <dig> antibody preceded the c-myc antibody .
in contrast, none of these promoters were detected when normal igg was used as the first antibody .
these results indicate that, at the indicated promoters , c-myc is a subunit of the sox2/oct3/4/nanog complex in undifferentiated bg01v cells.
to further test this hypothesis we immunodepleted sox <dig> from undifferentiated bg01v cells lysates  and then analyzed c-myc targets by chip.
fig.
6d shows which c-myc-bound promoters were detected in those sox2-depleted lysates and shows a significant reduction in the number of c-myc-associated promoters.
most of the promoters detected in fig.
6b top panel  were not detected by c-myc chip after sox <dig> depletion , including p <dig>  fgf <dig>  nanog, vegf, pten, bax, muc <dig>  oct3/ <dig> and suz <dig> .
collectively these results corroborate the applicability of the novel sequential chip assay.
in addition these results indicate that c-myc associates with the sox2/oct3/4/nanog complex in bg01v cells.
runx2/bmi-1/smad2/ <dig> complex co-occupies a second set of promoters in bg01v cells
we then sought to investigate the presence of other tf complexes in bg01v cells using the sequential chip assay.
in a screening with  <dig> potential target genes, we found that bmi- <dig>  runx <dig>  smad2/ <dig>  klf <dig> and olig <dig> bind to a similar set of promoters in bg01v cells .
therefore, we use this group of tfs in sequential chip assays.
seven of the promoters that were detected by most or none of those tfs were selected for this set of experiments.
our results indicate that ifng, e-cadherin, and bax promoters are co-occupied by a bmi-1/runx2/smad2/ <dig> complex, and that the sma promoter is co-occupied by bmi-1/smad2/ <dig> .
the gata <dig> promoter was not detected in any of the individual chip assays for this group of tfs  and was used as an additional negative control.
consistently gata <dig> was not detected by sequential chip using any combination of antibodies .
in addition, muc <dig> was not detected when bmi- <dig> was the first antibody in the sequential chip assay , as expected, since muc <dig> is not detected in bmi- <dig> chip assays .
furthermore, fig.
6e shows that snail was not detected when the bmi- <dig> antibody was followed by runx <dig> or smad2/ <dig> antibodies, which was also expected, since snail was detected in bmi- <dig> chip assays, but not detected in runx <dig> or smad2/ <dig> chip .
none of the above mentioned promoters were detected when the bmi- <dig> antibody was followed by the sox <dig> antibody  or by the klf <dig> or olig <dig> antibodies .
finally, none of the above mentioned promoters were detected when the smad2/ <dig> antibody was followed by the klf <dig> or olig <dig> antibodies .
overall, these experiments suggest the presence of a runx2/bmi-1/smad2/ <dig> complex at the above mentioned promoters in undifferentiated bg01v cells.
discussion
results shown here indicate that our simplified, basic chip and the novel sequential chip assays are suitable for systematic target gene identification and detection of tf interactions at specific dna sites.
the suitability of the basic chip assay was confirmed by our ability to rapidly identify known targets of sox2/oct3/4/nanog   <cit> , sox <dig>   <cit> , runx <dig>   <cit> , klf <dig>   <cit> , bmi- <dig>   <cit> , smad2/ <dig>   <cit> , and olig <dig>   <cit> .
the basic chip protocol used here is a simplified, combined version of fast chip protocols previously described  <cit> .
the main advantages of this basic chip assay over previously published conventional protocols  is the combination of speed  and sensitivity .
our basic chip protocol combines biotinylated antibodies, streptavidin magnetic beads, chelating resin for elimination of pcr inhibitors  <cit> , and dna purification with commercially available silica columns.
it differs from previously described fast chip protocols  <cit>  by not involving: antibody capture with protein a, cross-linking reversal, proteinase k treatment and phenol-chloroform-ethanol treatment for dna isolation.
in preliminary experiments we noticed that the combination of chelating resin  <cit>  and silica columns for dna purification leads to greater pcr products yields than with chelating resin alone .
the biotin-streptavidin interaction represents one of the strongest non-covalent interactions known  <cit>  and therefore it represents an advantage over protein a-igg interaction  <cit> .
our preliminary observations  and those reported by other investigators  <cit>  suggest that cross-linking reversal and proteinase k treatment can be avoided, although it might result in a slightly lower dna yield.
the boiling of the samples with chelating resin and subsequent dna purification with silica columns seem to replace the need for cross-linking reversal and proteinase k treatment.
the use of densitometric analysis of pcr results qualitatively matched the rtpcr results for all the antibodies used here since all above mentioned known targets were detected above background by both densitometry and rtpcr .
densitometric analysis with the imagej software, due to its simplicity, practicality and less time and resources used in preliminary calibration experiments, offers a viable alternative to rtpcr when absolute quantification is not required.
in addition, it can be applied for pre-screenings prior to rtpcr.
it was expected, however, that densitometric analysis with the imagej software would show a quantification limitation, probably coming from pixel saturation in dna bands from pcr reactions and consequent lower measurement capacity.
such limitation probably caused the lower levels of fold enrichment observed in the densitometric analysis as compared to rtpcr for some of the chip assays .
however, our comparisons indicate that when the goal is simply the identification of target genes, densitometric analysis provides a reliable alternative, besides, for some antibodies, such limitation was minimum .
we then analyzed promoter co-occupancy using the newly developed sequential chip assay .
this assay provides a significant improvement from previously described sequential chip assays  <cit>  and it differs from those in two main aspects.
first, during the sequential chip assay the antibody-antigen interaction is never disrupted by epitope-specific peptides or other treatments, and consequently immunoprecipitations can be carried-out in the same, intact samples.
this feature might contribute for lower background, higher specificity and the possibility of using smaller samples.
second, the modifications introduced in the basic chip assay to make it a straightforward, fast, sensitive assay, were maintained in the sequential chip protocol.
in addition, the sequential chip combines two methods of physical separation of the targeted dna fragments.
the first antibody, non-biotinylated, binds to agarose beads, via protein g-igg interaction, and is brought down by centrifugation.
the second antibody, this time a biotinylated one, binds to magnetic beads, via biotin-streptavidin interaction, and is separated from the rest of the sample with a magnet.
these two forms of physical separation  are quite complementary and exert minimum interference over each other.
the results are even visual with the reagents used here: when the antibodies used in the assay recognize tfs that co-occupy a significant number of target genes, as it is the case for sox2/oct3/4/nanog/c-myc, one can see the agarose beads slurry attaching to the magnet in the last steps of the assay, since they are carried by the streptavidin-magnetic beads.
this visual effect is not observed with any of the negative controls.
in proof-of-principle experiments we investigated promoter co-occupancy in undifferentiated bg01v cells using sox <dig>  oct3/ <dig> and nanog antibodies  and the obtained results are consistent with those obtained in chip-chip studies  <cit> .
furthermore, our sequential chip assay identified c-myc as a potential interacting partner of the sox2/oct3/4/nanog complex at several gene promoters in undifferentiated bg01v cells .
it has been suggested that c-myc is involved in the regulation of up to 15% of all genes.
it has been reported that c-myc modulates global chromatin structure via its interaction with histone acetyl-transferase  and swi/snf chromatin remodeling complexes  <cit> , which might be c-myc function in the sox2/oct3/4/nanog complex.
supporting this hypothesis, sox2/oct3/4/nanog and c-myc have been found to share a large number of target genes in chip-chip studies  <cit> .
furthermore, data shown in fig.
6e suggests the presence of a runx2/bmi-1/smad2/ <dig> complex in undifferentiated bg01v cells.
interestingly, the association of smad2/ <dig> with runx <dig> has been reported previously in b-lymphocytes and human breast cancer cells  <cit> .
bmi- <dig> is part of the polycomb-group complex  <cit>  and a known transcriptional repressor.
smad2/ <dig> and runx <dig> have been reported to act as both activators and repressors  <cit> .
accordingly, many of the genes associated with this runx2/bmi-1/smad2/ <dig> complex in bg01v cells  are not expressed in undifferentiated es cells  <cit> , suggesting that this runx2/bmi-1/smad2/ <dig> complex plays a transcriptional repressor role in undifferentiated bg01v cells.
CONCLUSIONS
in summary, our simplified basic chip and novel sequential chip protocols were successfully tested with a variety of antibodies and generated a number of novel observations for future studies.
the basic chip assay combines simplicity , speed  and sensitivity .
more importantly, promoter co-occupancy data obtained with our novel sequential chip protocol indicates the existence of two tf complexes in human es cells: sox2/oct3/4/nanog/c-myc and runx2/bmi-1/smad2/ <dig>  this novel sequential chip protocol provides a significant improvement from previously described sequential chip assays by combining two methods of physical separation of the targeted dna  and avoiding antibody-antigen interaction disruption during the assay.
both protocols might be useful for high-throughput chip-based assays, which are expensive, labor intensive, require complex statistical analysis, and might greatly benefit from faster and straightforward chip assay and analysis methodologies.

 regulatory transcription factors are encoded by approximately 10% of the human genome. the search for an accurate and complete list of target genes for thousands of tfs and the elucidation of their complex interactions at promoter sites, particularly in embryonic stem cells, has gained increasing interest. however, only a small fraction of the in vivo target genes and relatively few tf-tf interactions have been elucidated. chromatin immunoprecipitation and its derivatives have been widely used for the investigation of tf-dna interactions. high-throughput approaches, such as chip-chip and chip-sage, are necessary for genome-wide analysis and the systematic identification of new dna-binding sequences. real-time pcr remains extensively used for validation of genome-wide data and for analysis of chip results in general. high-throughput approaches are time-consuming, expensive, labor-intensive, involve multiple steps that facilitate error introduction, and require complex statistical analysis. therefore, advances in this field will greatly benefit from the development and use of faster and straightforward chip assay and analysis methodologies. here, we present data obtained with a simplified, basic chip assay and analysis protocol that allowed the rapid identification of known target genes for sox nanog, oct3/ sox klf runx olig smad2/ bmi- and c-myc in the human es cell line bg01v. we used rtpcr to initially validate the protocol/antibodies and densitometric analysis of pcr results with the imagej software as a more practical, less expansive, less time-consuming readout alternative. in addition, we developed a novel, non-disruptive, highly sensitive sequential chip protocol for the identification of promoter co-occupancy, based on our simplified basic chip protocol. the data obtained with this sequential chip protocol are consistent with data previously obtained with more labor-intensive, expensive, time-consuming chip-chip platforms. furthermore, sequential chip analysis led to the identification of two tf complexes in bg01v es cells: sox2/nanog/oct3/4/c-myc and runx2/bmi-1/smad2/ complexes. these two tf complexes associate with two different sets of target genes. the runx2/bmi-1/smad2/ complex is associated predominantly with genes not expressed in undifferentiated bg01v cells, consistent with the reported role of those tfs as transcriptional repressors. these simplified basic chip and novel sequential chip protocols were successfully tested with a variety of antibodies with bg01v es cells, generated a number of novel observations for future studies and might be useful for high-throughput chip-based assays.  development of an improved basic chip protocol we developed a simplified, basic chip protocol and test its usefulness with antibodies against tfs expressed in the human es cell line bg01v. these antibodies included those against sox nanog, oct3/ sox runx olig smad2/ klf bmi- and c-myc. this basic chip assay is characterized by the combination of simplicity, speed and sensitivity. recently described, commonly used protocols normally take longer time or lack one or more of those characteristics. chip assays were performed with previously characterized antibodies and known target genes and initially analyzed by rtpcr. we also analyzed the pcr results by densitometry using the imagej software, reducing time and resources for defining pcr parameters and, therefore, significantly decreasing experimental costs. target genes included fgf lefty, nanog, vegf, bcl gli e-cadherin, oct3/ c-myc, hesx zfp and suz, lama, b2r, hoxc, c-myc and gli, p and vegf and bax. all primer sets have been validated previously. normal igg and input dna were used as negative controls. * all others are human genes. the undifferentiated human es cell line bg01v, used throughout this study, shows nanog expression and nuclear localization, as expected. sox and oct3/ expression and nuclear localization was also verified. fig. 2b shows fold enrichment relative to input dna, as determined by rtpcr analysis, of three known sox2/oct3/4/nanog target genes from sox2/oct3/4/nanog chip assays. fig. 2c depicts pcr results using candidate targets and fig. 2d shows fold enrichment, as determined by densitometric analysis, of the pcr results, of the three abovementioned known targets and additional known sox2/oct3/4/nanog targets. figs. 2c and 2d also suggest hoxc pten and bax as novel putative sox2/oct3/4/nanog targets in undifferentiated bg01v cells. these results indicate that the described chip assay and analysis protocol are suitable for chip screening. moreover, the results also suggest that the densitometric quantification of pcr results using the imagej software can be used as an alternative chip readout, reducing experimental time and resources. to further confirm the usefulness of the protocol and test its applicability, chip assays were performed with additional es cell tfs and their known target genes. expression and nuclear localization of all these tfs were verified. sox plays critical roles in development regulation, stem cell function and is required for endoderm specification and maintenance in the embryo. as expected the mouse lama promoter was detected by rtpcr and densitometric analysis from sox chip assays using endoderm differentiated mouse d es cells. runx linked to bone development and maintenance, regulates vegf and bax, which were detected by rtpcr and densitometric analysis from runx chip assays using undifferentiated bg01v cells. klf implicated in transcriptional repression in smooth muscle cells, kidney development, stem cell self-renewal and pluripotency, regulates oct3/ nanog, and b2r, which were detected by rtpcr and densitometric analysis from klf chip assays using undifferentiated bg01v cells. bmi- implicated in cell proliferation and stem cell self-renewal, binds to the hoxc promoter, which was detected by rtpcr and densitometric analysis of bmi- chip assays using undifferentiated bg01v cells. smad2/ involved in epithelial-to-mesenchymal transition, neuronal differentiation, and chondrogenesis, have been shown to regulate c-myc and gli, which were also detected by rtpcr and densitometric analysis of smad2/ chip assays using undifferentiated bg01v cells. olig implicated in neuron, astrocyte and oligodendrocyte differentiation, binds to the p promoter, which was detected by rtpcr and densitometric analysis of olig chip assays using undifferentiated bg01v cells. as an additional negative control, an antibody against pdx which is involved in pancreatic organogenesis, was also used in chip assays. as expected pdx was not detected by immunofluorescence in undifferentiated bg01v cells. consistently, a know pdx target gene, insulin, could not be detected above background by rtpcr in pdx chip assays performed with undifferentiated bg01v cells. in addition, our basic chip protocol was directly compared to a previously described commonly used chip protocol, using three of the above mentioned antibodies and their target genes. fig. 4b shows that our basic chip protocol leads to higher fold enrichment, compared to igg controls, than the fold enrichment obtained with a previously described commonly used chip protocol. finally, to add experimental evidence for the applicability of the densitometric analysis of chip results using the imagej software, olig chip assays were performed with serially diluted template and primers. fig. 4c shows that imagej measurements of p promoter signals from these olig chip assays are within a logarithmic range. taken together, these results confirm that our simplified, basic chip assay can be used with a variety of antibodies and that the densitometric quantification of pcr results using the imagej software can be used as an alternative chip readout. sox2/oct3/4/nanog/c-myc complex co-occupies several promoters in bg01v cells we then sought to examine tf-tf interactions at promoter sites using a newly developed sequential chip assay, which is based on the simplified basic chip assay. chip-chip studies have identified numerous promoters co-occupied by the sox2/oct3/4/nanog complex. to validate the effectiveness of the sequential chip assay we selected a number of sox2/oct3/4/nanog known targets and subjected these targets to our new protocol. the data obtained with the sequential chip assay in undifferentiated bg01v cells is consistent with previously reported chip-chip data. first, these three tfs have been reported to co-occupy the oct3/ nanog, hesx and vegf promoters, and consistent with that, any combination of those tfs antibodies in the sequential chip assay led to the detection of those promoters. second, only sox and oct3/ co-occupy the suz promoter, and consistent with that, only immunoprecipitation with the sox antibody followed by the oct3/ antibody lead to the detection of the suz promoter, whereas other antibody combinations did not. a sox regular chip assay was used as a positive control. sox2/oct3/4nanog and c-myc have been found to share a large number of target genes in chip-chip studies. we then investigated if c-myc co-occupies promoters with the sox2/oct3/4/nanog complex. indeed, immunoprecipitation with the sox antibody followed by the c-myc antibody led to the detection of a number of promoters in undifferentiated bg01v cells. the same promoters were detected if the nanog antibody preceded the c-myc antibody or if the oct3/ antibody preceded the c-myc antibody. in contrast, none of these promoters were detected when normal igg was used as the first antibody. these results indicate that, at the indicated promoters, c-myc is a subunit of the sox2/oct3/4/nanog complex in undifferentiated bg01v cells. to further test this hypothesis we immunodepleted sox from undifferentiated bg01v cells lysates and then analyzed c-myc targets by chip. fig. 6d shows which c-myc-bound promoters were detected in those sox2-depleted lysates and shows a significant reduction in the number of c-myc-associated promoters. most of the promoters detected in fig. 6b top panel were not detected by c-myc chip after sox depletion, including p fgf nanog, vegf, pten, bax, muc oct3/ and suz. collectively these results corroborate the applicability of the novel sequential chip assay. in addition these results indicate that c-myc associates with the sox2/oct3/4/nanog complex in bg01v cells. runx2/bmi-1/smad2/ complex co-occupies a second set of promoters in bg01v cells we then sought to investigate the presence of other tf complexes in bg01v cells using the sequential chip assay. in a screening with potential target genes, we found that bmi- runx smad2/ klf and olig bind to a similar set of promoters in bg01v cells. therefore, we use this group of tfs in sequential chip assays. seven of the promoters that were detected by most or none of those tfs were selected for this set of experiments. our results indicate that ifng, e-cadherin, and bax promoters are co-occupied by a bmi-1/runx2/smad2/ complex, and that the sma promoter is co-occupied by bmi-1/smad2/. the gata promoter was not detected in any of the individual chip assays for this group of tfs and was used as an additional negative control. consistently gata was not detected by sequential chip using any combination of antibodies. in addition, muc was not detected when bmi- was the first antibody in the sequential chip assay, as expected, since muc is not detected in bmi- chip assays. furthermore, fig. 6e shows that snail was not detected when the bmi- antibody was followed by runx or smad2/ antibodies, which was also expected, since snail was detected in bmi- chip assays, but not detected in runx or smad2/ chip. none of the above mentioned promoters were detected when the bmi- antibody was followed by the sox antibody or by the klf or olig antibodies. finally, none of the above mentioned promoters were detected when the smad2/ antibody was followed by the klf or olig antibodies. overall, these experiments suggest the presence of a runx2/bmi-1/smad2/ complex at the above mentioned promoters in undifferentiated bg01v cells. discussion results shown here indicate that our simplified, basic chip and the novel sequential chip assays are suitable for systematic target gene identification and detection of tf interactions at specific dna sites. the suitability of the basic chip assay was confirmed by our ability to rapidly identify known targets of sox2/oct3/4/nanog, sox, runx, klf, bmi-, smad2/, and olig. the basic chip protocol used here is a simplified, combined version of fast chip protocols previously described. the main advantages of this basic chip assay over previously published conventional protocols is the combination of speed and sensitivity. our basic chip protocol combines biotinylated antibodies, streptavidin magnetic beads, chelating resin for elimination of pcr inhibitors, and dna purification with commercially available silica columns. it differs from previously described fast chip protocols by not involving: antibody capture with protein a, cross-linking reversal, proteinase k treatment and phenol-chloroform-ethanol treatment for dna isolation. in preliminary experiments we noticed that the combination of chelating resin and silica columns for dna purification leads to greater pcr products yields than with chelating resin alone. the biotin-streptavidin interaction represents one of the strongest non-covalent interactions known and therefore it represents an advantage over protein a-igg interaction. our preliminary observations and those reported by other investigators suggest that cross-linking reversal and proteinase k treatment can be avoided, although it might result in a slightly lower dna yield. the boiling of the samples with chelating resin and subsequent dna purification with silica columns seem to replace the need for cross-linking reversal and proteinase k treatment. the use of densitometric analysis of pcr results qualitatively matched the rtpcr results for all the antibodies used here since all above mentioned known targets were detected above background by both densitometry and rtpcr. densitometric analysis with the imagej software, due to its simplicity, practicality and less time and resources used in preliminary calibration experiments, offers a viable alternative to rtpcr when absolute quantification is not required. in addition, it can be applied for pre-screenings prior to rtpcr. it was expected, however, that densitometric analysis with the imagej software would show a quantification limitation, probably coming from pixel saturation in dna bands from pcr reactions and consequent lower measurement capacity. such limitation probably caused the lower levels of fold enrichment observed in the densitometric analysis as compared to rtpcr for some of the chip assays. however, our comparisons indicate that when the goal is simply the identification of target genes, densitometric analysis provides a reliable alternative, besides, for some antibodies, such limitation was minimum. we then analyzed promoter co-occupancy using the newly developed sequential chip assay. this assay provides a significant improvement from previously described sequential chip assays and it differs from those in two main aspects. first, during the sequential chip assay the antibody-antigen interaction is never disrupted by epitope-specific peptides or other treatments, and consequently immunoprecipitations can be carried-out in the same, intact samples. this feature might contribute for lower background, higher specificity and the possibility of using smaller samples. second, the modifications introduced in the basic chip assay to make it a straightforward, fast, sensitive assay, were maintained in the sequential chip protocol. in addition, the sequential chip combines two methods of physical separation of the targeted dna fragments. the first antibody, non-biotinylated, binds to agarose beads, via protein g-igg interaction, and is brought down by centrifugation. the second antibody, this time a biotinylated one, binds to magnetic beads, via biotin-streptavidin interaction, and is separated from the rest of the sample with a magnet. these two forms of physical separation are quite complementary and exert minimum interference over each other. the results are even visual with the reagents used here: when the antibodies used in the assay recognize tfs that co-occupy a significant number of target genes, as it is the case for sox2/oct3/4/nanog/c-myc, one can see the agarose beads slurry attaching to the magnet in the last steps of the assay, since they are carried by the streptavidin-magnetic beads. this visual effect is not observed with any of the negative controls. in proof-of-principle experiments we investigated promoter co-occupancy in undifferentiated bg01v cells using sox oct3/ and nanog antibodies and the obtained results are consistent with those obtained in chip-chip studies. furthermore, our sequential chip assay identified c-myc as a potential interacting partner of the sox2/oct3/4/nanog complex at several gene promoters in undifferentiated bg01v cells. it has been suggested that c-myc is involved in the regulation of up to 15% of all genes. it has been reported that c-myc modulates global chromatin structure via its interaction with histone acetyl-transferase and swi/snf chromatin remodeling complexes, which might be c-myc function in the sox2/oct3/4/nanog complex. supporting this hypothesis, sox2/oct3/4/nanog and c-myc have been found to share a large number of target genes in chip-chip studies. furthermore, data shown in fig. 6e suggests the presence of a runx2/bmi-1/smad2/ complex in undifferentiated bg01v cells. interestingly, the association of smad2/ with runx has been reported previously in b-lymphocytes and human breast cancer cells. bmi- is part of the polycomb-group complex and a known transcriptional repressor. smad2/ and runx have been reported to act as both activators and repressors. accordingly, many of the genes associated with this runx2/bmi-1/smad2/ complex in bg01v cells are not expressed in undifferentiated es cells, suggesting that this runx2/bmi-1/smad2/ complex plays a transcriptional repressor role in undifferentiated bg01v cells.  in summary, our simplified basic chip and novel sequential chip protocols were successfully tested with a variety of antibodies and generated a number of novel observations for future studies. the basic chip assay combines simplicity, speed and sensitivity. more importantly, promoter co-occupancy data obtained with our novel sequential chip protocol indicates the existence of two tf complexes in human es cells: sox2/oct3/4/nanog/c-myc and runx2/bmi-1/smad2/ this novel sequential chip protocol provides a significant improvement from previously described sequential chip assays by combining two methods of physical separation of the targeted dna and avoiding antibody-antigen interaction disruption during the assay. both protocols might be useful for high-throughput chip-based assays, which are expensive, labor intensive, require complex statistical analysis, and might greatly benefit from faster and straightforward chip assay and analysis methodologies. 
however, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. the runx2/bmi-1/smad2/ complex is associated predominantly with genes not expressed in undifferentiated bg01v cells, consistent with the reported role of those tfs as transcriptional repressors. using this sequential chip protocol we found that c-myc associates with the sox2/nanog/oct3/ complex and identified a novel runx2/bmi-1/smad2/ complex in bg01v cells. the investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. these two tf complexes associate with two distinct sets of target genes. it combines centrifugation of agarose beads and magnetic separation. chip analysis by real-time pcr was compared to analysis by densitometry with the imagej software. this protocol allowed the rapid identification of known target genes for sox nanog, oct3/ sox klf runx olig smad2/ bmi- and c-myc in a human embryonic stem cell line. advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward chip assay and analysis methodologies. high-throughput approaches based on chromatin immunoprecipitation have been widely used for this purpose. we initially developed a simplified, basic chip protocol that combines simplicity, speed and sensitivity. these simplified basic chip and novel sequential chip protocols were successfully tested with a variety of antibodies with human embryonic stem cells, generated a number of novel observations for future studies and might be useful for high-throughput chip-based assays. we then developed a novel sequential chip protocol to investigate in vivo promoter co-occupancy, which is basically characterized by the absence of antibody-antigen disruption during the assay.
 the investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. high-throughput approaches based on chromatin immunoprecipitation have been widely used for this purpose. however, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward chip assay and analysis methodologies.  we initially developed a simplified, basic chip protocol that combines simplicity, speed and sensitivity. chip analysis by real-time pcr was compared to analysis by densitometry with the imagej software. this protocol allowed the rapid identification of known target genes for sox nanog, oct3/ sox klf runx olig smad2/ bmi- and c-myc in a human embryonic stem cell line. we then developed a novel sequential chip protocol to investigate in vivo promoter co-occupancy, which is basically characterized by the absence of antibody-antigen disruption during the assay. it combines centrifugation of agarose beads and magnetic separation. using this sequential chip protocol we found that c-myc associates with the sox2/nanog/oct3/ complex and identified a novel runx2/bmi-1/smad2/ complex in bg01v cells. these two tf complexes associate with two distinct sets of target genes. the runx2/bmi-1/smad2/ complex is associated predominantly with genes not expressed in undifferentiated bg01v cells, consistent with the reported role of those tfs as transcriptional repressors.  these simplified basic chip and novel sequential chip protocols were successfully tested with a variety of antibodies with human embryonic stem cells, generated a number of novel observations for future studies and might be useful for high-throughput chip-based assays. 