BACKGROUND
development and survival of the b cell lineage is regulated by a number of important transcription factors, and dysregulation of the expression of these proteins can lead to impaired b cell function and disease such as leukemia  <cit> .
two highly related transcription factors involved in development of b cells are the e26-transformation specific  family members pu.
<dig>  and spi-b .
pu.
<dig> is expressed in hematopoietic stem cells and common lymphoid progenitors, and throughout b cell development  <cit> .
pu.1-null mice die during late gestation and do not generate progenitors for b cells, t cells, monocytes, or granulocytes  <cit> .
development of lymphoid progenitor cells requires pu.
<dig>  making it a key regulator of b-cell fate specification  <cit> .
in comparison, spi-b deficient mice are viable and develop mature b and t cells, although their b cells are functionally deficient and die in response to bcr signalling  <cit> .
therefore, pu.
<dig> cannot completely compensate for spi-b, as functionally deficient spi-b-/- b cells still express pu.
<dig> at wild-type levels  <cit> .
several lines of evidence suggest that pu.
<dig> and spi-b have at least some complementary function in the b cell lineage.
first, pu.
<dig> and spi-b have 43% overall amino acid identity and share 67% amino acid identity within their ets dna binding domain  <cit> .
the ets domains of pu.
<dig> and spi-b can interact with identical purine-rich 5′-ggaa-3′ motifs and can both activate transcription of a number of genes important for b cell differentiation and function through interaction with the same ets motifs.
genes with demonstrated regulation by both pu.
<dig> and spi-b include fcgr2b   <cit>  and blnk   <cit> .
at other genomic regions, binding to the ets domain may be preferred by one factor over another, particularly at combined ets-irf elements where spi-b is the principal partner for recruiting irf <dig> to regulatory regions  <cit> .
pu.
<dig> and spi-b exhibit differential dna binding at the c-fes promoter in some cell lines, which is predicted to be through the function of distinct activation domains outside of the ets-binding domain, particularly at the n terminus where there is low homology between the two proteins  <cit> .
pu.
<dig> and spi-b appear to have complementary function in the b cell lineage in vivo.
heterozygosity for spi <dig> encoding pu.
<dig> on a spib-/- mutant background leads to defects in bcr signalling and reduced frequencies of splenic follicular b cells that are more severe compared to spib-/- mice, demonstrating a degree functional overlap between pu.
<dig> and spi-b  <cit> .
conditional deletion of the spi <dig> gene after b cell commitment under the control of the b cell-specific cd19-cre leads to mild defects in b-cell development and function  <cit> .
however, conditional deletion of spi <dig> under the control of cd19-cre on a spib-/-  background leads to severely impaired b cell development and b cell acute lymphoblastic leukemia, which is partially attributed to loss of blnk  <cit> .
bruton tyrosine kinase  is also directly regulated by spi-b and pu.
<dig>  and induction of pu.
<dig> in cultured cd19-creΔpb pro-b cells restores btk expression and induces apoptosis  <cit> .
while decreased levels of pu.
<dig> and spi-b are associated with defects in lymphoid development and some forms of leukemia, elevated levels of pu.
<dig> and spi-b have been demonstrated in lymphoma  <cit> .
spib is amplified in activated b cell diffuse large b cell lymphomas  compared with other b cell lymphoma subtypes, and is translocated in the oci-ly <dig> abc-dlbcl cell line leading to over-expression of spib mrna compared with other lines  <cit> .
spi-b is required for the survival of abc-dlbcl cell lines, as depletion of spi-b using lenalidomide or rna interference leads to decreased survival in vitro  <cit> .
it is predicted that the requirement for spi-b and pu.
<dig> in lymphoma cells is due to an “addiction” to b cell receptor signaling, which is enforced by over-expression of these factors in activated lymphoma subtypes  <cit> .
next-generation sequencing  technologies allow for high-resolution analysis and detection of transcription factors across the entire genome.
by combining chromatin-immunoprecipitation with high-throughput sequencing, all regions within the genome bound by pu.
<dig> and spi-b can be identified.
based on the demonstrated complementary function of pu.
<dig> and spi-b, we hypothesize that pu.
<dig> and spi-b can interact with the same set of binding sites in the genome of b cells.
in this study, we report a genome-wide comparison of genomic regions of interaction by pu.
<dig> and spi-b in the murine lymphoma cell line wehi- <dig>  to remove bias introduced by different antibodies, expression levels, or gene regulation we expressed 3xflag-tagged pu.
<dig> and spi-b at similar levels in wehi- <dig> lymphoma cells.
chromatin immunoprecipitation was performed using anti-flag antibodies.
our results support the hypothesis that pu.
<dig> and spi-b occupy similar regions within the genome, but also identified a unique subset of regions only occupied by one factor.
additionally, motif analysis has suggested that these regions contain binding regions for different co-activator partner proteins.
in summary, these experiments provide biochemical insight into both the similarities and differences between the biological functions of pu.
<dig> and spi-b.
RESULTS
determination of target regions for spi-b and pu.1
to determine if the transcription factors pu.
<dig> and spi-b occupied identical regions within the mouse genome chip-seq was performed.
to ensure a fair comparison, wehi- <dig> b lymphoma clones expressing 3xflag-tagged full-length pu.
<dig> or spi-b protein were selected to ensure equivalent levels of protein expression  <cit> .
uninfected wehi- <dig> cells expressed spi <dig> and spib mrnas at a ratio of 1: <dig> , relative to the normalizer gene gapdh.
3xflag-pu.1-infected wehi- <dig> cells expressed spi <dig> mrna and spib mrna at ratio of  <dig> : <dig>  3xflag-spi-b-infected wehi- <dig> cells expressed spi <dig> mrna and spib mrna at a ratio of 1: <dig> .
these results suggested that spib mrna was modestly overexpressed relative to spi <dig> mrna.
however, protein levels of the flag-tagged products were similar between 3xflag-tagged pu.
<dig> and 3xflag-tagged spi-b, as described in xu et al.
<cit> .
this system is expected to provide the advantage of eliminating variability between antibody specificity and chip-seq yield, while allowing sustained expression of pu.
<dig> and spi-b in a mouse b cell line.
chip was performed on fixed wehi- <dig> cell lines using anti-flag mab and immunoprecipitated chromatin was validated by qpcr .figure  <dig>
chip sequencing of pu.
<dig> and spi-b in a mouse b lymphoma cell line.
a. workflow for generating chip sequencing data.
b. venn diagram for regions of significant chip binding determined by mm-chip.
regions with at least  <dig> base pair overlap were considered common to both factors.
c. venn diagram of peakset regions analyzed in diffbind.
the majority of regions in the matrix demonstrated less than  <dig> fold difference in enrichment between pu.
<dig> and spi-b.
d. scatter plot of chip signal between pu.
<dig> and spi-b binding.
binding of these transcription factors was similar across most of the genome.
e. de-novo motif analysis from chip.
from extracted dna sequences, frequently occurring motifs were discovered using meme-chip.
the most frequently occurring motif for each sample contained the canonical ets binding sequence f. representative chip-seq binding at a region considered to be regulated by pu.
<dig> and spi-b.
the histocompatibility  <dig> q region on chromosome  <dig> demonstrates high peaks for pu.
<dig> and spi-b at gene promoters.
pu.
<dig> or spi-b binding peaks were determined using replicate data from two experiments, sequenced by two independent sequencing core facilities with over  <dig> million reads per sample.
replicate chip-seq experiments were merged in mm-chip, which estimates sample-specific shift-size of chip-seq tags with macs modeling and then pools shifted tags from different samples to identify chip-peaks with a dynamic poisson model  <cit> .
this method allowed combining of separate datasets with different tag-sizes, noise features, and dynamic ranges.
using a model fold of  <dig>   <dig> and a p-value cut-off for peak detection of 1e- <dig>  mm-chip identified  <dig> high-stringency regions bound by pu.
<dig> and  <dig> high-stringency regions bound by spi-b.
the pu.1-bound regions called by mm-chip had an average size of 1469 bp, while the spi-b-bound regions called by mm-chip had an average size of 1460 bp.
pu.
<dig> and spi-b-bound regions were compared to determine if significantly enriched regions were common to both sets.
of these  <dig> regions,  <dig> had a minimum of 100 bp of overlap between the two sets of regions, and the average length of overlap between significantly enriched regions was  <dig> out of  <dig> base pairs  .
there were also a number of macs-defined regions that had a pu.
<dig> or spi-b peak in one sample, but not the other, but did not have a significant 2-fold difference in terms of number of aligned reads.
using diffbind,  <dig> pu.1/spi-b matrix peak regions were called.
<dig> high-confidence peak regions were found to differ significantly in tag count between the two conditions .
differentially bound regions were classified as pu.1-higher or spi-b higher, in which local enrichment of sequence tags for one factor was 2-fold higher than tags for the other.
of these  <dig> regions,  <dig> were considered to be pu.1-higher and  <dig> spi-b-higher .
the correlation coefficient on the genome scale was calculated using cis-regulatory element annotation system  application.
protein-dna binding signals  were correlated between two samples in 1 kb bins.
pu.
<dig> and spi-b signals demonstrated a high degree of correlation with an r <dig> correlation coefficient of  <dig>  .
these results demonstrated that 1 kb intervals of the genome with a high degree of pu.
<dig> binding were likely to have strong spi-b binding, and many regions containing significant chip-seq peaks for one factor also had a peak for the other.
taken together, these results showed that most regions in the genome of wehi- <dig> cells occupied by pu.
<dig> were also occupied by spi-b.
identification of enriched sequence motifs
members of the ets superfamily recognize similar purine-rich sequences with a 5′-ggaa-3′ core  <cit> .
to validate the enrichment of chip chromatin at ets motifs, sequences of chip-seq peak regions were screened for enriched motifs using meme, dreme and centrimo using meme-chip  <cit> .
the top motif for pu.1-bound regions was the spi <dig> motif, with an e-value of  <dig> e- <dig>  and a central canonical ets ggaa motif that was found in  <dig> % of all peaks.
the top motif for the  <dig> spi-b binding sites was also the spi <dig> motif with an e-value of  <dig> e- <dig>  this motif was centrally enriched contained a central ggaa sequence was found in  <dig> % of the peaks .
top motifs demonstrated central distribution within the sequences.
annotation and comparison of peak-associated genes
potential target genes of pu.
<dig> and spi-b were identified using beta-minus, which annotates binding data to predict target genes based on proximity to the transcription start site  <cit> .
to identify and visualize the function of genes potentially regulated by these transcription factors, the predicted target genes from beta-minus were functionally annotated using database for annotation, visualization and integrated discovery .
gene ontology analysis of predicted spi-b targets revealed significant  enrichment of antigen processing functions  immune response , intracellular signaling cascade  and regulation of lymphocyte activation .
histocompatibility  <dig> class ii loci, several fc receptors , as well as b cell markers cd <dig> and cd <dig> were included in multiple ontologies.
these gene lists suggest that spi-b has functions in lymphoid development, antigen processing, and b cell receptor-mediated signalling.
top processes for pu.
<dig> were highly similar to spi-b, consistent with previous studies that presumptive targets for pu.
<dig> are involved with b cell and myeloid lineages.
the gene ontology of processes for pu.
<dig> target genes included cell activation , leukocyte activation , and hematopoiesis .
top genes involved in all three pathways included bak <dig>  bcl <dig>  hdac <dig>  pou2f <dig>  spi <dig>  and vav <dig>  the region upstream of spi <dig> demonstrated a single peak at the -14 kb position .
the region at -14 kb of spi <dig> has been demonstrated to be occupied by pu.
<dig> in both b lymphocytes and myeloid cells, as opposed to a site at -12 kb that is occupied by pu.
<dig> only in myeloid cells  <cit> .
these gene lists are consistent with the known role for pu.
<dig> in both development and function of these lineages  <cit> .
peak to gene associations for pu.
<dig> and spi-b are available in additional file 1: table s <dig> and additional file 2: table s <dig>  respectively.
to predict genes regulated by either transcription factor, the  <dig> regions with more than 100 bp of overlap between pu.
<dig> and spi-b bound-regions were examined using beta-minus .
gene targets included the toll-like receptors tlr <dig>  tlr <dig>  tlr <dig>  and tlr <dig> in addition to well-documented targets such as btk and blnk.
top processes were immune response , multiple terms for antigen processing and presentation  and regulation of lymphocyte activation .
these pathways were considered most significant due to high ranking of mhc class ii genes in the associated gene list, including h2-aa, h2-q <dig>  h2-q <dig>  h2-q <dig> and h2-q <dig> .
spi-b and pu.
<dig> chip signal at genes highest at transcriptional start sites
in order to visualize genome-wide distribution of chip peaks, peaks were sorted according to their distribution around mouse refseq genes.
cis-regulatory element annotation revealed that regions bound by spi-b and pu.
<dig> were over-represented for transcription start sites with respect to the whole genome.
while  <dig> % of the mouse genome is located within 10000 bp of gene promoters,  <dig> % of spi-b bound regions were located within 10000 bp of gene promoters.
similarly,  <dig> % of pu.
<dig> bound regions were located within 10000 bp of gene promoters .figure  <dig>
distribution of chip-seq peaks within the genome.
a. pu.
<dig> and spi-b have similar binding patterns in relation to features in the genome.
b. profiles and associated heatmaps of chip-signal at the transcriptional start site of mouse refseq genes.
profiles represent the regions of strongest binding, which are clustered around the transcriptional start site.
c. all clusters of pu.
<dig> and spi-b signal in relation to mouse refseq genes.
five different patterns of binding were observed, where the largest cluster contained genes without transcription factor binding at the promoter.
clusters  <dig> and  <dig> contained genes with binding upstream or downstream of the promoter, although spi-b binding was often seen at the transcription start site.
cluster  <dig> contained regions with the highest log <dig> signal.
cluster  <dig> was gene with a broad region of transcription factor binding at the tss.
top row shows results from anti-flag chip-seq of 3xflag-pu.
<dig> in wehi- <dig> cells, second row shows results from anti-flag chip-seq of spi-b in wehi- <dig> cells, and third row shows results from anti-pu.
<dig> chip-seq in mouse splenic b cells .
d. profile of dnase genomic footprinting  at regions of significant chip enrichment for pu.
<dig> and spi-b.
regions of pu.
<dig> or spi-b binding demonstrated increased dnase sensitivity compared to regions outside the peaks.
e. profile of rna polymerase ii chip-signal in b cells at regions of chip enrichment for pu.
<dig> and spi-b.
polii data confirmed that significant pu.
<dig> and spi-b peaks frequently occur at sites of transcription initiation.
f. profile of the chromatin feature h3k36me <dig>  a marker of heterochromatin and gene bodies, at peak regions for pu.
<dig> and spi-b.
next, binding profiles of pu.
<dig> and spi-b relative to features in the mouse genome were determined using deeptools  <cit> .
log <dig> normalized read coverage of chip-seq data was clustered against regions of mouse refseq genes.
regions with the highest pu.
<dig> and spi-b binding clustered within 4 kb of the transcription start site of mouse genes, with the peak summit directed at the transcription start site and little binding within the gene body .
using k-means clustering, transcription factor binding at the start of mouse refseq genes in the genome demonstrated  <dig> distinct patterns .
cluster  <dig> contained the largest number of mouse genes, where little pu.
<dig> or spi-b binding was observed.
cluster  <dig> demonstrated an upstream binding modality, while cluster  <dig> demonstrated binding downstream from the transcription start site .
cluster  <dig> represented the peaks at the tss and had the highest signal.
cluster  <dig> represented broad binding across the tss, with lower signal intensity than in cluster  <dig>  importantly, reanalysis of pu.
<dig> chip-seq performed using primary splenic b-cells   <cit> , revealed a very similar enrichment at distal elements and promoters.
these clusters suggest that while pu.
<dig> and spi-b bind various locations within the genome, they have the highest degree of binding at distal elements and promoters of target genes.
in order to further characterize the chromatin environment of regions bound by pu.
<dig> or spi-b, significant peak regions were profiled against marks for transcription, gene body, and dnase sensitivity.
chip binding signal in b cells in the form of wig/bigwig files were downloaded from published geo datasets.
digital genomic footprinting  of dnase i hypersensitivity in mouse b cells from encode/university of washington  was used to predict regions of the genome containing cis-regulatory elements profiled against significantly chip-enriched regions in the genome .
regions of the genome bound by either spi-b or pu.
<dig> demonstrated higher digital genomic footprinting scores relative to regions 4 kb up or downstream.
in addition to using dnase footprints for predicted regions of transcription factor binding, promoter regions were further predicted using rna polymerase binding.
signal for published rna polymerase ii chip-seq on stimulated b lymphocytes  was profiled against chip-enriched regions for pu.
<dig> and spi-b.
similar to dgf profiling, regions of the genome bound by pu.
<dig> or spi-b demonstrated high enrichment of pol ii binding compared to regions 4 kb up or downstream .
to further characterize binding locations of spi-b and pu.
<dig> in relation to the gene body, peak intervals were clustered against histone modifications by chip-seq from encode/licr .
whole genome coverage of h3k36me <dig>  was inversely associated with regions of the genome bound by either pu.
<dig> or spi-b .
this mark has been observed to be strongest within the body of actively transcribed genes  <cit> .
pu.
<dig> or spi-b peak regions showed an inverse signal of binding, indicating that regions of pu.
<dig> and spi-b binding are unlikely to be within regions of the genome also bound by h3k36me <dig>  taken together, these profiles demonstrate that peak regions of pu.
<dig> and spi-b are characteristic of transcriptional activators.
unique binding sites of spi-b and pu.1
pu.
<dig> and spi-b demonstrated a consistent pattern of high chip signal at transcriptional start sites.
interestingly, binding sites unique to either pu.
<dig> or spi-b demonstrated distinct profiles.
unlike common peaks, which tended to be associated with tsss, unique pu.
<dig> or spi-b sites were frequently found in intergenic regions 10kb beyond the nearest gene promoter .
analysis of distribution within the genome using ceas demonstrated that unique pu.
<dig> peaks were not over-represented at the promoter compared to the background distribution within the genome.figure  <dig>
differential binding of pu.
<dig> and spi-b within the genome.
a. distribution of pu.
<dig> and spi-b unique chip-seq peaks within the genome.
unique sites for these factors are frequently located beyond 10kb of the transcription start site compared with the overall peak sets.
b. de-novo motif analysis by meme-chip of unique regions determined by diffbind.
the most frequently occurring motif for each sample is shown and compared with the most common motif found in all peaks for pu.
<dig> and spi-b.
pu.1-unique peaks retained similar motifs to those seen in all pu.
<dig> peaks, but just over half of these peaks also contained a pou2f <dig> motif.
spi-b unique peaks retained the ets-irf motif seen in all spi-b peaks, with higher conservation of the irf motif .
c. a unique pu.
<dig> binding peak was observed at the site of mir <dig> d. unique binding of pu.
<dig> within the gene nod <dig>
e. unique spi-b binding in the 3′ end of fgf <dig>  f. unique spi-b binding in the promoter of lat <dig>
differentially bound regions were analyzed with meme-chip for unique conserved binding motifs to identify potential differential associated binding partners.
regions uniquely bound by pu.
<dig> contained the ets motif.
a pou2f <dig>  motif was identified by meme chip, with an e-value of  <dig> e- <dig>  this motif was observed within 100 bp of  <dig> % of the ets motifs seen in pu.1-bound regions, but was overrepresented to  <dig> % of ets motifs in unique pu.
<dig> peaks.
there were  <dig> pou2f <dig> motif occurrences in unique pu.
<dig> peaks with a p-value less than  <dig> .
combined pou2f2-pu.
<dig> motifs were observed upon re-analysis of pu.
<dig> chip in primary splenic b-cells   <cit> , where a secondary pou2f <dig> was located in  <dig> % of all sequences also containing a gaggaa primary motif.
in contrast, regions bound by spi-b did not demonstrate a marked difference in pou2f <dig> motifs.
pou2f <dig> motifs were observed in  <dig> % of all spi-b bound regions and  <dig> % of regions uniquely bound by spi-b but not pu.
<dig>
remarkably, de-novo motif finding with spi-b unique regions revealed that 100% of unique spi-b regions contained an ets–irf composite element  with an e-value of  <dig> e- <dig> .
this combined motif was similar to the binding sequence for all significant spi-b peaks, but the irf element was more conserved.
the primary ggaanngaaa motif was found in  <dig> % of spi-b bound sites.
sites uniquely bound by pu.
<dig> did not demonstrate the same increase in eices; ggaanngaaa motifs were observed in  <dig> % of all pu.
<dig> peaks and reduced to  <dig> % in unique pu.1-bound regions.
this composite element was observed in spi-b unique peaks and provides evidence that irf factors might bind within a few base pairs of the spi-b binding sequence.
to determine whether the uniquely-bound spi-b sites containing irf motifs might interact with irf <dig>  we compared our sites with irf <dig> chip-seq regions from mouse b cells   <cit> .
of these sites,  <dig> % were located within 100 bp of an irf4-bound site in unstimulated b cells or b cells stimulated with il- <dig>   <dig> % of ets-irf sites were located within 100 bp of an irf <dig> peak under both conditions.
these findings suggest that unique spi-b binding is associated with local deposition of irf <dig> in b cells.
in order to determine what genes were regulated at differentially bound regions, gene function for differential gene targets was determined using gene ontology.
peak to gene associations were determined using ceas and annotated using david.
genes uniquely bound by pu.
<dig> were predominantly associated with leukocyte activation during immune response  and cell activation during immune response  .
these pathways were both highly ranked due to inclusion of interleukin  <dig> and 12b, cx3cr <dig>  as well as a unique peak in an intron of nfkb <dig> not observed in spi-b.
pu.
<dig> unique peaks were found to be associated with genes related to mrna processing  and with a number of mirnas; mir-155/ay <dig>  mir-3108/zfhx <dig>  mir-1938/ifrd <dig>  mir-3102/arhgef <dig>  and mir-5130/kctd <dig>  of these, mir- <dig> has a known oncogenic role in lymphomas  <cit> .
furthermore, mir- <dig> directly targets pu.
<dig> and leads to a down-regulation of pu.
<dig> protein in b cells  <cit> .
pu.
<dig> unique peaks were observed in the promoter region of nod <dig>  a nod-like receptor involved in b cell proliferation and activation  <cit> .
typical examples of these regions are represented in figure 3c,d.
gene targets of differentially bound spi-b peaks were associated with immune effector process  and b cell activation  that included bnip3l, lat <dig> and pou2f <dig>  multiple go terms associated with both positive and negative regulation of transcription and gene expression  were also identified due to the inclusion of fgf <dig>  myc and pou2f <dig> .
a large unique peak of spi-b binding was observed at a combined ets-irf motif within the 3′ end of fgf <dig> .
fgf <dig> is involved in the regulation of phosphate concentration in plasma, and has been found to be elevated in the serum of patients with b cell neoplasms  <cit> .
spi-b unique binding was also observed at the promoter of sema4b.
sema4b abundantly expressed in lymphocytes and been demonstrated to be a novel regulator of immunological memory responses and homeostatic t helper 1/t helper  <dig> balance in mice  <cit> .
lat <dig>   demonstrated significant spi-b binding directly at the promoter, which was not observed in the pu.
<dig> chip .
lat <dig> is expressed in the spleen, peripheral blood lymphocytes, and various human b cell lines, and is tyrosine-phosphorylated in response to b cell receptor signalling  <cit> .
lat <dig> expression has been observed in monocytes  and b cells   <cit> .
these analyses demonstrate that while pu.
<dig> and spi-b frequently occur at the regulatory regions of genes predicted to have b cell function, a small subset of regions and genes that are uniquely bound by only of these transcription factors.
these factors have unique requirements for associated binding partners, and are involved in different functions.
discussion
to investigate genome-wide binding of spi-b and pu.
<dig>  we conducted next generation sequencing of chromatin immunoprecipitated with anti-3xflag-tagged spi-b or pu.
<dig>  previous studies have reported chip-seq analysis of pu.
<dig> binding sites in erythroid, myeloid, and lymphoid cells but none have compared pu.
<dig> to spi-b sites in mouse cells due to technical constraints  <cit> .
detailed analysis of transcription factor binding revealed that these two ets transcription factors had similar binding regions within the genome of murine wehi- <dig> lymphoma cells.
we determined that both transcription factors demonstrated the highest degree of binding near the tss and regions of open chromatin, although peaks were also observed within introns and upstream of genes beyond 10 kb.
characterization of the sequences most strongly bound by pu.
<dig> and spi-b has confirmed that the primary target sequence of both factors contained the purine-rich core motif 5′-gaggaa-3′.
this motif was identified within regions of significant chip enrichment, and determined to be overrepresented by de-novo motif enrichment analysis.
peak-to-gene associations confirmed occupancy of both factors within  <dig>  bp of the tss for genes involved in immune system regulation and processes.
gene ontology analysis confirmed that pu.
<dig> and spi-b are involved in the regulation of genes required for b cell development and function.
the top go pathways determined by david for pu.
<dig>  spi-b, and the common peaks were cell activation, immune response and immune response, respectively.
an important finding of this study is that of the genomic regions occupied by pu.
<dig>  a majority were also occupied by spi-b.
this fraction was 1900/ <dig> regions using mm-chip, and 5378/ <dig> regions  using diffbind.
this result supports our hypothesis that pu.
<dig> and spi-b can interact with the same regulatory regions in the genome of b cells.
this result is consistent with previous reports that pu.
<dig> and spi-b have similar dna binding specificities  <cit> .
we suggest that similar dna binding preferences and similar biochemical activity are the explanation for why pu.
<dig> and spi-b have complementary biological activity in the b cell lineage  <cit> .
fewer regions of the genome were found to be differentially occupied by pu.
<dig> or spi-b in the genome of wehi- <dig> b cells.
one important aspect of next-generation sequencing demonstrated in this study is the importance of mathematical modeling and input sequencing in identifying differential peaks.
while direct subtraction of regions called by peak callers revealed several peak regions that did not overlap between samples, affinity  data analysis using diffbind determined that only several hundred regions demonstrated significant differences between factors.
using diffbind to identify differential peaks in combined data most likely under-predicted gene targets, but provided high-stringency lists.
therefore, while there remains a possibility that other differential regions exist within the genome, this method provides the most accurate and specific regions.
therefore, subsequent analysis of these high-stringency peaks highlights the most significant differences between spi-b and pu.
<dig> binding within the genome.
regions identified that were occupied uniquely by pu.
<dig> or spi-b may reveal insights into biological functions unique to each factor.
genes associated with unique pu.
<dig> bound regions were related to immune development using gene ontology analysis.
regions unique to pu.
<dig> contained a number of microrna genes, while no microrna genes were observed at unique spi-b peaks.
the unique binding of pu.
<dig> to the sequence of mir- <dig> may have a role in regulation of pu.
<dig> itself, as expression this micro-rna has been shown by others to down-regulate pu.
<dig>  <cit> .
binding at this region suggests that pu.
<dig> may be involved in the regulation of mir- <dig>  the identification of an oct <dig> binding motif within unique pu.
<dig> regions implies a potential co-activator role, as pu.
<dig> and oct <dig> have previously been shown to bind concomitantly at the vκ <dig> promoter in b lymphocytes, and point mutations in the binding sites for either factor can lead to diminished immunoglobulin production  <cit> .
oct <dig> binding has been shown to be covariant with pu.
<dig> binding activity  <cit>  and oct <dig> preferentially binds to pu.
<dig> regions in b cells compared with macrophages, suggesting a functional role for pu.
<dig> to recruit oct <dig> to regulatory regions of the genome and induce cellular reprogramming in b cells  <cit> .
unique spi-b associated genes were associated with roles in b-cell activation and immune function.
all unique spi-b peaks had an associated irf sites in wehi- <dig> lymphoma cells, while ets-irf sites were not overrepresented in unique pu.
<dig> peaks.
this suggests that in cases where genes are under the regulation of an ets-irf composite element , spi-b and not pu.
<dig> is the preferential irf partner.
our results are consistent with a recent study showing that combined eices were dominantly occupied by spi-b in b lymphoma cell lines, and that pu.
<dig> could not fully compensate for spi-b in recruiting irf <dig> to target regions  <cit> .
rnai-mediated down-regulation of either spi-b or irf <dig> leads to rapid death of cultured lymphoma cells, irrespective of pu.
<dig> co-occupancy  <cit> .
therefore, spi-b may be involved in a unique regulatory network where it is more frequently associated with irf <dig> in b cell lymphomas, and may be directed to these sites by irf <dig> or function to alter irf <dig> occupancy within the genome.
our model of pu.
<dig> and spi-b binding is based on lymphoma cells over-expressing the proteins, which bears similarity to other b cell studies in terms of binding near target genes and associated binding motifs.
while the changes in the expression level of pu.
<dig> can enhance binding of other factors to influence local chromatin structure and dictate eventual cell fate  <cit> , our tagged-expression model does not appear to have substantially altered binding of pu.
<dig> or spi-b, since identified peaks were very similar to those identified in a study of mouse splenic b cells  <cit> .
peak-associated gene lists support our hypothesis that pu.
<dig> and spi-b are involved in the regulation of lymphoid development and immune function, and we report fewer than  <dig>  peaks per factor, possibly due to the restricted lineage of the lymphoma cell line.
CONCLUSIONS
in conclusion, we report genome-wide binding of the ets transcription factors spi-b and pu.
<dig> to similar regions of the genome, with high affinity for transcriptional start sites containing ets motifs.
these regions are frequently located near the regulatory domains of genes involved in b cell development and function.
in addition to these common regions, there exists a small set of unique regions where pu.
<dig> and spi-b demonstrate differential preference for associated factors based on sequence identity, target gene function, and potential co-activators.
our findings provide novel insight into differences between the two factors, and offers novel biochemical pathways involving spi-b as a molecular targeting therapy in b cell malignancies.

 development and survival of the b cell lineage is regulated by a number of important transcription factors, and dysregulation of the expression of these proteins can lead to impaired b cell function and disease such as leukemia. two highly related transcription factors involved in development of b cells are the e26-transformation specific family members pu. and spi-b. pu. is expressed in hematopoietic stem cells and common lymphoid progenitors, and throughout b cell development. pu.1-null mice die during late gestation and do not generate progenitors for b cells, t cells, monocytes, or granulocytes. development of lymphoid progenitor cells requires pu. making it a key regulator of b-cell fate specification. in comparison, spi-b deficient mice are viable and develop mature b and t cells, although their b cells are functionally deficient and die in response to bcr signalling. therefore, pu. cannot completely compensate for spi-b, as functionally deficient spi-b-/- b cells still express pu. at wild-type levels. several lines of evidence suggest that pu. and spi-b have at least some complementary function in the b cell lineage. first, pu. and spi-b have 43% overall amino acid identity and share 67% amino acid identity within their ets dna binding domain. the ets domains of pu. and spi-b can interact with identical purine-rich 5′-ggaa-3′ motifs and can both activate transcription of a number of genes important for b cell differentiation and function through interaction with the same ets motifs. genes with demonstrated regulation by both pu. and spi-b include fcgr2b and blnk. at other genomic regions, binding to the ets domain may be preferred by one factor over another, particularly at combined ets-irf elements where spi-b is the principal partner for recruiting irf to regulatory regions. pu. and spi-b exhibit differential dna binding at the c-fes promoter in some cell lines, which is predicted to be through the function of distinct activation domains outside of the ets-binding domain, particularly at the n terminus where there is low homology between the two proteins. pu. and spi-b appear to have complementary function in the b cell lineage in vivo. heterozygosity for spi encoding pu. on a spib-/- mutant background leads to defects in bcr signalling and reduced frequencies of splenic follicular b cells that are more severe compared to spib-/- mice, demonstrating a degree functional overlap between pu. and spi-b. conditional deletion of the spi gene after b cell commitment under the control of the b cell-specific cd19-cre leads to mild defects in b-cell development and function. however, conditional deletion of spi under the control of cd19-cre on a spib-/- background leads to severely impaired b cell development and b cell acute lymphoblastic leukemia, which is partially attributed to loss of blnk. bruton tyrosine kinase is also directly regulated by spi-b and pu. and induction of pu. in cultured cd19-creΔpb pro-b cells restores btk expression and induces apoptosis. while decreased levels of pu. and spi-b are associated with defects in lymphoid development and some forms of leukemia, elevated levels of pu. and spi-b have been demonstrated in lymphoma. spib is amplified in activated b cell diffuse large b cell lymphomas compared with other b cell lymphoma subtypes, and is translocated in the oci-ly abc-dlbcl cell line leading to over-expression of spib mrna compared with other lines. spi-b is required for the survival of abc-dlbcl cell lines, as depletion of spi-b using lenalidomide or rna interference leads to decreased survival in vitro. it is predicted that the requirement for spi-b and pu. in lymphoma cells is due to an “addiction” to b cell receptor signaling, which is enforced by over-expression of these factors in activated lymphoma subtypes. next-generation sequencing technologies allow for high-resolution analysis and detection of transcription factors across the entire genome. by combining chromatin-immunoprecipitation with high-throughput sequencing, all regions within the genome bound by pu. and spi-b can be identified. based on the demonstrated complementary function of pu. and spi-b, we hypothesize that pu. and spi-b can interact with the same set of binding sites in the genome of b cells. in this study, we report a genome-wide comparison of genomic regions of interaction by pu. and spi-b in the murine lymphoma cell line wehi- to remove bias introduced by different antibodies, expression levels, or gene regulation we expressed 3xflag-tagged pu. and spi-b at similar levels in wehi- lymphoma cells. chromatin immunoprecipitation was performed using anti-flag antibodies. our results support the hypothesis that pu. and spi-b occupy similar regions within the genome, but also identified a unique subset of regions only occupied by one factor. additionally, motif analysis has suggested that these regions contain binding regions for different co-activator partner proteins. in summary, these experiments provide biochemical insight into both the similarities and differences between the biological functions of pu. and spi-b.  determination of target regions for spi-b and pu.1 to determine if the transcription factors pu. and spi-b occupied identical regions within the mouse genome chip-seq was performed. to ensure a fair comparison, wehi- b lymphoma clones expressing 3xflag-tagged full-length pu. or spi-b protein were selected to ensure equivalent levels of protein expression. uninfected wehi- cells expressed spi and spib mrnas at a ratio of 1:, relative to the normalizer gene gapdh. 3xflag-pu.1-infected wehi- cells expressed spi mrna and spib mrna at ratio of: 3xflag-spi-b-infected wehi- cells expressed spi mrna and spib mrna at a ratio of 1:. these results suggested that spib mrna was modestly overexpressed relative to spi mrna. however, protein levels of the flag-tagged products were similar between 3xflag-tagged pu. and 3xflag-tagged spi-b, as described in xu et al.. this system is expected to provide the advantage of eliminating variability between antibody specificity and chip-seq yield, while allowing sustained expression of pu. and spi-b in a mouse b cell line. chip was performed on fixed wehi- cell lines using anti-flag mab and immunoprecipitated chromatin was validated by qpcr.figure chip sequencing of pu. and spi-b in a mouse b lymphoma cell line. a. workflow for generating chip sequencing data. b. venn diagram for regions of significant chip binding determined by mm-chip. regions with at least base pair overlap were considered common to both factors. c. venn diagram of peakset regions analyzed in diffbind. the majority of regions in the matrix demonstrated less than fold difference in enrichment between pu. and spi-b. d. scatter plot of chip signal between pu. and spi-b binding. binding of these transcription factors was similar across most of the genome. e. de-novo motif analysis from chip. from extracted dna sequences, frequently occurring motifs were discovered using meme-chip. the most frequently occurring motif for each sample contained the canonical ets binding sequence f. representative chip-seq binding at a region considered to be regulated by pu. and spi-b. the histocompatibility q region on chromosome demonstrates high peaks for pu. and spi-b at gene promoters. pu. or spi-b binding peaks were determined using replicate data from two experiments, sequenced by two independent sequencing core facilities with over million reads per sample. replicate chip-seq experiments were merged in mm-chip, which estimates sample-specific shift-size of chip-seq tags with macs modeling and then pools shifted tags from different samples to identify chip-peaks with a dynamic poisson model. this method allowed combining of separate datasets with different tag-sizes, noise features, and dynamic ranges. using a model fold of and a p-value cut-off for peak detection of 1e- mm-chip identified high-stringency regions bound by pu. and high-stringency regions bound by spi-b. the pu.1-bound regions called by mm-chip had an average size of 1469 bp, while the spi-b-bound regions called by mm-chip had an average size of 1460 bp. pu. and spi-b-bound regions were compared to determine if significantly enriched regions were common to both sets. of these regions, had a minimum of 100 bp of overlap between the two sets of regions, and the average length of overlap between significantly enriched regions was out of base pairs. there were also a number of macs-defined regions that had a pu. or spi-b peak in one sample, but not the other, but did not have a significant 2-fold difference in terms of number of aligned reads. using diffbind, pu.1/spi-b matrix peak regions were called. high-confidence peak regions were found to differ significantly in tag count between the two conditions. differentially bound regions were classified as pu.1-higher or spi-b higher, in which local enrichment of sequence tags for one factor was 2-fold higher than tags for the other. of these regions, were considered to be pu.1-higher and spi-b-higher. the correlation coefficient on the genome scale was calculated using cis-regulatory element annotation system application. protein-dna binding signals were correlated between two samples in 1 kb bins. pu. and spi-b signals demonstrated a high degree of correlation with an r correlation coefficient of. these results demonstrated that 1 kb intervals of the genome with a high degree of pu. binding were likely to have strong spi-b binding, and many regions containing significant chip-seq peaks for one factor also had a peak for the other. taken together, these results showed that most regions in the genome of wehi- cells occupied by pu. were also occupied by spi-b. identification of enriched sequence motifs members of the ets superfamily recognize similar purine-rich sequences with a 5′-ggaa-3′ core. to validate the enrichment of chip chromatin at ets motifs, sequences of chip-seq peak regions were screened for enriched motifs using meme, dreme and centrimo using meme-chip. the top motif for pu.1-bound regions was the spi motif, with an e-value of e- and a central canonical ets ggaa motif that was found in % of all peaks. the top motif for the spi-b binding sites was also the spi motif with an e-value of e- this motif was centrally enriched contained a central ggaa sequence was found in % of the peaks. top motifs demonstrated central distribution within the sequences. annotation and comparison of peak-associated genes potential target genes of pu. and spi-b were identified using beta-minus, which annotates binding data to predict target genes based on proximity to the transcription start site. to identify and visualize the function of genes potentially regulated by these transcription factors, the predicted target genes from beta-minus were functionally annotated using database for annotation, visualization and integrated discovery. gene ontology analysis of predicted spi-b targets revealed significant enrichment of antigen processing functions immune response, intracellular signaling cascade and regulation of lymphocyte activation. histocompatibility class ii loci, several fc receptors, as well as b cell markers cd and cd were included in multiple ontologies. these gene lists suggest that spi-b has functions in lymphoid development, antigen processing, and b cell receptor-mediated signalling. top processes for pu. were highly similar to spi-b, consistent with previous studies that presumptive targets for pu. are involved with b cell and myeloid lineages. the gene ontology of processes for pu. target genes included cell activation, leukocyte activation, and hematopoiesis. top genes involved in all three pathways included bak bcl hdac pou2f spi and vav the region upstream of spi demonstrated a single peak at the -14 kb position. the region at -14 kb of spi has been demonstrated to be occupied by pu. in both b lymphocytes and myeloid cells, as opposed to a site at -12 kb that is occupied by pu. only in myeloid cells. these gene lists are consistent with the known role for pu. in both development and function of these lineages. peak to gene associations for pu. and spi-b are available in additional file 1: table s and additional file 2: table s respectively. to predict genes regulated by either transcription factor, the regions with more than 100 bp of overlap between pu. and spi-b bound-regions were examined using beta-minus. gene targets included the toll-like receptors tlr tlr tlr and tlr in addition to well-documented targets such as btk and blnk. top processes were immune response, multiple terms for antigen processing and presentation and regulation of lymphocyte activation. these pathways were considered most significant due to high ranking of mhc class ii genes in the associated gene list, including h2-aa, h2-q h2-q h2-q and h2-q. spi-b and pu. chip signal at genes highest at transcriptional start sites in order to visualize genome-wide distribution of chip peaks, peaks were sorted according to their distribution around mouse refseq genes. cis-regulatory element annotation revealed that regions bound by spi-b and pu. were over-represented for transcription start sites with respect to the whole genome. while % of the mouse genome is located within 10000 bp of gene promoters, % of spi-b bound regions were located within 10000 bp of gene promoters. similarly, % of pu. bound regions were located within 10000 bp of gene promoters.figure distribution of chip-seq peaks within the genome. a. pu. and spi-b have similar binding patterns in relation to features in the genome. b. profiles and associated heatmaps of chip-signal at the transcriptional start site of mouse refseq genes. profiles represent the regions of strongest binding, which are clustered around the transcriptional start site. c. all clusters of pu. and spi-b signal in relation to mouse refseq genes. five different patterns of binding were observed, where the largest cluster contained genes without transcription factor binding at the promoter. clusters and contained genes with binding upstream or downstream of the promoter, although spi-b binding was often seen at the transcription start site. cluster contained regions with the highest log signal. cluster was gene with a broad region of transcription factor binding at the tss. top row shows results from anti-flag chip-seq of 3xflag-pu. in wehi- cells, second row shows results from anti-flag chip-seq of spi-b in wehi- cells, and third row shows results from anti-pu. chip-seq in mouse splenic b cells. d. profile of dnase genomic footprinting at regions of significant chip enrichment for pu. and spi-b. regions of pu. or spi-b binding demonstrated increased dnase sensitivity compared to regions outside the peaks. e. profile of rna polymerase ii chip-signal in b cells at regions of chip enrichment for pu. and spi-b. polii data confirmed that significant pu. and spi-b peaks frequently occur at sites of transcription initiation. f. profile of the chromatin feature h3k36me a marker of heterochromatin and gene bodies, at peak regions for pu. and spi-b. next, binding profiles of pu. and spi-b relative to features in the mouse genome were determined using deeptools. log normalized read coverage of chip-seq data was clustered against regions of mouse refseq genes. regions with the highest pu. and spi-b binding clustered within 4 kb of the transcription start site of mouse genes, with the peak summit directed at the transcription start site and little binding within the gene body. using k-means clustering, transcription factor binding at the start of mouse refseq genes in the genome demonstrated distinct patterns. cluster contained the largest number of mouse genes, where little pu. or spi-b binding was observed. cluster demonstrated an upstream binding modality, while cluster demonstrated binding downstream from the transcription start site. cluster represented the peaks at the tss and had the highest signal. cluster represented broad binding across the tss, with lower signal intensity than in cluster importantly, reanalysis of pu. chip-seq performed using primary splenic b-cells, revealed a very similar enrichment at distal elements and promoters. these clusters suggest that while pu. and spi-b bind various locations within the genome, they have the highest degree of binding at distal elements and promoters of target genes. in order to further characterize the chromatin environment of regions bound by pu. or spi-b, significant peak regions were profiled against marks for transcription, gene body, and dnase sensitivity. chip binding signal in b cells in the form of wig/bigwig files were downloaded from published geo datasets. digital genomic footprinting of dnase i hypersensitivity in mouse b cells from encode/university of washington was used to predict regions of the genome containing cis-regulatory elements profiled against significantly chip-enriched regions in the genome. regions of the genome bound by either spi-b or pu. demonstrated higher digital genomic footprinting scores relative to regions 4 kb up or downstream. in addition to using dnase footprints for predicted regions of transcription factor binding, promoter regions were further predicted using rna polymerase binding. signal for published rna polymerase ii chip-seq on stimulated b lymphocytes was profiled against chip-enriched regions for pu. and spi-b. similar to dgf profiling, regions of the genome bound by pu. or spi-b demonstrated high enrichment of pol ii binding compared to regions 4 kb up or downstream. to further characterize binding locations of spi-b and pu. in relation to the gene body, peak intervals were clustered against histone modifications by chip-seq from encode/licr. whole genome coverage of h3k36me was inversely associated with regions of the genome bound by either pu. or spi-b. this mark has been observed to be strongest within the body of actively transcribed genes. pu. or spi-b peak regions showed an inverse signal of binding, indicating that regions of pu. and spi-b binding are unlikely to be within regions of the genome also bound by h3k36me taken together, these profiles demonstrate that peak regions of pu. and spi-b are characteristic of transcriptional activators. unique binding sites of spi-b and pu.1 pu. and spi-b demonstrated a consistent pattern of high chip signal at transcriptional start sites. interestingly, binding sites unique to either pu. or spi-b demonstrated distinct profiles. unlike common peaks, which tended to be associated with tsss, unique pu. or spi-b sites were frequently found in intergenic regions 10kb beyond the nearest gene promoter. analysis of distribution within the genome using ceas demonstrated that unique pu. peaks were not over-represented at the promoter compared to the background distribution within the genome.figure differential binding of pu. and spi-b within the genome. a. distribution of pu. and spi-b unique chip-seq peaks within the genome. unique sites for these factors are frequently located beyond 10kb of the transcription start site compared with the overall peak sets. b. de-novo motif analysis by meme-chip of unique regions determined by diffbind. the most frequently occurring motif for each sample is shown and compared with the most common motif found in all peaks for pu. and spi-b. pu.1-unique peaks retained similar motifs to those seen in all pu. peaks, but just over half of these peaks also contained a pou2f motif. spi-b unique peaks retained the ets-irf motif seen in all spi-b peaks, with higher conservation of the irf motif. c. a unique pu. binding peak was observed at the site of mir d. unique binding of pu. within the gene nod e. unique spi-b binding in the 3′ end of fgf f. unique spi-b binding in the promoter of lat differentially bound regions were analyzed with meme-chip for unique conserved binding motifs to identify potential differential associated binding partners. regions uniquely bound by pu. contained the ets motif. a pou2f motif was identified by meme chip, with an e-value of e- this motif was observed within 100 bp of % of the ets motifs seen in pu.1-bound regions, but was overrepresented to % of ets motifs in unique pu. peaks. there were pou2f motif occurrences in unique pu. peaks with a p-value less than. combined pou2f2-pu. motifs were observed upon re-analysis of pu. chip in primary splenic b-cells, where a secondary pou2f was located in % of all sequences also containing a gaggaa primary motif. in contrast, regions bound by spi-b did not demonstrate a marked difference in pou2f motifs. pou2f motifs were observed in % of all spi-b bound regions and % of regions uniquely bound by spi-b but not pu. remarkably, de-novo motif finding with spi-b unique regions revealed that 100% of unique spi-b regions contained an ets–irf composite element with an e-value of e-. this combined motif was similar to the binding sequence for all significant spi-b peaks, but the irf element was more conserved. the primary ggaanngaaa motif was found in % of spi-b bound sites. sites uniquely bound by pu. did not demonstrate the same increase in eices; ggaanngaaa motifs were observed in % of all pu. peaks and reduced to % in unique pu.1-bound regions. this composite element was observed in spi-b unique peaks and provides evidence that irf factors might bind within a few base pairs of the spi-b binding sequence. to determine whether the uniquely-bound spi-b sites containing irf motifs might interact with irf we compared our sites with irf chip-seq regions from mouse b cells. of these sites, % were located within 100 bp of an irf4-bound site in unstimulated b cells or b cells stimulated with il- % of ets-irf sites were located within 100 bp of an irf peak under both conditions. these findings suggest that unique spi-b binding is associated with local deposition of irf in b cells. in order to determine what genes were regulated at differentially bound regions, gene function for differential gene targets was determined using gene ontology. peak to gene associations were determined using ceas and annotated using david. genes uniquely bound by pu. were predominantly associated with leukocyte activation during immune response and cell activation during immune response. these pathways were both highly ranked due to inclusion of interleukin and 12b, cx3cr as well as a unique peak in an intron of nfkb not observed in spi-b. pu. unique peaks were found to be associated with genes related to mrna processing and with a number of mirnas; mir-155/ay mir-3108/zfhx mir-1938/ifrd mir-3102/arhgef and mir-5130/kctd of these, mir- has a known oncogenic role in lymphomas. furthermore, mir- directly targets pu. and leads to a down-regulation of pu. protein in b cells. pu. unique peaks were observed in the promoter region of nod a nod-like receptor involved in b cell proliferation and activation. typical examples of these regions are represented in figure 3c,d. gene targets of differentially bound spi-b peaks were associated with immune effector process and b cell activation that included bnip3l, lat and pou2f multiple go terms associated with both positive and negative regulation of transcription and gene expression were also identified due to the inclusion of fgf myc and pou2f. a large unique peak of spi-b binding was observed at a combined ets-irf motif within the 3′ end of fgf. fgf is involved in the regulation of phosphate concentration in plasma, and has been found to be elevated in the serum of patients with b cell neoplasms. spi-b unique binding was also observed at the promoter of sema4b. sema4b abundantly expressed in lymphocytes and been demonstrated to be a novel regulator of immunological memory responses and homeostatic t helper 1/t helper balance in mice. lat demonstrated significant spi-b binding directly at the promoter, which was not observed in the pu. chip. lat is expressed in the spleen, peripheral blood lymphocytes, and various human b cell lines, and is tyrosine-phosphorylated in response to b cell receptor signalling. lat expression has been observed in monocytes and b cells. these analyses demonstrate that while pu. and spi-b frequently occur at the regulatory regions of genes predicted to have b cell function, a small subset of regions and genes that are uniquely bound by only of these transcription factors. these factors have unique requirements for associated binding partners, and are involved in different functions. discussion to investigate genome-wide binding of spi-b and pu. we conducted next generation sequencing of chromatin immunoprecipitated with anti-3xflag-tagged spi-b or pu. previous studies have reported chip-seq analysis of pu. binding sites in erythroid, myeloid, and lymphoid cells but none have compared pu. to spi-b sites in mouse cells due to technical constraints. detailed analysis of transcription factor binding revealed that these two ets transcription factors had similar binding regions within the genome of murine wehi- lymphoma cells. we determined that both transcription factors demonstrated the highest degree of binding near the tss and regions of open chromatin, although peaks were also observed within introns and upstream of genes beyond 10 kb. characterization of the sequences most strongly bound by pu. and spi-b has confirmed that the primary target sequence of both factors contained the purine-rich core motif 5′-gaggaa-3′. this motif was identified within regions of significant chip enrichment, and determined to be overrepresented by de-novo motif enrichment analysis. peak-to-gene associations confirmed occupancy of both factors within  bp of the tss for genes involved in immune system regulation and processes. gene ontology analysis confirmed that pu. and spi-b are involved in the regulation of genes required for b cell development and function. the top go pathways determined by david for pu. spi-b, and the common peaks were cell activation, immune response and immune response, respectively. an important finding of this study is that of the genomic regions occupied by pu. a majority were also occupied by spi-b. this fraction was 1900/ regions using mm-chip, and 5378/ regions using diffbind. this result supports our hypothesis that pu. and spi-b can interact with the same regulatory regions in the genome of b cells. this result is consistent with previous reports that pu. and spi-b have similar dna binding specificities. we suggest that similar dna binding preferences and similar biochemical activity are the explanation for why pu. and spi-b have complementary biological activity in the b cell lineage. fewer regions of the genome were found to be differentially occupied by pu. or spi-b in the genome of wehi- b cells. one important aspect of next-generation sequencing demonstrated in this study is the importance of mathematical modeling and input sequencing in identifying differential peaks. while direct subtraction of regions called by peak callers revealed several peak regions that did not overlap between samples, affinity data analysis using diffbind determined that only several hundred regions demonstrated significant differences between factors. using diffbind to identify differential peaks in combined data most likely under-predicted gene targets, but provided high-stringency lists. therefore, while there remains a possibility that other differential regions exist within the genome, this method provides the most accurate and specific regions. therefore, subsequent analysis of these high-stringency peaks highlights the most significant differences between spi-b and pu. binding within the genome. regions identified that were occupied uniquely by pu. or spi-b may reveal insights into biological functions unique to each factor. genes associated with unique pu. bound regions were related to immune development using gene ontology analysis. regions unique to pu. contained a number of microrna genes, while no microrna genes were observed at unique spi-b peaks. the unique binding of pu. to the sequence of mir- may have a role in regulation of pu. itself, as expression this micro-rna has been shown by others to down-regulate pu.. binding at this region suggests that pu. may be involved in the regulation of mir- the identification of an oct binding motif within unique pu. regions implies a potential co-activator role, as pu. and oct have previously been shown to bind concomitantly at the vκ promoter in b lymphocytes, and point mutations in the binding sites for either factor can lead to diminished immunoglobulin production. oct binding has been shown to be covariant with pu. binding activity and oct preferentially binds to pu. regions in b cells compared with macrophages, suggesting a functional role for pu. to recruit oct to regulatory regions of the genome and induce cellular reprogramming in b cells. unique spi-b associated genes were associated with roles in b-cell activation and immune function. all unique spi-b peaks had an associated irf sites in wehi- lymphoma cells, while ets-irf sites were not overrepresented in unique pu. peaks. this suggests that in cases where genes are under the regulation of an ets-irf composite element, spi-b and not pu. is the preferential irf partner. our results are consistent with a recent study showing that combined eices were dominantly occupied by spi-b in b lymphoma cell lines, and that pu. could not fully compensate for spi-b in recruiting irf to target regions. rnai-mediated down-regulation of either spi-b or irf leads to rapid death of cultured lymphoma cells, irrespective of pu. co-occupancy. therefore, spi-b may be involved in a unique regulatory network where it is more frequently associated with irf in b cell lymphomas, and may be directed to these sites by irf or function to alter irf occupancy within the genome. our model of pu. and spi-b binding is based on lymphoma cells over-expressing the proteins, which bears similarity to other b cell studies in terms of binding near target genes and associated binding motifs. while the changes in the expression level of pu. can enhance binding of other factors to influence local chromatin structure and dictate eventual cell fate, our tagged-expression model does not appear to have substantially altered binding of pu. or spi-b, since identified peaks were very similar to those identified in a study of mouse splenic b cells. peak-associated gene lists support our hypothesis that pu. and spi-b are involved in the regulation of lymphoid development and immune function, and we report fewer than peaks per factor, possibly due to the restricted lineage of the lymphoma cell line.  in conclusion, we report genome-wide binding of the ets transcription factors spi-b and pu. to similar regions of the genome, with high affinity for transcriptional start sites containing ets motifs. these regions are frequently located near the regulatory domains of genes involved in b cell development and function. in addition to these common regions, there exists a small set of unique regions where pu. and spi-b demonstrate differential preference for associated factors based on sequence identity, target gene function, and potential co-activators. our findings provide novel insight into differences between the two factors, and offers novel biochemical pathways involving spi-b as a molecular targeting therapy in b cell malignancies. 
 binding included pou2f while unique motifs in the spi-b regions contained a combined ets-irf motif. analysis for high-stringency enriched genomic regions demonstrated that pu. and spi-b are both expressed in b cells, and have been demonstrated to redundantly activate transcription of genes required for b cell differentiation and function. were frequently located immediately upstream of genes associated with immune response and activation of b cells. and spi-b may be explained by their interaction with a similar set of regions in the genome of b cells. our results suggest that complementary biological functions of pu. are highly related members of the e26-transformation-specific family of transcription factors that have similar, but not identical, roles in b cell development. motif-finding revealed that both transcription factors were predominantly located at the ets core domain, however, other unique motifs were identified when examining regions associated with only one of the two factors.
 spi-b and pu. are highly related members of the e26-transformation-specific family of transcription factors that have similar, but not identical, roles in b cell development. pu. and spi-b are both expressed in b cells, and have been demonstrated to redundantly activate transcription of genes required for b cell differentiation and function. it was hypothesized that spi-b and pu. occupy a similar set of regions within the genome of a b lymphoma cell line.  to compare binding regions of spi-b and pu. murine wehi- lymphoma cells were infected with retroviral vectors encoding 3xflag-tagged pu. or spi-b. anti-flag chromatin immunoprecipitation followed by next generation sequencing was performed. analysis for high-stringency enriched genomic regions demonstrated that pu. occupied regions and spi-b occupied regions. the majority of regions occupied by spi-b were also occupied by pu. regions bound by spi-b and pu. were frequently located immediately upstream of genes associated with immune response and activation of b cells. motif-finding revealed that both transcription factors were predominantly located at the ets core domain, however, other unique motifs were identified when examining regions associated with only one of the two factors. motifs associated with unique pu. binding included pou2f while unique motifs in the spi-b regions contained a combined ets-irf motif.  our results suggest that complementary biological functions of pu. and spi-b may be explained by their interaction with a similar set of regions in the genome of b cells. however, sites uniquely occupied by pu. or spi-b provide insight into their unique functions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pu.1spi-bchip-seqmotifb cellgene regulationissue-copyright-statement© the author 2015 