BACKGROUND
of the roughly  <dig>  protein-coding  genes  <cit> , there are approximately  <dig> to  <dig>  genes expressed at a significant level in a given cell type.
the gene regulatory code consists of the dna sequence-dependent binding specificities of a prominent class of dna binding proteins.
these so called transcription factors  are able to read the regulatory information and transmit the information encoded in primary dna sequence to the transcriptional machinery, thereby regulating the synthesis of rna transcripts  <cit> .
differential tissue-specific gene expression is an essential component of gene regulation in humans, which restricts the expression of certain genes to given stages of development.
importantly, the erroneous reading of the gene regulatory code is often accompanied by the formation of tumors or the manifestation of other diseases associated with developmental disorders.
therefore, a detailed understanding of the spatial and temporal gene expression patterns is of fundamental importance for understanding the complexity of the human genome.
this is by far not a trivial task because the cis-regulatory elements are usually ambiguous and context-dependent and can function both in the immediate vicinity of genes and as enhancer elements  <cit>  thousands of dna base pairs away from their transcriptional start site.
since the cis-regulatory elements and associated transcription factors are believed to be responsible for maintaining the conserved gene expression patterns between related organisms, the elucidation of the gene regulatory code brings back into focus a simple question: “which transcription factors  do bind to a cis-regulatory element in a given cell type?”
traditionally, tf binding to a putative cis-element could be identified through the combination of classical biochemical fractionations coupled to eletrophoretic mobility shift assay  and amino acid sequencing of the purified transcription factors.
in order to significantly improve the traditional approach, it is essential to link the isolation and enrichment of the tf directly to the detection of its sequence specificity and its identification.
recently, we  <cit>  and others  <cit>  have developed scalable methodologies  <cit>  that are fulfilling this task.
our silac-based dna protein interaction screen  <cit>  makes use of the fact that one can introduce mutational changes in the dna sequence to destroy the function of a cis-element thereby preventing the binding of its regulatory tfs.
hence, a direct quantitative proteomic comparison of the protein binding pattern between the natural and the mutated dna sequence via nanolc-ms will provide the identity of the tfs that have the potential to interact with the regulatory element in vivo.
here we apply this technology to investigate an evolutionary hyperconserved promoter-associated cis-element, termed m <dig> that until now has been poorly studied.
briefly, a modern bioinformatic whole genome alignment comparison of mammalian genomes  led to the discovery of several novel, evolutionarily conserved cis-elements, whose function was in most cases poorly characterized  <cit> .
among them the best conserved novel element found was the m <dig> motif with the dna consensus sequence “rractacanntcccrrnrnrc.” this was partially described as a binding site for the non-lymphoid transcription factors thap <dig>  staf/zfn <dig> and rbp-j  <cit> .
as suggested by the fact that genes harboring m <dig> motifs exhibit increased expression levels in lymphoid and some leukemia or lymphoma cells  <cit> , it implied a putative role of m <dig> motif in the lymphoid system or in hematopoietic malignancies.
in the present study, by using silac-based quantitative proteomics, we show that the lymphocyte specific ikaros family proteins , the nf-kappa b proteins nfkb <dig> and nfkb <dig> , the pluripotency factor ronin/thap <dig> and host cell factor- <dig>  are able to bind specifically, either directly or indirectly, to the m <dig> motif in vitro.
importantly, the tf m4-dna interactions of ikaros, nfkb <dig> , thap <dig> and hcf- <dig> are confirmed by chip assays in living cells, thus providing evidence that the silac-based dna protein interaction screen can work as a surrogate for a bona fide reverse chip technology.
notably, our data also suggest that the conserved m <dig> motif is a composite cis-element containing two tf binding modules, which can operate independently from each other.
we provide strong evidence that thap <dig> interacts directly with the actayr part of m <dig> thereby engaging in a ternary complex with hcf- <dig>  whereas ikaros binds to the cccrrnrnrc sequence of m <dig>  the latter interaction is essential for recruiting nfkb <dig>  to the m <dig> motif.
transient reporter assays performed in hek  <dig> cells and sh-rna mediated loss-of-function assays in molt <dig> t cells unequivocally demonstrate binding of ikaros and thap <dig> to genes containing m4-promoters as functional and therefore biologically relevant.
our current model, utilizing transient reporter assays and thap <dig> shrna knockdown in molt <dig> t cells, mainly suggests an activator function of thap11/hcf- <dig> for m4-genes in these cells.
unlike thap <dig>  the apparent role of ikaros is to modulate the transcriptional output of endogenous m4-harboring genes by either repressing or activating them.
RESULTS
emsa assay reveals presence of m <dig> binding factors in lymphocyte nuclear extracts
although several studies have shown the binding of some tfs to the m <dig> motif , none reported m <dig> as the binding site for a heamatopoietic tf regardless of the fact that genes harboring the m <dig> motif exhibit higher expression levels in many cell lines derived from the immune system  <cit> .
emsa was performed using a radioactively labeled double-stranded oligodeoxynucleotide bearing the m <dig> motif that was incubated with either raji burkitt’s lymphoma or hela nuclear extracts followed by electrophoretic separation on a native polyacrylamide gel in order to reveal potential m <dig> binders that are specifically expressed in hematopoietic cells.
the band shift data from fig.
<dig> show the presence of lymphocyte-specific  m <dig> binding factors that are hardly expressed in cervical cancer hela cells .
importantly, the raji lymphoma specific band shifts are still formed even in the presence of a hundred-fold excess of an unlabeled competitor oligonucleotide bearing a sequence unrelated to m <dig> .
conversely, the formation of band shifts is completely abolished if a hundred-fold excess of non-radioactive m <dig> oligonucleotide is added .
this suggests that raji burkitt’s lymphoma cells express proteins that can selectively interact with m <dig> motif in vitro.fig.
<dig> emsa assay to uncover lymphocyte-specific transcription factors interacting with m <dig> motif bearing dna.
radioactive γ-atp labeled m <dig> oligo was incubated either with raji burkitt’s lymphoma nuclear extracts , or hela nuclear extract .
emsa complexes formed in assays using raji burkitt’s lymphoma nuclear extract  are virtually absent in hela nuclear extract .
those band shifts were significantly faded away when an increasing amount of cold m <dig> motif was added  but still preserved  if the cold m <dig> motif containing oligo was replaced by an m <dig> scrambled counterpart.
lanes  <dig>   <dig> are control assays using m <dig> oligo in the absence of nuclear extracts
ms-based proteomic screening to identify lymphocyte-specific m <dig> interacting factors
to identify m <dig> interacting factors, raji burkitt’s lymphoma cells were metabolically encoded with either “heavy” or “light”  isotopic variants of lysine .
the corresponding nuclear extracts  were prepared and used as an input for subsequent silac-based dna affinity pull-down screening  <cit>  comparing the protein binding profile of m <dig> wild-type  versus m <dig> mutant baits .
following trypsin digestion and nanolc-ms  <dig> out of more than  <dig> identified proteins were quantified by the msquant software  <cit>  utilizing lysine-containing tryptic peptides.
most of the isotopic variants of quantified peptides were present in almost equal amounts .
this unequivocally demonstrated that most of the quantified proteins do bind unspecifically to either m <dig> wt or m <dig> mt columns.
however, there were seven candidate factors , exhibiting silac ratios greater than two that were termed m4-binding candidates .fig.
<dig> silac-based dna protein interaction sreen reveals m <dig> binding candidates.
a distribution of silac ratio against quantified proteins identified in tandem m <dig> dna affinity chromatography experiments using raji burkitt’s lymphoma nuclear extracts: the x-axis present the random code of quantified proteins originating from the mass spectrometric screening of m <dig> dna affinity chromatography experiments; the  present silac ratios of individually quantified proteins; b mass spectra of selected m <dig> interacting partners’ derived peptides: mass spectra of individual peptides comprising both light isotope  and heavy peaks .
those derived from specific m <dig> binders were specified by higher intensity of heavy isotope encoded peaks.
in contrast, the ones descending from unspecific binders are presented by equal intensity of both light and heavy isotope encoded peaks
nfkb <dig>  and thap <dig> to the mono m <dig> motif-containing dna affinity column.
a prominent putative m <dig> binding candidate factors.
b dna affinity chromatography: m <dig> wt and control mono m <dig> mt were immobilized as ligand, raji burkitt’s lymphoma nuclear extract was used as input.
protein-dna complexes from two columns were eluted, resolved by sds-page separately for western blotting analysis.
specific antibodies were used to detect the retention of suspected m <dig> binding candidates.
the unspecific dna binding factor tpb was adopted as loading control:
immunoblot analysis confirms the in vitro interaction of m <dig> binding candidates with m4
the m <dig> sequence of the oligodesoxynucleotide baits used in the proteomic screening experiment were derived from the dffa gene  promoter, which contains two copies of the m <dig> motif in close vicinity ; however, most human m <dig> bearing genes contain only one copy of the m <dig> motif in their promoter  <cit> .
to exclude the possibility that a duplicated m <dig> motif could induce some avidity binding effects in respect to putative m <dig> interacting factors, the dna affinity chromatography was repeated using columns that harbor only one copy of either wt or mutant m <dig> motif.
separate eluates from wt and mt dna columns were resolved by sds-page and subjected to immunoblot analysis for validating the putative m4-binding candidates.
as shown in fig.
<dig>  antibodies directed against ikaros, host cell factor  <dig> , nfkb <dig> , and thap <dig> confirmed the specific binding of these proteins to the wt m <dig> motif .
m4-binding proteins interact with promoter regions containing the m <dig> motif in vivo
to demonstrate the potential biological function of m <dig> motif binding candidates, classical formaldehyde crosslinking chromatin immunoprecipitation  assays were performed, followed by qrt-pcr of bound dna.
in this assay, the three m <dig> bearing promoters of the genes skp <dig>  cdc25a, and g9a were selected for two reasons: first, gene ontology analysis of m4-genes reveal that genes involved in mitotic cell cycle regulation and chromatin function are statistically overrepresented amongst m <dig> bearing genes .
second, expression levels of the three aformentioned genes were significantly affected upon ectopic over-expression of the dominant negative ik <dig> isoform  <cit>  of ikaros in raji burkitt’s lymphoma cells as well as molt <dig> t-cells .
initial attempts to establish chip conditions for ikaros in raji burkitt’s lymphoma cells employing antibodies directed against the ikaros c-terminus were difficult and could be ascribed to the fact that raji burkitt’s lymphoma cells exhibit an abundant expression of small ikaros isoforms , which are not able to bind dna directly but can multimerize with ik <dig> and ik <dig>  isoforms.
such higher-order complexes cannot be efficiently recovered by the standard chip protocol, which works under stringent denaturing conditions strongly favouring the recovery of dna sequences that are directly crosslinked to the immunoprecipitated protein.
to circumvent this, we decided to focus on t-lymphoma molt <dig> cells because these cells do not express the small ikaros isoforms .
furthermore, to standardize our ip conditions, c-terminally ha-tagged ikaros that is known to recognize canonical ikaros target genes  <cit>  was constructed and retrovirally transduced into molt <dig> cells .
a similar procedure was adopted for the generation of molt4-thap11-ha cells, which ectopically expresses c-terminally ha-tagged thap <dig> protein.
importantly, like its endogenous counterpart, thap-11-ha demonstrates robust m4-specific binding in a dna affinity pulldown assay .
the two ha-tagged transgenic molt <dig> t-cell lines were later used for either ikaros or thap <dig> chip assay using a high affinity rat-anti-ha antibody .
as seen in fig.
4a, m <dig> motif-bearing dna was specifically co-precipitated by anti-ikaros/ha ip , demonstrating specificity of the ip protocol.
strikingly, binding of ikaros to chromatin was selectively enriched on dna fragments containing the m <dig> motif in all three promoters examined .
therefore, it can be concluded that ikaros interacts with the m <dig> cis-element in living cells.
recapitulating the same experimental strategy, we were able to show the specific and selective enrichments of nfkb <dig>  , thap11-ha  and hcf- <dig>  on the promoters of the m <dig> bearing genes skp <dig>  cdc25a, and g9a.fig.
<dig> chromatin immunoprecipitation  analysis of ikaros, nfkb <dig> , thap <dig>  and hcf- <dig> binding to m4-bearing promoters of the genes skp <dig>  cdc25a, and g9a.
formaldehyde crosslinked chromatin of molt-ik-ha or molt4-thap11-ha cells were sheared by ultrasonication into an average length of  <dig> to 1000 bps to be subjected to immuno-precipitation assays using anti-nf-kappa b p <dig>  anti-hcf- <dig>  anti-ha/ikaros, anti-ha/thap <dig>  or preimmune igg sera .
the tf-associated chromatin were analyzed via quantitative real time pcr  using primer sets flanking the suspected tf binding site and background binding control regions: promoter , exon , ±2000 bp .
enrichment of tf-bound dna amount were normalized to corresponding amount in input.
chip results  for ikaros , nfkb <dig>  , thap <dig>  and hcf- <dig>  are shown
intriguingly, chip enrichment of nfkb <dig>  bound interleukin- <dig> promoter dna, which represents a well established nf-kappa b target gene harboring a classical palindromic kappa b binding site  <cit> , was comparable to the enrichment of nfkb <dig>  bound m4-promoter dna .
this suggested that the affinity of nfkb <dig>  towards m4-bearing promoters was not substantially different compared to a well studied classical nf-kappa b target gene.
m <dig> motif is a composite element, comprising two distinct non-overlapping binding sites for both thap <dig> and ikaros
according to our studies the m <dig> motif consists of two highly conserved parts.
while the “upstream core” rractayr is similiar to the ronin  binding site, the “downstream core”  is similar to the previously described murine ikaros binding site consensus   <cit> .
this raises the question if the m <dig> motif is indeed a composite element that can be partitioned into two sub-consensus sequences.
the latter can be best answered by investigating if the suspected direct sequence-specific interaction partners of the m <dig> cis-element, thap <dig> and ikaros, are binding synergistically or independently to m <dig>
therefore, we hypothesized that the “downstream core”  will be sufficient to recruit ikaros, whereas the “upstream core” rractayr would be indispensable for engaging other m <dig> binders namely thap <dig>  to verify this, oligo-pulldown assays were conducted using three modified m <dig> motif-bearing dna sequences as a ligand: mod-m <dig> , f-mt-m <dig> , and r-mt-m <dig> .
as can be seen in fig.
<dig>  the “flanking” mutations did not interfer with the binding of either m <dig> binding factors to the m <dig> motif.
in contrast, the mutation of the m <dig> “upstream core” rractayr was sufficient to simultaneously abrogate the binding of thap <dig> and hcf- <dig> to m <dig> motif bearing dna, while still preserving the interaction of both ikaros and nfkb <dig>  with the m <dig> motif.
consequently, mutation of the “downstream” part of the m4-motif abolished recruitment of ikaros and nfkb <dig>  to m <dig> dna, but did virtually not alter the binding of thap <dig> and hcf- <dig> to m <dig>  for this reason we conclude from our in vitro binding data that m <dig> is a composite tf binding module.
it consists of two tf binding sites; the “upstream core” rractayr that is essential for recruiting thap11/hcf- <dig> to the m <dig> cis-element and the “downstream core” tcccrrnr that is required for establishing m4-ikaros/nf-kappa b interactions.fig.
<dig> the m <dig> motif is a bipartite composite element, consisting of two independent binding site modules for thap <dig> and ikaros.
a dna ligands used in affinity chromatography: mono-m <dig> wild-type , mono-m <dig> mutant , mod-m <dig> , f-mt-m <dig>  and r-mt-m <dig> .
b immunblot analysis of dna affinity chromatography experiments using five dna ligands: input  and eluates from three columns were resolved by sds-page in parallel and subjected to western blotting.
specific antibodies detected the retention of suspected m <dig> binding factors
nfkb <dig>  interaction with m <dig> is critically dependent on ikaros
the m <dig> motif is neither a palindromic sequence nor analogous to any known kappa b site  <cit> .
however, the “downstream core”  that mediates nfkb <dig> association is highly similar to the murine ikaros binding site.
therefore, one of two scenarios can be envisioned: i) ikaros and nfkb <dig> compete in binding to the “downstream core”  of the m <dig> motif or ii) nfkb <dig> is recruited to the m <dig> motif in an ikaros-dependent manner.
to investigate this a stable shrna ikaros knock-down molt <dig> cell line  was generated by retroviral transduction, in which ikaros protein expression was almost completely abrogated .
dna affinity chromatography experiments using mono-m <dig> wt and mono-m <dig> mt beads in combination with nuclear extracts from molt <dig> , molt4-ik-kd or cells that do not express ikaros  were conducted and subjected to immunblot analysis as described before.
notably, even though protein expression levels of nfkb <dig>  were virtually not affected by our highly efficient ikaros knockdown, its binding to the m <dig> column was completely abolished.
however, upon ectopic expression of ikaros in hek <dig> cells , the recruitment of nfkb <dig>  to m <dig> containing dna was reestablished.
in contrast, the binding of the two other m4-binders  was not affected either by the absence  or presence  of ikaros expression.
this implies that ikaros expression is absolutely crucial for the binding of nfkb <dig>  to dna harboring the m <dig> sequence in vitro.
nevertheless, it remained to be resolved if nfkb <dig>  was recruited to m <dig> by a more direct interaction with ikaros.
to address this, we have carried out a coimmunoprecipitation assay using anti-nfkb <dig>  antibody.
the results clearly demonstrate that ikaros is part of a nfkb <dig>  protein complex, which is resistant to efficient  dna and rna nuclease  digestion .
of note, the latter indicates that the ikaros containing nfkb <dig> complex is not formed or stabilized by random protein nucleic-acid interactions.fig.
<dig> nfkb <dig>  interacts with ikaros and this interaction is essential for recruiting rel-nf-kappa b family tfs to m <dig> motif harboring dna.
a immunoblot analysis of dna affinity chromatography experiments using two dna ligands: mono-m <dig> wt and mono-m <dig> mt.
molt <dig> control or molt4-ikaros kd nuclear extracts served as input.
in the absence of ikaros, protein levels of nfkb <dig>  were virtually not affected  but its binding to the m <dig> column was completely abrogated .
in contrast, the binding of the two other established m4-binders  were robustly maintained .
b anti-nf-kappa b p <dig>  ip  co-precipitated ikaros in the presence  and absence  of the highly efficient nuclease bezonase.
c cross-linked streptactin pulldown experiment demonstrates co-purification of strepii-tagged ikaros  with nfkb <dig>  protein , whereas the nuclear protein kinase cdk <dig> that was reported to interact with nfkb <dig>   was not present in the cross-linked complex .
d benzonase nuclease digestion control assay: the control 40 bp duplex dna or control salmon sperm dna were mixed with molt <dig> nuclear extracts in the presence or absence of benzonase at 4 °c .
the whole samples were analyzed by an 8 %  native tbe-page gel that allows separation of dna down to the size of 10 bps
to exclude the possibility that ikaros might unspecifically associate with the anti-nfkb <dig>  serum, a reverse immunoprecipitation assay using either anti-ikaros or anti-ha  antibodies was conducted.
however, co-precipitation of nfkb <dig>  was not achieved .
since both the anti-ikaros and anti-ha antibody recognize epitopes at the c-terminus of ikaros, which is known to mediate protein-protein interactions  <cit> , we reasoned that antibody binding to the ikaros carboxyterminus might disturb the ikaros-nfkb <dig>  interaction.
to circumvent this, an aminoterminal tag was fused to the cdna of human ikaros .
the tandem-strepii-tag  was selected as it exhibits an extremely fast on-rate during binding to streptactin beads and chromatography can be performed under very stringent conditions employing formaldehyde crosslinking.
in order to pursue this, a molt <dig> cell line stably expressing strepii-tagged ikaros  was created via retroviral transduction.
to confirm that the heterologously expressed strepii-tagged ikaros protein is functional, chip experiments were conducted as described above.
the chip enrichement values for selected m4-bearing gene promoters  were almost identical  to the ones obtained with ikaros-ha protein  suggesting that ectopically expressed strepii-tagged ikaros retains its biological function.
as seen in fig.
6e, the crosslinked streptactin pulldown experiment reveals a moderate  co-purification of nfkb <dig>  protein with strepii-tagged ikaros, whereas the nuclear protein kinase cdk <dig> that was reported to directly interact with nfkb <dig>   <cit>  was not present in the crosslinked complex.
this demonstrates high specificity of the formaldehyde crosslinking approach .
since the benzonase treatment proved to be very efficient in removing nucleic acids from the input fraction , it could be inferred that the ikaros-nfkb <dig>  complex formation most likely involves protein-protein contacts and is not unspecifically mediated by random dna or rna.
hence, we conclude  that a minor fraction of nuclear nfkb <dig>  is able to engage with ikaros and that this interaction is most likely essential for recruiting nfkb <dig> to m <dig> motif containing dna in vitro.
importantly, the data do not rule out the possibility that the ikaros-nfkb <dig>  interaction can be enforced upon binding to m <dig> dna, either by an allosteric effect transmitted through a conformational change of ikaros upon dna binding or by  dna-contacts mediated by the rel homology domain of nfkb <dig> .
ikaros knock-down does not influence the m <dig> motif binding of thap <dig> and hcf- <dig> in vivo
to confirm that the m <dig> motif does also operate in vivo as a composite element, which assumes that the ikaros/ nfkb <dig>  complex does not influcence the binding of thap11/hcf- <dig> to m4-gene promoters, we performed chip in molt <dig> and molt-ik-kd cells, respectively .
in the absence of ikaros expression , the enrichment of nfkb <dig>  at three representative m <dig> bearing promoters  was totally abrogated , whereas binding of either thap <dig> or hcf- <dig> did not change substantially.
consequently, these data together with the results depicted in fig.
<dig> lead us to conclude that the m <dig> motif also constitutes in vivo a composite cis-element, with distinct and independent thap11/hcf- <dig> and ikaros/ nfkb <dig> binding modules.fig.
<dig> chromatin immunoprecipitation  analysis of nfkb <dig> , thap <dig> and hcf- <dig> interaction with m <dig> genes in ikaros knockdown cells.
formaldehyde crosslinked chromatin of molt <dig> or molt4-ik-kd cells was sheared by ultrasonication into an average length of  <dig> to 1000 bps and ultimately subjected to immuno-precipitation assays using a anti-thap <dig> antibody, b anti-nf-kappa b-p <dig>  antibody, c anti hcf- <dig>  or preimmune igg sera .
the tf-associated chromatin were analyzed via quantitative real time pcr  using primer sets flanking the promoters , and gene body control regions  of m4-bearing skp <dig>  cdc25a, and g9a genes; d the known nfkb <dig>  target genes vcam- <dig>  hla-b were also included as control.
enrichment of tf-bound dna amount were normalized to corresponding amount in input.
chip results  for nfkb1/nf-kappa b p <dig> , thap <dig> , and hcf- <dig>  were shown
thap <dig> interacts with hcf- <dig> via its c-terminal domain, which is required for tethering hcf- <dig> to m <dig> motif containing dna
towards this end we could show that hcf- <dig> is a bona fide component of m <dig> motif-binding complexes.
however, hcf- <dig> is not known to be a sequence-specific dna binding factor but primarily contacts dna through association with other tfs, such as the oct <dig> protein  <cit>  and ronin-the mouse orthologue of thap <dig>  <cit> .
in order to examine if this interaction is also taking place in the context of human proteins, a gst-pulldown assay was carried out using molt <dig> nuclear extracts as input.
briefly, the cdna encoding either full-length or the carboxyterminal part of human thap <dig>  were cloned in-frame downstream of a glutathione-s-transferase gene  cassette and expressed in e. coli.
subsequently, the purified gst-fusion proteins were used as bait for identifying thap11-interacting partners from molt <dig> nuclear extracts in gst pull-down assays.
in this context glutathione-sepharose beads loaded with gst only served as a negative control.
as illustrated in additional file 1: figure s <dig>  homogenously purified gst-fusion proteins were obtained  that efficiently and specifically captured hcf- <dig> protein as visualized by immunoblot analysis .
this result could also be confirmed in an independent proteomic experiment using lc-ms-based protein identification .
therefore, we infer that the carboxyterminal domain of thap <dig> interacts quite strongly with hcf- <dig> in human cells, an interaction potentially crucial for tethering hcf- <dig> to m <dig> motif-bearing dna.
binding of ikaros and thap <dig> to a m <dig> motif containing promoter is functional in transient reporter assays
until now we demonstrated that in lymphocytes, thap <dig> and ikaros independently interact with m <dig> cis-elements in vitro as well as in living cells.
however, it is equally important to assess if m4-binding plays a functional role in regulating gene expression of m4-harboring genes.
as a functional readout, we first established a reporter assay system using hek  <dig> cells, in which, a set of artificial m <dig> and m <dig> mutant-bearing promoters could be studied for their regulatory potential to drive expression of a downstream-conjugated luciferase gene reporter.
as discussed above hek <dig> cells do not possess any detectable ikaros expression but ectopic expression is easily accomplished by transient transfection.
as already described in fig.
6b and c, ectopically expressed ikaros displays specific m <dig> binding in vitro and additionally induces the recruitment of nfkb <dig>  to m <dig> wt baits.
to assay the transcriptional activity of an m4-bearing promoter, a well-characterized luciferase reporter  was selected, which accommodates a functionally validated binding site for the ubiquitiously expressed transcriptional activator staf   <cit> .
the latter fits to the staf consensus binding sequence  ywcccrnmatscmyyrcr .
this sequence was fused at its 5′-position with the sequence of the m <dig> motif, which is frequently overlapping with sbs in mammalian promoters  <cit> .
hence, the resulting synthetic promoter sequence  comprises the following regulatory modules: actaca , tcccatta  and tcccattatgccccgcg .
additionally, each reporter construct contains two copies of the regulatory module that encompasses either the m <dig> wt motif  or mutated m <dig> variants .fig.
<dig> a luciferase m4-reporter assay system to study the functional role of ikaros for m4-harboring genes.
a scheme illustrating the modular m4-reporter gene.
the m <dig> motif is composed of a thap <dig> binding site  and an ikaros binding site , yellow) which overlaps with the binding site of the transcriptional activator staf/znf <dig> .
b m4-reporter gene activity  of m4-wt compared to mutated m4-variant promoters in hek  <dig> and hek293-ikaros cells, which are stably expressing heterologous ikaros.
transcriptional activity of corresponding m4-promoters in the absence  or presence  of ikaros.
immunoblot analysis measuring ikaros protein expression levels and input loading control  c nucleotide sequence presentation of the m4-modules and its variants used for m4-reporter gene assays.
d transient luciferase reporter assay employing m4-reporter q and r .
ikaros is repressing m4-reporter q in a dose-dependent manner
as seen in fig.
8b, ikaros expression  correlates with a twofold reduction in luciferase activity .
importantly, the repressive activity is absolutely dependent on the presence of an intact ikaros binding site .
to confirm this observation, the two m4-reporter constructs that harbor mutated  staf/znf <dig> binding sites, namely reporter q  and sequence r  were selected in order to alleviate the profound transcription stimulatory activity of staf/znf <dig> .
then a transient reporter assay was performed expressing increasing amounts of ikaros .
these data  clearly demonstrate that ikaros is repressing reporter gene activity in a strictly dose-dependent manner because ikaros protein expression level was inversely correlated to transcriptional activity of the m4-reporter q .
conversely, upon ikaros binding site mutation  the repressive effect of ikaros was completely abrogated .
mutation of the thap <dig> binding site diminished reporter gene activity by at least two-fold, both in the presence and absence of ikaros protein .
in summary, in the context of our transient-reporter assays, ikaros acts as a transcriptional repressor, whereas thap <dig> does function as an activator.
ikaros knockdown results in de-regulated expression of m4-harboring genes
so far, it was demonstrated that ikaros is able to regulate the transcriptional output of m4-bearing genes in the context of a transient reporter assay.
however, it is not known if ikaros might play a critical role in gene regulation of endogenous m4-harboring genes.
to determine this, the rna from molt <dig> ikaros knockdown cells  and molt <dig> control cells  were used for quantitative rt-pcr experiments in order to monitor the changes in gene expression of selected m <dig> bearing genes.
as seen in fig.
<dig>  the knockdown of ikaros did not affect thap <dig> mrna levels significantly.
in contrast, it substantially increased the mrna amount of some well established ikaros target genes , like the b cell-specific lambda <dig>  <cit>  or t cell-specific hes1-gene, for which ikaros is known to function as a transcriptional repressor  <cit> .
in parallel, the ikaros knockdown dramatically reduces the expression level of the ikaros family gene aiolos, for which ikaros serves as an important transcriptional activator  <cit> .
apart from affecting known ikaros target genes, depletion of ikaros caused both up-regulation  and down-regulation  of selected m4-harboring genes .
in addition, the m4-genes g9a and mll <dig> did not virtually change expression.
similar results were obtained from a comparative microarray analysis of molt-ik-kd versus molt4-mock cells .
in conclusion we propose that ikaros can modulate the activity of m4-harboring genes in living cells.fig.
<dig> ikaros loss-of-function  leads to de-regulated expression of m <dig> bearing genes.
the mrna level  fold change of known ikaros target genes  and of selected m <dig> bearing  genes is shown
thap <dig> knockdown leads to weak but significant changes in gene expression of selected m <dig> motif harboring genes
finally, for deciphering the biological and functional relevance of the thap11-m <dig> motif interaction in living cells, a stable  thap <dig> knockdown cell was generated .
however, it seems that thap <dig> might be essential for cell viability, because selection of clones that exhibited both a complete ablation of thap <dig> mrna levels and a substantial decrease in thap <dig> expression was not possible .
it was noted that thap <dig> localizes to both the cytoplasm and nucleus .
surprisingly, the knockdown mainly affected its cytosolic fraction while keeping a reduced, but still substantial amount of thap <dig> in the nucleus .
as for this reason, strong effects of the thap <dig> knockdown on m <dig> target gene expression were not expected.
to provide reproducible data it was decided to conduct biological replicate experiments in which the extraction of molt4-sh-thap <dig> total mrna was carried out at two different time points  after selecting the clone.
these samples are termed biological replicate  <dig> and biological replicate  <dig>  respectively.fig.
<dig> thap <dig> knockdown leads to weak but significant changes in selected m <dig> bearing gene expression.
a left panel shows fold reduction of thap <dig> mrna upon shrna-thap <dig> knockdown, right panel shows western blot analysis of thap <dig> protein from whole cell extract  of molt <dig> thap <dig> knockdown  or "mock" molt <dig> cells ; the amount of dna polymerase delta cofactor pcna was visualized by its specific antibody and served as sds-page “loading control”.
b cytosol and nuclear extract of molt <dig> thap <dig> knockdown  or "mock" molt <dig> cells was subjected to immunoblot analysis to investigate the reduction of thap <dig> protein level c fold change of selected m4-gene expression levels upon thap <dig> knockdown.
violet bar: m4-gene mrna levels of control cells  is normalized to one.
purple bar: fold change of m4-gene mrna levels of molt <dig> thap <dig> knockdown cells extracted right after establishing the shthap <dig> clone .
yellow bar: fold change of m4-gene mrna levels measured from biological replicate  <dig>  of molt <dig> thap <dig> knockdown cells
as seen in fig.
10c, the biological replicate  <dig>  of molt4-sh-thap <dig> transcript exhibits a ten-fold reduction of thap <dig> mrna that correlates with the down-regulation of most selected m <dig> bearing genes.
this is in accordance with previous observations made in transient reporter assays  and suggests that thap <dig> works as a transcriptional activator in controlling m <dig> motif-bearing genes.
two weeks after the first mrna extraction, biological replicate  <dig>  of molt4-sh-thap <dig> mrna preparation was performed.
the quantitative pcr  analysis of the second mrna extraction revealed reproducible effects of thap <dig> protein reduction on the transcript levels of cdc25a, skp <dig>  cdk <dig>  mll <dig>  and g9a, whereas the influence of thap <dig> on gene expression levels of hexim <dig> and aamp was quite variable.
discussion and
CONCLUSIONS
the motif m <dig> actayrnnncccr  was discovered by xie and colleagues in  <dig>  <cit>  as a putative cis-regulatory element.
this motif was described to be highly conserved in a window from less than 2500 bps upstream and 500 bps downstream of transcription start sites  of many annotated genes of human, mouse, rat, and dog with a peak centered around − 89 bp from tsss.
hence, the hyperconserved m <dig> motif can be considered as a core-promoter associated cis-element that can be found on  <dig> human genes, implying a potential important role in regulating multiple cellular processes.
recently, by using chip-seq assays combined with bioinformatics, two independent groups  <cit>  have demonstrated m <dig> motifs as binding site for rbp-j and icn <dig> , as well as staf/znf <dig>  and thap <dig> , mostly in context of non-lymphoid cells like hela, hek <dig>  k <dig> and murine es cells.
however, according to the initial publication by xie and colleagues, genes bearing the m <dig> motif are preferentially expressed in human cells of hematopoietic origin , which prompted us to revisit and comprehensively characterize the m <dig> tf binding landscape in lymphoid cell lines.
in order to pursue this we employed our silac-based dna protein interaction screening method, which we showed to be technically capable of uncovering direct and indirect  tf cis-element interactions  <cit> .
in this respect, our motivation was twofold.
first of all, we wanted to make use of the partially studied m <dig> model with the aim of demonstrating that our quantitative proteomic methodology  <cit>  is working as a surrogate for a bona fide reverse chip technology.
second, we intended to screen for m4-binders in an unbiased manner exclusively utilizing the m <dig> sequence logo  <cit>  information derived from xie and colleagues  <cit> , thereby avoiding issues with the overlap of m <dig> with sbs .
building on that, in this proteomic study, we successfully identified the nuclear factors thap <dig>  hcf- <dig>  ikaros, aiolos, helios, nfkb <dig>  and nfkb <dig>  as interacting partners of the m <dig> motif in the context of a human lymphocyte nuclear extract .
limited by the availability of reagents we conducted follow-up experiments for thap <dig>  hcf- <dig>  ikaros and nfkb <dig>  importantly, we demonstrated that at least in lymphocyte cell lines, m <dig> is functioning as a bipartite composite element, in which the “upstream” actayr part constitutes a binding site for the tf thap <dig>  while the “downstream” cccr module provides the direct dna contact for the lymphocyte specific tf ikaros.
most probable and similar to ikaros, helios and aiolos might also play a modulatory role for certain m <dig> genes, but it is very difficult to investigate their function because ikaros itself serves as a master regulator of aiolos and helios gene transcription .
nevertheless, it would be interesting to investigate this further because ikaros and helios can have opposite functions at the same target gene  <cit> .
in our model  and building on our data discussed below the epigenetic factor hcf- <dig> is recruited to m <dig> motif dna by virtue of its interaction with the thap <dig> c-terminal domain , whereas the rel/nf-kappa b-factors  are strongly believed to be tethered to the m <dig> element in an ikaros-dependent manner .
these findings indeed verify the power of the silac-based dna protein interaction screening methodas a comprehensive approach, which can capture direct and indirect dna-tf assemblies.
in contrast, the limitation of emsa is seen in fig.
<dig>  for which emsa was carried out with raji nuclear extracts.
the latter showed at least two strongly retarded  bands that are virtually absent in hela nuclear extracts, albeit thap <dig> and hcf- <dig> are equally well expressed in hela cells .
this is surprising because bacterially expressed thap <dig>  <cit>  is able to retard radioactive-labelled dna sequences bearing the m <dig> motif in conjuction with the sbs element.
this phenomenon could be explained by the lack of lymphocyte specific factors in hela nuclear extracts that together with thap <dig> and hcf- <dig> might be responsible for higher order complex formation.fig.
<dig> working model for the assembly of m <dig> binding factors on promoters of m <dig> genes.
the m <dig> motif is composed of two highly conserved elements: an “upstream” actacr module  and a “downstream” module tcccrr , which constitute bipartite binding sites for thap <dig> and ikaros, respectively.
interaction between thap <dig> and hcf- <dig> is required for recruiting hcf- <dig> to the m <dig> motif, whereas an interaction of ikaros with nfkb <dig>  or nfkb <dig>  is essential for tethering these rel tf family proteins to the m <dig> motif
ikaros and nf-kappa b proteins are direct dna binding factors whose canonical binding or half sites  <cit>  are similar to the cccr module of m <dig> motifs.
however in nuclear extracts prepared from cells that do not express ikaros  the binding of nfkb <dig>  to m <dig> could not be detected.
interestingly, the binding of nfkb <dig> to the “downstream” module of m <dig> was clearly restored upon heterologous expression of ikaros in hek <dig> cells , suggesting an essential role of ikaros protein in tethering nf-kappa b proteins to m <dig>  this was further corroborated in experiments that utilized an ikaros knockdown molt <dig> cell line, which does not express any ikaros as assessed by western blotting .
employing nuclear extracts from these kd cells revealed that recruitment of nfkb <dig>  to m <dig> dna was completely abrogated, while binding of thap <dig> and hcf- <dig> was essentially not affected .
beyond this, ikaros kd cells do also loose expression of helios and aiolos at least at the mrna level .
therefore, it will be interesting to test in future experiments if helios and aiolos are also capable to promote nfkb <dig> binding to m <dig>  the observations discussed above were further recapitulated by chip assays performed in molt <dig> ikaros kd cells.
more precisely, the presence of nfkb <dig>  at m <dig> motif harboring promoters was severely compromised , whereas recruitment of thap <dig> and hcf- <dig> was still preserved .
it could therefore be inferred that nfkb <dig>  is recruited to m4-genes in an ikaros dependent manner.
mechanistically, this is probably taking place by the formation of ikaros-nfkb <dig> protein complexes that we detected by coimmunoprecipitation of ikaros employing an anti-nfkb <dig> antibody and copurification of nfkb <dig>  with strepii-tagged ikaros, respectively .
since we did not carry out in vitro binding studies with recombinant ikaros and nfkb <dig> proteins it is formally still possible that their interaction is either indirect or heavily dependent on the presence of m <dig> containing dna/chromatin.
furthermore, we cannot rule out the possibility that ikaros kd has an additional influence on nf-kappa b signaling, eventually resulting in a decreased ability of nfkb <dig> to bind to dna/chromatin .
based on the results obtained from reporter assays and ikaros loss-of-function  experiments we are confident that the association of ikaros with hyperconserved m <dig> elements is functionally relevant .
however, an elucidation of the detailed mechanistic action of ikaros  in governing the transcriptional output of m <dig> bearing genes might be gene-specific  <cit>  and was outside the scope of this manuscript.
as described for the negative cross-talk between staf/znf <dig> and rbp-j-icn <dig> heteroconjugates  <cit> , ikaros-nfkb <dig> complexes could similarly compete with m <dig> binding of staf/znf <dig> and/or rbp-j, especially in light of the fact that at the genome-wide level ikaros and rbp-j are generally sharing the same short binding consensus  <cit> .
together this could also mean that the transcriptional output of m <dig> genes can be modulated by a complex interplay of relative expression levels of ikaros, helios, aiolos and rbp-j that is further orchestrated by the status of notch <dig> and nf-kappa b pathway signaling inputs.
at this instance, it has been reported that nfkb <dig> , which in contrast to rela/nfkb <dig>  is lacking a classical transactivation domain, can be found in n-cor corepressor complexes.
upon interleukin- <dig> beta stimulation the promoter occupancy of n-cor corepressor complex at nfkb <dig> bound promoters is abolished, which is followed by recruitment of tip <dig> coactivator complexes and a subsequent change in the gene expression program  <cit> .
interestingly, our ikaros interactome from molt <dig> cells  contains various corepressors  along with nfkb <dig> and nfkb <dig>  while thap <dig> and hcf- <dig> are absent.
collectively, we would like to propose the current working model for m <dig> function in lympocytes that is depicted in fig.
<dig>  in accordance to previous findings  <cit>  thap <dig>  whose binding is required for the recruitment of hcf- <dig> binds directly to the “upstream” actacr module of the m <dig> motif.
conversely, ikaros interacts with the “downstream” module  and is either directly or indirectly responsible for the association of the rel family tfs nfkb <dig>  and nfkb <dig> .
as discussed above other tfs like staf/znf <dig> and rbp-j   <cit>  whose binding sites do also comprise the “downstream” module ycccr would also bind to m <dig> in non-lymphoid cells.

 of the roughly protein-coding genes, there are approximately to genes expressed at a significant level in a given cell type. the gene regulatory code consists of the dna sequence-dependent binding specificities of a prominent class of dna binding proteins. these so called transcription factors are able to read the regulatory information and transmit the information encoded in primary dna sequence to the transcriptional machinery, thereby regulating the synthesis of rna transcripts. differential tissue-specific gene expression is an essential component of gene regulation in humans, which restricts the expression of certain genes to given stages of development. importantly, the erroneous reading of the gene regulatory code is often accompanied by the formation of tumors or the manifestation of other diseases associated with developmental disorders. therefore, a detailed understanding of the spatial and temporal gene expression patterns is of fundamental importance for understanding the complexity of the human genome. this is by far not a trivial task because the cis-regulatory elements are usually ambiguous and context-dependent and can function both in the immediate vicinity of genes and as enhancer elements thousands of dna base pairs away from their transcriptional start site. since the cis-regulatory elements and associated transcription factors are believed to be responsible for maintaining the conserved gene expression patterns between related organisms, the elucidation of the gene regulatory code brings back into focus a simple question: “which transcription factors do bind to a cis-regulatory element in a given cell type?” traditionally, tf binding to a putative cis-element could be identified through the combination of classical biochemical fractionations coupled to eletrophoretic mobility shift assay and amino acid sequencing of the purified transcription factors. in order to significantly improve the traditional approach, it is essential to link the isolation and enrichment of the tf directly to the detection of its sequence specificity and its identification. recently, we and others have developed scalable methodologies that are fulfilling this task. our silac-based dna protein interaction screen makes use of the fact that one can introduce mutational changes in the dna sequence to destroy the function of a cis-element thereby preventing the binding of its regulatory tfs. hence, a direct quantitative proteomic comparison of the protein binding pattern between the natural and the mutated dna sequence via nanolc-ms will provide the identity of the tfs that have the potential to interact with the regulatory element in vivo. here we apply this technology to investigate an evolutionary hyperconserved promoter-associated cis-element, termed m that until now has been poorly studied. briefly, a modern bioinformatic whole genome alignment comparison of mammalian genomes led to the discovery of several novel, evolutionarily conserved cis-elements, whose function was in most cases poorly characterized. among them the best conserved novel element found was the m motif with the dna consensus sequence “rractacanntcccrrnrnrc.” this was partially described as a binding site for the non-lymphoid transcription factors thap staf/zfn and rbp-j. as suggested by the fact that genes harboring m motifs exhibit increased expression levels in lymphoid and some leukemia or lymphoma cells, it implied a putative role of m motif in the lymphoid system or in hematopoietic malignancies. in the present study, by using silac-based quantitative proteomics, we show that the lymphocyte specific ikaros family proteins, the nf-kappa b proteins nfkb and nfkb, the pluripotency factor ronin/thap and host cell factor- are able to bind specifically, either directly or indirectly, to the m motif in vitro. importantly, the tf m4-dna interactions of ikaros, nfkb, thap and hcf- are confirmed by chip assays in living cells, thus providing evidence that the silac-based dna protein interaction screen can work as a surrogate for a bona fide reverse chip technology. notably, our data also suggest that the conserved m motif is a composite cis-element containing two tf binding modules, which can operate independently from each other. we provide strong evidence that thap interacts directly with the actayr part of m thereby engaging in a ternary complex with hcf- whereas ikaros binds to the cccrrnrnrc sequence of m the latter interaction is essential for recruiting nfkb to the m motif. transient reporter assays performed in hek cells and sh-rna mediated loss-of-function assays in molt t cells unequivocally demonstrate binding of ikaros and thap to genes containing m4-promoters as functional and therefore biologically relevant. our current model, utilizing transient reporter assays and thap shrna knockdown in molt t cells, mainly suggests an activator function of thap11/hcf- for m4-genes in these cells. unlike thap the apparent role of ikaros is to modulate the transcriptional output of endogenous m4-harboring genes by either repressing or activating them.  emsa assay reveals presence of m binding factors in lymphocyte nuclear extracts although several studies have shown the binding of some tfs to the m motif, none reported m as the binding site for a heamatopoietic tf regardless of the fact that genes harboring the m motif exhibit higher expression levels in many cell lines derived from the immune system. emsa was performed using a radioactively labeled double-stranded oligodeoxynucleotide bearing the m motif that was incubated with either raji burkitt’s lymphoma or hela nuclear extracts followed by electrophoretic separation on a native polyacrylamide gel in order to reveal potential m binders that are specifically expressed in hematopoietic cells. the band shift data from fig.  show the presence of lymphocyte-specific m binding factors that are hardly expressed in cervical cancer hela cells. importantly, the raji lymphoma specific band shifts are still formed even in the presence of a hundred-fold excess of an unlabeled competitor oligonucleotide bearing a sequence unrelated to m. conversely, the formation of band shifts is completely abolished if a hundred-fold excess of non-radioactive m oligonucleotide is added. this suggests that raji burkitt’s lymphoma cells express proteins that can selectively interact with m motif in vitro.fig. emsa assay to uncover lymphocyte-specific transcription factors interacting with m motif bearing dna. radioactive γ-atp labeled m oligo was incubated either with raji burkitt’s lymphoma nuclear extracts, or hela nuclear extract. emsa complexes formed in assays using raji burkitt’s lymphoma nuclear extract are virtually absent in hela nuclear extract. those band shifts were significantly faded away when an increasing amount of cold m motif was added but still preserved if the cold m motif containing oligo was replaced by an m scrambled counterpart. lanes are control assays using m oligo in the absence of nuclear extracts ms-based proteomic screening to identify lymphocyte-specific m interacting factors to identify m interacting factors, raji burkitt’s lymphoma cells were metabolically encoded with either “heavy” or “light” isotopic variants of lysine. the corresponding nuclear extracts were prepared and used as an input for subsequent silac-based dna affinity pull-down screening comparing the protein binding profile of m wild-type versus m mutant baits. following trypsin digestion and nanolc-ms out of more than identified proteins were quantified by the msquant software utilizing lysine-containing tryptic peptides. most of the isotopic variants of quantified peptides were present in almost equal amounts. this unequivocally demonstrated that most of the quantified proteins do bind unspecifically to either m wt or m mt columns. however, there were seven candidate factors, exhibiting silac ratios greater than two that were termed m4-binding candidates.fig. silac-based dna protein interaction sreen reveals m binding candidates. a distribution of silac ratio against quantified proteins identified in tandem m dna affinity chromatography experiments using raji burkitt’s lymphoma nuclear extracts: the x-axis present the random code of quantified proteins originating from the mass spectrometric screening of m dna affinity chromatography experiments; the present silac ratios of individually quantified proteins; b mass spectra of selected m interacting partners’ derived peptides: mass spectra of individual peptides comprising both light isotope and heavy peaks. those derived from specific m binders were specified by higher intensity of heavy isotope encoded peaks. in contrast, the ones descending from unspecific binders are presented by equal intensity of both light and heavy isotope encoded peaks nfkb and thap to the mono m motif-containing dna affinity column. a prominent putative m binding candidate factors. b dna affinity chromatography: m wt and control mono m mt were immobilized as ligand, raji burkitt’s lymphoma nuclear extract was used as input. protein-dna complexes from two columns were eluted, resolved by sds-page separately for western blotting analysis. specific antibodies were used to detect the retention of suspected m binding candidates. the unspecific dna binding factor tpb was adopted as loading control: immunoblot analysis confirms the in vitro interaction of m binding candidates with m4 the m sequence of the oligodesoxynucleotide baits used in the proteomic screening experiment were derived from the dffa gene promoter, which contains two copies of the m motif in close vicinity; however, most human m bearing genes contain only one copy of the m motif in their promoter. to exclude the possibility that a duplicated m motif could induce some avidity binding effects in respect to putative m interacting factors, the dna affinity chromatography was repeated using columns that harbor only one copy of either wt or mutant m motif. separate eluates from wt and mt dna columns were resolved by sds-page and subjected to immunoblot analysis for validating the putative m4-binding candidates. as shown in fig.  antibodies directed against ikaros, host cell factor, nfkb, and thap confirmed the specific binding of these proteins to the wt m motif. m4-binding proteins interact with promoter regions containing the m motif in vivo to demonstrate the potential biological function of m motif binding candidates, classical formaldehyde crosslinking chromatin immunoprecipitation assays were performed, followed by qrt-pcr of bound dna. in this assay, the three m bearing promoters of the genes skp cdc25a, and g9a were selected for two reasons: first, gene ontology analysis of m4-genes reveal that genes involved in mitotic cell cycle regulation and chromatin function are statistically overrepresented amongst m bearing genes. second, expression levels of the three aformentioned genes were significantly affected upon ectopic over-expression of the dominant negative ik isoform of ikaros in raji burkitt’s lymphoma cells as well as molt t-cells. initial attempts to establish chip conditions for ikaros in raji burkitt’s lymphoma cells employing antibodies directed against the ikaros c-terminus were difficult and could be ascribed to the fact that raji burkitt’s lymphoma cells exhibit an abundant expression of small ikaros isoforms, which are not able to bind dna directly but can multimerize with ik and ik isoforms. such higher-order complexes cannot be efficiently recovered by the standard chip protocol, which works under stringent denaturing conditions strongly favouring the recovery of dna sequences that are directly crosslinked to the immunoprecipitated protein. to circumvent this, we decided to focus on t-lymphoma molt cells because these cells do not express the small ikaros isoforms. furthermore, to standardize our ip conditions, c-terminally ha-tagged ikaros that is known to recognize canonical ikaros target genes was constructed and retrovirally transduced into molt cells. a similar procedure was adopted for the generation of molt4-thap11-ha cells, which ectopically expresses c-terminally ha-tagged thap protein. importantly, like its endogenous counterpart, thap-11-ha demonstrates robust m4-specific binding in a dna affinity pulldown assay. the two ha-tagged transgenic molt t-cell lines were later used for either ikaros or thap chip assay using a high affinity rat-anti-ha antibody. as seen in fig. 4a, m motif-bearing dna was specifically co-precipitated by anti-ikaros/ha ip, demonstrating specificity of the ip protocol. strikingly, binding of ikaros to chromatin was selectively enriched on dna fragments containing the m motif in all three promoters examined. therefore, it can be concluded that ikaros interacts with the m cis-element in living cells. recapitulating the same experimental strategy, we were able to show the specific and selective enrichments of nfkb, thap11-ha and hcf- on the promoters of the m bearing genes skp cdc25a, and g9a.fig. chromatin immunoprecipitation analysis of ikaros, nfkb, thap and hcf- binding to m4-bearing promoters of the genes skp cdc25a, and g9a. formaldehyde crosslinked chromatin of molt-ik-ha or molt4-thap11-ha cells were sheared by ultrasonication into an average length of to 1000 bps to be subjected to immuno-precipitation assays using anti-nf-kappa b p anti-hcf- anti-ha/ikaros, anti-ha/thap or preimmune igg sera. the tf-associated chromatin were analyzed via quantitative real time pcr using primer sets flanking the suspected tf binding site and background binding control regions: promoter, exon, ±2000 bp. enrichment of tf-bound dna amount were normalized to corresponding amount in input. chip results for ikaros, nfkb, thap and hcf- are shown intriguingly, chip enrichment of nfkb bound interleukin- promoter dna, which represents a well established nf-kappa b target gene harboring a classical palindromic kappa b binding site, was comparable to the enrichment of nfkb bound m4-promoter dna. this suggested that the affinity of nfkb towards m4-bearing promoters was not substantially different compared to a well studied classical nf-kappa b target gene. m motif is a composite element, comprising two distinct non-overlapping binding sites for both thap and ikaros according to our studies the m motif consists of two highly conserved parts. while the “upstream core” rractayr is similiar to the ronin binding site, the “downstream core” is similar to the previously described murine ikaros binding site consensus. this raises the question if the m motif is indeed a composite element that can be partitioned into two sub-consensus sequences. the latter can be best answered by investigating if the suspected direct sequence-specific interaction partners of the m cis-element, thap and ikaros, are binding synergistically or independently to m therefore, we hypothesized that the “downstream core” will be sufficient to recruit ikaros, whereas the “upstream core” rractayr would be indispensable for engaging other m binders namely thap to verify this, oligo-pulldown assays were conducted using three modified m motif-bearing dna sequences as a ligand: mod-m, f-mt-m, and r-mt-m. as can be seen in fig.  the “flanking” mutations did not interfer with the binding of either m binding factors to the m motif. in contrast, the mutation of the m “upstream core” rractayr was sufficient to simultaneously abrogate the binding of thap and hcf- to m motif bearing dna, while still preserving the interaction of both ikaros and nfkb with the m motif. consequently, mutation of the “downstream” part of the m4-motif abolished recruitment of ikaros and nfkb to m dna, but did virtually not alter the binding of thap and hcf- to m for this reason we conclude from our in vitro binding data that m is a composite tf binding module. it consists of two tf binding sites; the “upstream core” rractayr that is essential for recruiting thap11/hcf- to the m cis-element and the “downstream core” tcccrrnr that is required for establishing m4-ikaros/nf-kappa b interactions.fig. the m motif is a bipartite composite element, consisting of two independent binding site modules for thap and ikaros. a dna ligands used in affinity chromatography: mono-m wild-type, mono-m mutant, mod-m, f-mt-m and r-mt-m. b immunblot analysis of dna affinity chromatography experiments using five dna ligands: input and eluates from three columns were resolved by sds-page in parallel and subjected to western blotting. specific antibodies detected the retention of suspected m binding factors nfkb interaction with m is critically dependent on ikaros the m motif is neither a palindromic sequence nor analogous to any known kappa b site. however, the “downstream core” that mediates nfkb association is highly similar to the murine ikaros binding site. therefore, one of two scenarios can be envisioned: i) ikaros and nfkb compete in binding to the “downstream core” of the m motif or ii) nfkb is recruited to the m motif in an ikaros-dependent manner. to investigate this a stable shrna ikaros knock-down molt cell line was generated by retroviral transduction, in which ikaros protein expression was almost completely abrogated. dna affinity chromatography experiments using mono-m wt and mono-m mt beads in combination with nuclear extracts from molt, molt4-ik-kd or cells that do not express ikaros were conducted and subjected to immunblot analysis as described before. notably, even though protein expression levels of nfkb were virtually not affected by our highly efficient ikaros knockdown, its binding to the m column was completely abolished. however, upon ectopic expression of ikaros in hek cells, the recruitment of nfkb to m containing dna was reestablished. in contrast, the binding of the two other m4-binders was not affected either by the absence or presence of ikaros expression. this implies that ikaros expression is absolutely crucial for the binding of nfkb to dna harboring the m sequence in vitro. nevertheless, it remained to be resolved if nfkb was recruited to m by a more direct interaction with ikaros. to address this, we have carried out a coimmunoprecipitation assay using anti-nfkb antibody. the results clearly demonstrate that ikaros is part of a nfkb protein complex, which is resistant to efficient dna and rna nuclease digestion. of note, the latter indicates that the ikaros containing nfkb complex is not formed or stabilized by random protein nucleic-acid interactions.fig. nfkb interacts with ikaros and this interaction is essential for recruiting rel-nf-kappa b family tfs to m motif harboring dna. a immunoblot analysis of dna affinity chromatography experiments using two dna ligands: mono-m wt and mono-m mt. molt control or molt4-ikaros kd nuclear extracts served as input. in the absence of ikaros, protein levels of nfkb were virtually not affected but its binding to the m column was completely abrogated. in contrast, the binding of the two other established m4-binders were robustly maintained. b anti-nf-kappa b p ip co-precipitated ikaros in the presence and absence of the highly efficient nuclease bezonase. c cross-linked streptactin pulldown experiment demonstrates co-purification of strepii-tagged ikaros with nfkb protein, whereas the nuclear protein kinase cdk that was reported to interact with nfkb was not present in the cross-linked complex. d benzonase nuclease digestion control assay: the control 40 bp duplex dna or control salmon sperm dna were mixed with molt nuclear extracts in the presence or absence of benzonase at 4 °c. the whole samples were analyzed by an 8 % native tbe-page gel that allows separation of dna down to the size of 10 bps to exclude the possibility that ikaros might unspecifically associate with the anti-nfkb serum, a reverse immunoprecipitation assay using either anti-ikaros or anti-ha antibodies was conducted. however, co-precipitation of nfkb was not achieved. since both the anti-ikaros and anti-ha antibody recognize epitopes at the c-terminus of ikaros, which is known to mediate protein-protein interactions, we reasoned that antibody binding to the ikaros carboxyterminus might disturb the ikaros-nfkb interaction. to circumvent this, an aminoterminal tag was fused to the cdna of human ikaros. the tandem-strepii-tag was selected as it exhibits an extremely fast on-rate during binding to streptactin beads and chromatography can be performed under very stringent conditions employing formaldehyde crosslinking. in order to pursue this, a molt cell line stably expressing strepii-tagged ikaros was created via retroviral transduction. to confirm that the heterologously expressed strepii-tagged ikaros protein is functional, chip experiments were conducted as described above. the chip enrichement values for selected m4-bearing gene promoters were almost identical to the ones obtained with ikaros-ha protein suggesting that ectopically expressed strepii-tagged ikaros retains its biological function. as seen in fig. 6e, the crosslinked streptactin pulldown experiment reveals a moderate co-purification of nfkb protein with strepii-tagged ikaros, whereas the nuclear protein kinase cdk that was reported to directly interact with nfkb was not present in the crosslinked complex. this demonstrates high specificity of the formaldehyde crosslinking approach. since the benzonase treatment proved to be very efficient in removing nucleic acids from the input fraction, it could be inferred that the ikaros-nfkb complex formation most likely involves protein-protein contacts and is not unspecifically mediated by random dna or rna. hence, we conclude that a minor fraction of nuclear nfkb is able to engage with ikaros and that this interaction is most likely essential for recruiting nfkb to m motif containing dna in vitro. importantly, the data do not rule out the possibility that the ikaros-nfkb interaction can be enforced upon binding to m dna, either by an allosteric effect transmitted through a conformational change of ikaros upon dna binding or by dna-contacts mediated by the rel homology domain of nfkb. ikaros knock-down does not influence the m motif binding of thap and hcf- in vivo to confirm that the m motif does also operate in vivo as a composite element, which assumes that the ikaros/ nfkb complex does not influcence the binding of thap11/hcf- to m4-gene promoters, we performed chip in molt and molt-ik-kd cells, respectively. in the absence of ikaros expression, the enrichment of nfkb at three representative m bearing promoters was totally abrogated, whereas binding of either thap or hcf- did not change substantially. consequently, these data together with the results depicted in fig.  lead us to conclude that the m motif also constitutes in vivo a composite cis-element, with distinct and independent thap11/hcf- and ikaros/ nfkb binding modules.fig. chromatin immunoprecipitation analysis of nfkb, thap and hcf- interaction with m genes in ikaros knockdown cells. formaldehyde crosslinked chromatin of molt or molt4-ik-kd cells was sheared by ultrasonication into an average length of to 1000 bps and ultimately subjected to immuno-precipitation assays using a anti-thap antibody, b anti-nf-kappa b-p antibody, c anti hcf- or preimmune igg sera. the tf-associated chromatin were analyzed via quantitative real time pcr using primer sets flanking the promoters, and gene body control regions of m4-bearing skp cdc25a, and g9a genes; d the known nfkb target genes vcam- hla-b were also included as control. enrichment of tf-bound dna amount were normalized to corresponding amount in input. chip results for nfkb1/nf-kappa b p, thap, and hcf- were shown thap interacts with hcf- via its c-terminal domain, which is required for tethering hcf- to m motif containing dna towards this end we could show that hcf- is a bona fide component of m motif-binding complexes. however, hcf- is not known to be a sequence-specific dna binding factor but primarily contacts dna through association with other tfs, such as the oct protein and ronin-the mouse orthologue of thap. in order to examine if this interaction is also taking place in the context of human proteins, a gst-pulldown assay was carried out using molt nuclear extracts as input. briefly, the cdna encoding either full-length or the carboxyterminal part of human thap were cloned in-frame downstream of a glutathione-s-transferase gene cassette and expressed in e. coli. subsequently, the purified gst-fusion proteins were used as bait for identifying thap11-interacting partners from molt nuclear extracts in gst pull-down assays. in this context glutathione-sepharose beads loaded with gst only served as a negative control. as illustrated in additional file 1: figure s homogenously purified gst-fusion proteins were obtained that efficiently and specifically captured hcf- protein as visualized by immunoblot analysis. this result could also be confirmed in an independent proteomic experiment using lc-ms-based protein identification. therefore, we infer that the carboxyterminal domain of thap interacts quite strongly with hcf- in human cells, an interaction potentially crucial for tethering hcf- to m motif-bearing dna. binding of ikaros and thap to a m motif containing promoter is functional in transient reporter assays until now we demonstrated that in lymphocytes, thap and ikaros independently interact with m cis-elements in vitro as well as in living cells. however, it is equally important to assess if m4-binding plays a functional role in regulating gene expression of m4-harboring genes. as a functional readout, we first established a reporter assay system using hek cells, in which, a set of artificial m and m mutant-bearing promoters could be studied for their regulatory potential to drive expression of a downstream-conjugated luciferase gene reporter. as discussed above hek cells do not possess any detectable ikaros expression but ectopic expression is easily accomplished by transient transfection. as already described in fig. 6b and c, ectopically expressed ikaros displays specific m binding in vitro and additionally induces the recruitment of nfkb to m wt baits. to assay the transcriptional activity of an m4-bearing promoter, a well-characterized luciferase reporter was selected, which accommodates a functionally validated binding site for the ubiquitiously expressed transcriptional activator staf. the latter fits to the staf consensus binding sequence ywcccrnmatscmyyrcr. this sequence was fused at its 5′-position with the sequence of the m motif, which is frequently overlapping with sbs in mammalian promoters. hence, the resulting synthetic promoter sequence comprises the following regulatory modules: actaca, tcccatta and tcccattatgccccgcg. additionally, each reporter construct contains two copies of the regulatory module that encompasses either the m wt motif or mutated m variants.fig. a luciferase m4-reporter assay system to study the functional role of ikaros for m4-harboring genes. a scheme illustrating the modular m4-reporter gene. the m motif is composed of a thap binding site and an ikaros binding site, yellow) which overlaps with the binding site of the transcriptional activator staf/znf. b m4-reporter gene activity of m4-wt compared to mutated m4-variant promoters in hek and hek293-ikaros cells, which are stably expressing heterologous ikaros. transcriptional activity of corresponding m4-promoters in the absence or presence of ikaros. immunoblot analysis measuring ikaros protein expression levels and input loading control c nucleotide sequence presentation of the m4-modules and its variants used for m4-reporter gene assays. d transient luciferase reporter assay employing m4-reporter q and r. ikaros is repressing m4-reporter q in a dose-dependent manner as seen in fig. 8b, ikaros expression correlates with a twofold reduction in luciferase activity. importantly, the repressive activity is absolutely dependent on the presence of an intact ikaros binding site. to confirm this observation, the two m4-reporter constructs that harbor mutated staf/znf binding sites, namely reporter q and sequence r were selected in order to alleviate the profound transcription stimulatory activity of staf/znf. then a transient reporter assay was performed expressing increasing amounts of ikaros. these data clearly demonstrate that ikaros is repressing reporter gene activity in a strictly dose-dependent manner because ikaros protein expression level was inversely correlated to transcriptional activity of the m4-reporter q. conversely, upon ikaros binding site mutation the repressive effect of ikaros was completely abrogated. mutation of the thap binding site diminished reporter gene activity by at least two-fold, both in the presence and absence of ikaros protein. in summary, in the context of our transient-reporter assays, ikaros acts as a transcriptional repressor, whereas thap does function as an activator. ikaros knockdown results in de-regulated expression of m4-harboring genes so far, it was demonstrated that ikaros is able to regulate the transcriptional output of m4-bearing genes in the context of a transient reporter assay. however, it is not known if ikaros might play a critical role in gene regulation of endogenous m4-harboring genes. to determine this, the rna from molt ikaros knockdown cells and molt control cells were used for quantitative rt-pcr experiments in order to monitor the changes in gene expression of selected m bearing genes. as seen in fig.  the knockdown of ikaros did not affect thap mrna levels significantly. in contrast, it substantially increased the mrna amount of some well established ikaros target genes, like the b cell-specific lambda or t cell-specific hes1-gene, for which ikaros is known to function as a transcriptional repressor. in parallel, the ikaros knockdown dramatically reduces the expression level of the ikaros family gene aiolos, for which ikaros serves as an important transcriptional activator. apart from affecting known ikaros target genes, depletion of ikaros caused both up-regulation and down-regulation of selected m4-harboring genes. in addition, the m4-genes g9a and mll did not virtually change expression. similar results were obtained from a comparative microarray analysis of molt-ik-kd versus molt4-mock cells. in conclusion we propose that ikaros can modulate the activity of m4-harboring genes in living cells.fig. ikaros loss-of-function leads to de-regulated expression of m bearing genes. the mrna level fold change of known ikaros target genes and of selected m bearing genes is shown thap knockdown leads to weak but significant changes in gene expression of selected m motif harboring genes finally, for deciphering the biological and functional relevance of the thap11-m motif interaction in living cells, a stable thap knockdown cell was generated. however, it seems that thap might be essential for cell viability, because selection of clones that exhibited both a complete ablation of thap mrna levels and a substantial decrease in thap expression was not possible. it was noted that thap localizes to both the cytoplasm and nucleus. surprisingly, the knockdown mainly affected its cytosolic fraction while keeping a reduced, but still substantial amount of thap in the nucleus. as for this reason, strong effects of the thap knockdown on m target gene expression were not expected. to provide reproducible data it was decided to conduct biological replicate experiments in which the extraction of molt4-sh-thap total mrna was carried out at two different time points after selecting the clone. these samples are termed biological replicate and biological replicate respectively.fig. thap knockdown leads to weak but significant changes in selected m bearing gene expression. a left panel shows fold reduction of thap mrna upon shrna-thap knockdown, right panel shows western blot analysis of thap protein from whole cell extract of molt thap knockdown or "mock" molt cells; the amount of dna polymerase delta cofactor pcna was visualized by its specific antibody and served as sds-page “loading control”. b cytosol and nuclear extract of molt thap knockdown or "mock" molt cells was subjected to immunoblot analysis to investigate the reduction of thap protein level c fold change of selected m4-gene expression levels upon thap knockdown. violet bar: m4-gene mrna levels of control cells is normalized to one. purple bar: fold change of m4-gene mrna levels of molt thap knockdown cells extracted right after establishing the shthap clone. yellow bar: fold change of m4-gene mrna levels measured from biological replicate of molt thap knockdown cells as seen in fig. 10c, the biological replicate of molt4-sh-thap transcript exhibits a ten-fold reduction of thap mrna that correlates with the down-regulation of most selected m bearing genes. this is in accordance with previous observations made in transient reporter assays and suggests that thap works as a transcriptional activator in controlling m motif-bearing genes. two weeks after the first mrna extraction, biological replicate of molt4-sh-thap mrna preparation was performed. the quantitative pcr analysis of the second mrna extraction revealed reproducible effects of thap protein reduction on the transcript levels of cdc25a, skp cdk mll and g9a, whereas the influence of thap on gene expression levels of hexim and aamp was quite variable. discussion and  the motif m actayrnnncccr was discovered by xie and colleagues in as a putative cis-regulatory element. this motif was described to be highly conserved in a window from less than 2500 bps upstream and 500 bps downstream of transcription start sites of many annotated genes of human, mouse, rat, and dog with a peak centered around − 89 bp from tsss. hence, the hyperconserved m motif can be considered as a core-promoter associated cis-element that can be found on human genes, implying a potential important role in regulating multiple cellular processes. recently, by using chip-seq assays combined with bioinformatics, two independent groups have demonstrated m motifs as binding site for rbp-j and icn, as well as staf/znf and thap, mostly in context of non-lymphoid cells like hela, hek k and murine es cells. however, according to the initial publication by xie and colleagues, genes bearing the m motif are preferentially expressed in human cells of hematopoietic origin, which prompted us to revisit and comprehensively characterize the m tf binding landscape in lymphoid cell lines. in order to pursue this we employed our silac-based dna protein interaction screening method, which we showed to be technically capable of uncovering direct and indirect tf cis-element interactions. in this respect, our motivation was twofold. first of all, we wanted to make use of the partially studied m model with the aim of demonstrating that our quantitative proteomic methodology is working as a surrogate for a bona fide reverse chip technology. second, we intended to screen for m4-binders in an unbiased manner exclusively utilizing the m sequence logo information derived from xie and colleagues, thereby avoiding issues with the overlap of m with sbs. building on that, in this proteomic study, we successfully identified the nuclear factors thap hcf- ikaros, aiolos, helios, nfkb and nfkb as interacting partners of the m motif in the context of a human lymphocyte nuclear extract. limited by the availability of reagents we conducted follow-up experiments for thap hcf- ikaros and nfkb importantly, we demonstrated that at least in lymphocyte cell lines, m is functioning as a bipartite composite element, in which the “upstream” actayr part constitutes a binding site for the tf thap while the “downstream” cccr module provides the direct dna contact for the lymphocyte specific tf ikaros. most probable and similar to ikaros, helios and aiolos might also play a modulatory role for certain m genes, but it is very difficult to investigate their function because ikaros itself serves as a master regulator of aiolos and helios gene transcription. nevertheless, it would be interesting to investigate this further because ikaros and helios can have opposite functions at the same target gene. in our model and building on our data discussed below the epigenetic factor hcf- is recruited to m motif dna by virtue of its interaction with the thap c-terminal domain, whereas the rel/nf-kappa b-factors are strongly believed to be tethered to the m element in an ikaros-dependent manner. these findings indeed verify the power of the silac-based dna protein interaction screening methodas a comprehensive approach, which can capture direct and indirect dna-tf assemblies. in contrast, the limitation of emsa is seen in fig.  for which emsa was carried out with raji nuclear extracts. the latter showed at least two strongly retarded bands that are virtually absent in hela nuclear extracts, albeit thap and hcf- are equally well expressed in hela cells. this is surprising because bacterially expressed thap is able to retard radioactive-labelled dna sequences bearing the m motif in conjuction with the sbs element. this phenomenon could be explained by the lack of lymphocyte specific factors in hela nuclear extracts that together with thap and hcf- might be responsible for higher order complex formation.fig. working model for the assembly of m binding factors on promoters of m genes. the m motif is composed of two highly conserved elements: an “upstream” actacr module and a “downstream” module tcccrr, which constitute bipartite binding sites for thap and ikaros, respectively. interaction between thap and hcf- is required for recruiting hcf- to the m motif, whereas an interaction of ikaros with nfkb or nfkb is essential for tethering these rel tf family proteins to the m motif ikaros and nf-kappa b proteins are direct dna binding factors whose canonical binding or half sites are similar to the cccr module of m motifs. however in nuclear extracts prepared from cells that do not express ikaros the binding of nfkb to m could not be detected. interestingly, the binding of nfkb to the “downstream” module of m was clearly restored upon heterologous expression of ikaros in hek cells, suggesting an essential role of ikaros protein in tethering nf-kappa b proteins to m this was further corroborated in experiments that utilized an ikaros knockdown molt cell line, which does not express any ikaros as assessed by western blotting. employing nuclear extracts from these kd cells revealed that recruitment of nfkb to m dna was completely abrogated, while binding of thap and hcf- was essentially not affected. beyond this, ikaros kd cells do also loose expression of helios and aiolos at least at the mrna level. therefore, it will be interesting to test in future experiments if helios and aiolos are also capable to promote nfkb binding to m the observations discussed above were further recapitulated by chip assays performed in molt ikaros kd cells. more precisely, the presence of nfkb at m motif harboring promoters was severely compromised, whereas recruitment of thap and hcf- was still preserved. it could therefore be inferred that nfkb is recruited to m4-genes in an ikaros dependent manner. mechanistically, this is probably taking place by the formation of ikaros-nfkb protein complexes that we detected by coimmunoprecipitation of ikaros employing an anti-nfkb antibody and copurification of nfkb with strepii-tagged ikaros, respectively. since we did not carry out in vitro binding studies with recombinant ikaros and nfkb proteins it is formally still possible that their interaction is either indirect or heavily dependent on the presence of m containing dna/chromatin. furthermore, we cannot rule out the possibility that ikaros kd has an additional influence on nf-kappa b signaling, eventually resulting in a decreased ability of nfkb to bind to dna/chromatin. based on the results obtained from reporter assays and ikaros loss-of-function experiments we are confident that the association of ikaros with hyperconserved m elements is functionally relevant. however, an elucidation of the detailed mechanistic action of ikaros in governing the transcriptional output of m bearing genes might be gene-specific and was outside the scope of this manuscript. as described for the negative cross-talk between staf/znf and rbp-j-icn heteroconjugates, ikaros-nfkb complexes could similarly compete with m binding of staf/znf and/or rbp-j, especially in light of the fact that at the genome-wide level ikaros and rbp-j are generally sharing the same short binding consensus. together this could also mean that the transcriptional output of m genes can be modulated by a complex interplay of relative expression levels of ikaros, helios, aiolos and rbp-j that is further orchestrated by the status of notch and nf-kappa b pathway signaling inputs. at this instance, it has been reported that nfkb, which in contrast to rela/nfkb is lacking a classical transactivation domain, can be found in n-cor corepressor complexes. upon interleukin- beta stimulation the promoter occupancy of n-cor corepressor complex at nfkb bound promoters is abolished, which is followed by recruitment of tip coactivator complexes and a subsequent change in the gene expression program. interestingly, our ikaros interactome from molt cells contains various corepressors along with nfkb and nfkb while thap and hcf- are absent. collectively, we would like to propose the current working model for m function in lympocytes that is depicted in fig.  in accordance to previous findings thap whose binding is required for the recruitment of hcf- binds directly to the “upstream” actacr module of the m motif. conversely, ikaros interacts with the “downstream” module and is either directly or indirectly responsible for the association of the rel family tfs nfkb and nfkb. as discussed above other tfs like staf/znf and rbp-j whose binding sites do also comprise the “downstream” module ycccr would also bind to m in non-lymphoid cells. 
further analysis reveals that m is a bipartite composite cis-element, which is recognized by thap via binding to the actayr sequence module, thereby promoting ternary complex formation with hcf- similarly, ikaros binds to the cccr module of the m motif and this interaction is crucial for recruiting nfkb to m harboring genes. here, we present a quantitative proteomic investigation of the m motif tf binding landscape in lymphoid cell lines that is further validated by chip experiments and functional assays. apart from rbp-j that is well known to recruit icn, the functional role of the hyperconserved m cis-element in the context of transcriptional regulation of m4-genes in lymphoid cells remains poorly defined. human m4-promoter genes show enhanced expression in cells of hematopoietic origin, especially in b lymphoblasts and peripheral blood b and t cells. the motif actayrnnncccr, called m was discovered as a putative cis-regulatory element, present times in human promoter regions. transient reporter assays in hek and loss-of-function experiments in molt t cells unequivocally demonstrate that binding of ikaros and/or thap to m bearing promoters is functionally important and therefore biologically relevant. accordingly, this study validates our silac-based dna protein interaction screening methodology as a valuable surrogate for a bona fide reverse chip technology.
 the motif actayrnnncccr, called m was discovered as a putative cis-regulatory element, present times in human promoter regions. of these, are conserved within promoter sequences of four related organisms: human, mouse, rat, and dog. recent genome-wide studies have described m as a transcription factor binding site for thap that does often overlap with sbs a second core-promoter associated tf binding module, which associates with the tfs staf/znf and rbp-j. human m4-promoter genes show enhanced expression in cells of hematopoietic origin, especially in b lymphoblasts and peripheral blood b and t cells. apart from rbp-j that is well known to recruit icn, the functional role of the hyperconserved m cis-element in the context of transcriptional regulation of m4-genes in lymphoid cells remains poorly defined.  here, we present a quantitative proteomic investigation of the m motif tf binding landscape in lymphoid cell lines that is further validated by chip experiments and functional assays. our data strongly suggest that thap and ikaros interact directly, while nfkb and hcf- are binding indirectly to m4-promoters in vitro and in living cells. further analysis reveals that m is a bipartite composite cis-element, which is recognized by thap via binding to the actayr sequence module, thereby promoting ternary complex formation with hcf- similarly, ikaros binds to the cccr module of the m motif and this interaction is crucial for recruiting nfkb to m harboring genes. transient reporter assays in hek and loss-of-function experiments in molt t cells unequivocally demonstrate that binding of ikaros and/or thap to m bearing promoters is functionally important and therefore biologically relevant. accordingly, this study validates our silac-based dna protein interaction screening methodology as a valuable surrogate for a bona fide reverse chip technology.  the m motif is a functional regulatory bipartite cis-element, which engages a thap11/hcf- complex via binding to the actayr module, while the cccrrnrnrc subsequence part constitutes a binding platform for ikaros and nfkb electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. http://dx.doi.org/ /501100004189max-planck-gesellschaftissue-copyright-statement© the author 2016 