BACKGROUND
runx proteins are transcription factors that function critically in gene regulatory networks that control cell proliferation and differentiation during animal development  <cit> .
the runx family is defined by a highly conserved  <dig> amino acid sequence known as the runt domain, which binds specifically to the dna sequence tgt/cggt  <cit> .
the sea urchin runx transcription factor sprunt- <dig> was discovered biochemically as a protein that binds this sequence in the cis-regulatory domain of the aboral ectoderm differentiation gene cyiiia, an interaction that contributes to transcriptional activation of cyiiia  <cit> .
sprunt- <dig> transcripts are globally expressed in the early embryo, and then become localized predominantly to regions of continued growth and cell proliferation within larval oral ectoderm and endomesoderm  <cit> .
morpholino antisense-mediated knockdown of sprunt- <dig> has shown that it is required throughout the embryo for cell differentiation  <cit>  and survival  <cit> .
the latter function is mediated by a positively-acting interaction between sprunt- <dig> and its target gene sppkc <dig>  which encodes a conventional protein kinase c  <cit> .
runx proteins heterodimerize with another protein commonly referred to as core binding factor beta , a non-dna binding protein that allosterically enhances the dna binding of the runt domain  <cit> .
mammalian cbfβ also increases the half-life of runx <dig> by protecting it from ubiquitin-mediated proteolysis  <cit> .
since runx <dig> contributes to cell cycle control and its protein levels are cell cycle regulated  <cit> , this suggests that cbfβ may be a regulatory subunit that modulates runx activity during the developmental transition from proliferation to terminal differentiation.
knockout of cbfb in mouse leads to embryonic lethality caused by a failure of hematopoiesis  <cit> , a phenotype very similar to that caused by knockout of runx <dig>  as with runx <dig>  chromosomal translocations involving cbfb are commonly associated with human leukemia  <cit> .
like runx <dig>  cbfb has also been shown to be important for osteogenesis, although its loss-of-function phenotype is not identical to that caused by runx <dig> knockout since some osteoblast differentiation does occur  <cit> .
drosophila has two cbfβ genes, brother and big brother, the products of which contribute redundantly to the functionality of the runx proteins runt and lozenge  <cit> .
these studies all indicate that cbfβ is important for runx function; however, to date, no studies have definitively addressed the question of whether the runx-cbfβ interaction is constitutive  or facultative .
the latter possibility is suggested by the facts that  the interaction is inhibited by sequences within mammalian runx <dig>  <cit>  and the c-terminal domains of some runx <dig> isoforms  <cit> , and  runx proteins can bind dna as a monomer, albeit less effectively than as a heterodimer  <cit> .
mouse cbfβ, which is ubiquitously expressed, has been shown to interact specifically with another protein termed crl- <dig>  which is expressed in subsets of neuronal cells  <cit> .
finally, avian cbfb displays a complex pattern of expression in early development that is not identical to the pattern of runx <dig> expression, with some regions of the embryo expressing only one or the other gene  <cit> .
taken together, these observations invite speculation that runx proteins might interact with only a subset of target genes  as a heterodimer with cbfβ, and that the choice between these alternative states may in part be determined by the specific runx isoform as well as protein-protein interactions specified by the context of each particular tissue, cell type, and cis-regulatory system.
here we describe the cloning and initial characterization of spcbfβ, the heterodimeric runx partner from the sea urchin strongylocentrotus purpuratus.
we show that spcbfβ is expressed in a pattern that is similar but not identical to that of sprunt- <dig>  moreover, while spcbfβ participates in the runx-dependent activation of several genes including cyiiia, it is not required for or involved in runx-dependent activation of sppkc <dig>  which we showed previously to be critical for cell survival in the embryo  <cit> .
therefore, heterodimerization of sprunt- <dig> with spcbfβ occurs facultatively in the sea urchin, and is a context-dependent aspect of runx-mediated transcriptional control.
RESULTS
spcbfβ expression during embryogenesis
sequences encoding the sea urchin homologue of cbfβ were amplified from s. purpuratus blastula stage cdna by polymerase chain reaction  using degenerate primers corresponding to highly conserved regions of the protein.
the resulting amplicon was cloned and sequenced, and the sequence was used to obtain the full-length spcbfβ mrna by 5' and 3' race.
the spcbfβ mrna encodes a protein with a predicted molecular weight of ~ <dig> kda.
as shown in fig.
1a, the deduced amino acid sequence of spcbfβ is highly similar to that of its homologues from other phyla, and as would be expected, is more similar to the vertebrate representatives than to those from the non-deuterostome invertebrates.
spcbfbeta gene sequences were located in the s. purpuratus genome database  <cit> , and the exon-intron structure of the gene was deduced from alignment with the cdna.
spcbfbeta appears to be the only homologue in the s. purpuratus genome, and contains four introns, the positions of which are conserved in vertebrates .
in contrast, the drosophila genes bro and bgb have no introns, while the c. elegans homologue is missing introns  <dig> and  <dig> .
electrophoretic mobility shift analysis  was used to test whether spcbfβ enhances the dna binding activity of sprunt- <dig>  recombinant sprunt- <dig> protein was reacted with a fluorescently labeled oligonucleotide probe containing the sprunt- <dig> binding site from cyiiia  <cit> , either with or without pre-incubation with recombinant spcbfβ.
dna binding of full-length sprunt- <dig>  or a fragment containing only the runt domain thereof, is substantially enhanced in the presence of spcbfβ, which forms a complex with the sprunt- <dig> protein , whereas spcbfβ alone does not bind dna .
it is likely that native sprunt- <dig> forms a heterodimeric complex with spcbfβ in the embryo, and consistent with this, affinity purified sprunt- <dig> protein from blastula stage nuclear extracts was initially identified as a heterodimer containing a  <dig> kda subunit  <cit> .
to confirm this, an antibody generated against recombinant spcbfβ was used to probe an immunoblot of whole nuclear extract, nuclear extract immunodepleted with an anti-sprunt- <dig> antibody, and the resulting immunoprecipitate.
a  <dig> kda spcbfβ-immunoreactive band present in blastula stage nuclear extract  is specifically immunodepleted from the extract by the sprunt- <dig> antibody , and pulled down in the sprunt- <dig> immuoprecipitate , whereas the band is not similarly precipitated by non-specific igg .
these data suggest that spcbfβ exists in a complex with sprunt- <dig> in vivo.
rna blot analysis shows that spcbfβ is represented by a single species of transcript that is virtually absent in the egg and early embryo, and which accumulates dramatically during blastula and gastrula stages .
the temporal expression of spcbfβ was further examined by quantitative reverse transcription-coupled pcr .
spcbfβ transcripts are present at low levels during cleavage, accumulate approximately 25-fold between  <dig> and  <dig> hours post-fertilization , and thereafter decline in abundance .
this pattern parallels that of sprunt- <dig>  <cit> , although possibly with a slight temporal lag.
immunoblot analysis  reveals that there is no maternal spcbfβ protein, and that zygotic accumulation of spcbfβ protein is similar to that of its mrna, although unlike the mrna the protein levels do not increase significantly between  <dig> and  <dig> h, suggesting that there may be translational or post-translational regulation during this interval.
quantitative rt-pcr was also used to measure the abundance of spcbfβ transcripts.
toward this end a standard curve was constructed from in vitro synthesized spcbfβ mrna .
we used the same method to measure the abundance of sprunt- <dig> transcripts, which were previously measured by an rnaase protection method  <cit> .
the measurements of sprunt- <dig> were in agreement with our previous measurements, indicating  <dig> – <dig>  transcripts per blastula or gastrula stage embryo.
in the mid-gastrula stage embryo, when spcbfβ is maximally expressed, it is present at slightly higher levels than is sprunt- <dig>  determined by qrt-pcr to be  <dig>  transcripts per embryo.
thereafter, spcbfβ transcripts decline in abundance to levels approximating those of sprunt- <dig>
the spatial pattern of spcbfβ expression was examined by whole mount in situ hybridization  .
as with its temporal expression, spcbfβ is expressed initially in a spatial pattern that is similar to that of sprunt-1: at mesenchyme blastula stage, transcripts are globally distributed , and by late gastrula stage, they start to become enriched in the oral ectoderm and endomesderm .
in the 2-day old gastrula endoderm expression is highest in a ring of cells just inside the blastopore .
expression is also enhanced in the oral ectoderm  and on the oral side of the hindgut region .
in the mature  pluteus, spcbfβ transcripts are expressed most prominently in the differentiating midgut and hindgut of the endoderm , whereas in the ciliated band, expression is confined to the tips of the anal arm buds .
this is somewhat different from the larval expression pattern of sprunt- <dig>  which is also expressed in the endoderm but most prominently in the larval foregut region, as well as throughout the ciliated band  <cit> .
spcbfβ is dispensable for the pro-survival function of sprunt-1
a morpholino antisense oligonucleotide  was designed to target the translational start site in the spcbfβ mrna.
introduction of this maso into embryos produces a distinctive phenotype that is milder than that obtained with sprunt- <dig> masos .
while spcbfβ morphants display morphological defects, most notably a failure of skeletogenesis and a poorly differentiated ectoderm , unlike sprunt- <dig> morphants, they gastrulate .
the spcbfβ morphants also have a secondary axis and appear to form bilateral clusters of skeletogenic mesenchyme cells , which normally occurs in response to signals from the oral ectoderm  <cit> , suggesting that the ectodermal and skeletogenic differentiation defects are not caused by a failure of regional specification, but rather of subsequent cell differentiation.
that the spcbfβ maso effectively depletes embryos of spcbfβ is shown by immunoblot analysis of endogenous protein .
the gastrulation defective phenotype associated with sprunt- <dig> deficiency is largely a secondary effect of extensive apoptosis that occurs throughout the post-blastula stage embryo  <cit> .
since spcbfβ morphants gastrulate, we hypothesized that they do not undergo the extensive apoptosis characteristic of sprunt- <dig> morphants.
this is indeed the case: unlike embryos injected with the sprunt- <dig> maso , those injected with the spcbfβ maso display negligible tunel signal .
this suggests that spcbfβ is dispensable for sprunt- <dig> function in promoting cell survival.
sprunt- <dig> derived from exogenous mrna rescues cell survival in sprunt- <dig> morphants  <cit> .
if spcbfβ is dispensable for the survival function of sprunt- <dig>  then a sprunt- <dig> protein that has been mutated so that it cannot interact with spcbfβ should retain the ability to rescue cell survival in sprunt- <dig> morphants.
to test this, we constructed a sprunt- <dig> mutant protein wherein an arginine is substituted for a glycine residue that is essential for the interaction between the runt domain and cbfβ .
electrophoretic mobility shift analysis verifies that the runt domain from sprunt-1-g115r retains the ability to bind dna as a monomer, but does not heterodimerize with spcbfβ .
importantly, sprunt-1-g115r rescues cell survival in sprunt- <dig> morphants , indicating that heterodimerization with spcbfβ is not required for the anti-apoptotic function of sprunt- <dig>
sppkc <dig> is a spcbfβ-independent runx target
the apoptotic phenotype in sprunt- <dig> deficient embryos is specifically caused by a deficit in the expression of sppkc <dig>  a direct regulatory target of sprunt- <dig> that encodes a conventional protein kinase c  <cit> .
the fact that spcbfβ-deficient embryos do not display an apoptotic phenotype suggests that loss of spcbfβ function does not adversely affect the expression of sppkc <dig>  to test this we used reverse transcription-coupled pcr to measure the relative levels of sppkc <dig> transcripts in either sprunt- <dig> morphants or spcbfβ morphants with respect to controls.
whereas sppkc <dig> is ~7-fold under-expressed in sprunt-1-deficient gastrula stage embryos, its expression at that stage is not significantly affected by knockdown of spcbfβ .
in contrast, expression of the aboral ectoderm differentiation marker cyiiia is diminished to an equivalent extent by knockdown of either sprunt- <dig> or spcbfβ, as are expression of two other putative sprunt- <dig> targets  <cit> , spcyclind and spdri  .
these results indicate that at gastrula stage, sprunt-1-mediated activation of cyiiia, spcyclind and spdri requires heterodimerization with spcbfβ, whereas sprunt-1-mediated activation of sppkc <dig> does not.
interestingly, cyiiia expression is not as strongly affected by sprunt- <dig> depletion as is sppkc <dig> expression; this could relate to the fact that the splice-blocking maso does not completely deplete sprunt- <dig> protein  <cit> , some of which is provided maternally  <cit> .
these data suggest that sppkc <dig> activity is particularly sensitive to sprunt- <dig> protein levels, which might occur if the architectural context of the sppkc <dig> cis-regulatory system confers selectivity for zygotically-expressed sprunt- <dig> and/or sprunt- <dig> isoforms that cannot heterodimerize with spcbfβ.
while these results indicate that spcbfβ is not required for the transcriptional activation of sppkc <dig>  they do not rule out the possibility that spcbfβ nonetheless heterodimerizes with sprunt- <dig> on its target sequences in the sppkc <dig> promoter.
to address this issue, we performed chromatin immunoprecipitation  of chromatin prepared from late gastrula stage embryos using antibodies to either sprunt- <dig> or spcbfβ.
consistent with the expression data indicating that cyiiia activation requires the heterodimer, sequences from the cyiiia promoter are recovered by chip using both antibodies, as are sequences from spcyclind .
in contrast, whereas sequences from the sppkc <dig> promoter are recovered by chip using the sprunt- <dig> antibody, they are not recovered by the spcbfβ antibody .
we conclude that in the gastrula stage embryo, spcbfβ does not exist in a heterodimeric complex with sprunt- <dig> within the context of the sppkc <dig> promoter.
CONCLUSIONS
our data are consistent with the proposition that cbfβ is a facultative runx partner that participates in the regulation of a subset of runx target genes.
it will be important to analyze the sppkc <dig> cis-regulatory system to learn the contextual rules that facilitate cbfβ-independent runx function, and to determine the biological rationale for runx-mediated transcriptional regulation that does not involve cbfβ.
one intriguing possibility is that genes that need to be rapidly responsive to physiological signals require runx-dna binding complexes that are less stable and/or have shorter half lives.
addressing this issue will require identification and comparison of the cis-regulatory systems from additional cfbβ-dependent and -independent runx target genes.

 runx proteins are transcription factors that function critically in gene regulatory networks that control cell proliferation and differentiation during animal development. the runx family is defined by a highly conserved amino acid sequence known as the runt domain, which binds specifically to the dna sequence tgt/cggt. the sea urchin runx transcription factor sprunt- was discovered biochemically as a protein that binds this sequence in the cis-regulatory domain of the aboral ectoderm differentiation gene cyiiia, an interaction that contributes to transcriptional activation of cyiiia. sprunt- transcripts are globally expressed in the early embryo, and then become localized predominantly to regions of continued growth and cell proliferation within larval oral ectoderm and endomesoderm. morpholino antisense-mediated knockdown of sprunt- has shown that it is required throughout the embryo for cell differentiation and survival. the latter function is mediated by a positively-acting interaction between sprunt- and its target gene sppkc which encodes a conventional protein kinase c. runx proteins heterodimerize with another protein commonly referred to as core binding factor beta, a non-dna binding protein that allosterically enhances the dna binding of the runt domain. mammalian cbfβ also increases the half-life of runx by protecting it from ubiquitin-mediated proteolysis. since runx contributes to cell cycle control and its protein levels are cell cycle regulated, this suggests that cbfβ may be a regulatory subunit that modulates runx activity during the developmental transition from proliferation to terminal differentiation. knockout of cbfb in mouse leads to embryonic lethality caused by a failure of hematopoiesis, a phenotype very similar to that caused by knockout of runx as with runx chromosomal translocations involving cbfb are commonly associated with human leukemia. like runx cbfb has also been shown to be important for osteogenesis, although its loss-of-function phenotype is not identical to that caused by runx knockout since some osteoblast differentiation does occur. drosophila has two cbfβ genes, brother and big brother, the products of which contribute redundantly to the functionality of the runx proteins runt and lozenge. these studies all indicate that cbfβ is important for runx function; however, to date, no studies have definitively addressed the question of whether the runx-cbfβ interaction is constitutive or facultative. the latter possibility is suggested by the facts that the interaction is inhibited by sequences within mammalian runx and the c-terminal domains of some runx isoforms, and runx proteins can bind dna as a monomer, albeit less effectively than as a heterodimer. mouse cbfβ, which is ubiquitously expressed, has been shown to interact specifically with another protein termed crl- which is expressed in subsets of neuronal cells. finally, avian cbfb displays a complex pattern of expression in early development that is not identical to the pattern of runx expression, with some regions of the embryo expressing only one or the other gene. taken together, these observations invite speculation that runx proteins might interact with only a subset of target genes as a heterodimer with cbfβ, and that the choice between these alternative states may in part be determined by the specific runx isoform as well as protein-protein interactions specified by the context of each particular tissue, cell type, and cis-regulatory system. here we describe the cloning and initial characterization of spcbfβ, the heterodimeric runx partner from the sea urchin strongylocentrotus purpuratus. we show that spcbfβ is expressed in a pattern that is similar but not identical to that of sprunt- moreover, while spcbfβ participates in the runx-dependent activation of several genes including cyiiia, it is not required for or involved in runx-dependent activation of sppkc which we showed previously to be critical for cell survival in the embryo. therefore, heterodimerization of sprunt- with spcbfβ occurs facultatively in the sea urchin, and is a context-dependent aspect of runx-mediated transcriptional control.  spcbfβ expression during embryogenesis sequences encoding the sea urchin homologue of cbfβ were amplified from s. purpuratus blastula stage cdna by polymerase chain reaction using degenerate primers corresponding to highly conserved regions of the protein. the resulting amplicon was cloned and sequenced, and the sequence was used to obtain the full-length spcbfβ mrna by 5' and 3' race. the spcbfβ mrna encodes a protein with a predicted molecular weight of ~ kda. as shown in fig. 1a, the deduced amino acid sequence of spcbfβ is highly similar to that of its homologues from other phyla, and as would be expected, is more similar to the vertebrate representatives than to those from the non-deuterostome invertebrates. spcbfbeta gene sequences were located in the s. purpuratus genome database, and the exon-intron structure of the gene was deduced from alignment with the cdna. spcbfbeta appears to be the only homologue in the s. purpuratus genome, and contains four introns, the positions of which are conserved in vertebrates. in contrast, the drosophila genes bro and bgb have no introns, while the c. elegans homologue is missing introns and. electrophoretic mobility shift analysis was used to test whether spcbfβ enhances the dna binding activity of sprunt- recombinant sprunt- protein was reacted with a fluorescently labeled oligonucleotide probe containing the sprunt- binding site from cyiiia, either with or without pre-incubation with recombinant spcbfβ. dna binding of full-length sprunt- or a fragment containing only the runt domain thereof, is substantially enhanced in the presence of spcbfβ, which forms a complex with the sprunt- protein, whereas spcbfβ alone does not bind dna. it is likely that native sprunt- forms a heterodimeric complex with spcbfβ in the embryo, and consistent with this, affinity purified sprunt- protein from blastula stage nuclear extracts was initially identified as a heterodimer containing a kda subunit. to confirm this, an antibody generated against recombinant spcbfβ was used to probe an immunoblot of whole nuclear extract, nuclear extract immunodepleted with an anti-sprunt- antibody, and the resulting immunoprecipitate. a kda spcbfβ-immunoreactive band present in blastula stage nuclear extract is specifically immunodepleted from the extract by the sprunt- antibody, and pulled down in the sprunt- immuoprecipitate, whereas the band is not similarly precipitated by non-specific igg. these data suggest that spcbfβ exists in a complex with sprunt- in vivo. rna blot analysis shows that spcbfβ is represented by a single species of transcript that is virtually absent in the egg and early embryo, and which accumulates dramatically during blastula and gastrula stages. the temporal expression of spcbfβ was further examined by quantitative reverse transcription-coupled pcr. spcbfβ transcripts are present at low levels during cleavage, accumulate approximately 25-fold between and hours post-fertilization, and thereafter decline in abundance. this pattern parallels that of sprunt-, although possibly with a slight temporal lag. immunoblot analysis reveals that there is no maternal spcbfβ protein, and that zygotic accumulation of spcbfβ protein is similar to that of its mrna, although unlike the mrna the protein levels do not increase significantly between and h, suggesting that there may be translational or post-translational regulation during this interval. quantitative rt-pcr was also used to measure the abundance of spcbfβ transcripts. toward this end a standard curve was constructed from in vitro synthesized spcbfβ mrna. we used the same method to measure the abundance of sprunt- transcripts, which were previously measured by an rnaase protection method. the measurements of sprunt- were in agreement with our previous measurements, indicating – transcripts per blastula or gastrula stage embryo. in the mid-gastrula stage embryo, when spcbfβ is maximally expressed, it is present at slightly higher levels than is sprunt- determined by qrt-pcr to be transcripts per embryo. thereafter, spcbfβ transcripts decline in abundance to levels approximating those of sprunt- the spatial pattern of spcbfβ expression was examined by whole mount in situ hybridization. as with its temporal expression, spcbfβ is expressed initially in a spatial pattern that is similar to that of sprunt-1: at mesenchyme blastula stage, transcripts are globally distributed, and by late gastrula stage, they start to become enriched in the oral ectoderm and endomesderm. in the 2-day old gastrula endoderm expression is highest in a ring of cells just inside the blastopore. expression is also enhanced in the oral ectoderm and on the oral side of the hindgut region. in the mature pluteus, spcbfβ transcripts are expressed most prominently in the differentiating midgut and hindgut of the endoderm, whereas in the ciliated band, expression is confined to the tips of the anal arm buds. this is somewhat different from the larval expression pattern of sprunt- which is also expressed in the endoderm but most prominently in the larval foregut region, as well as throughout the ciliated band. spcbfβ is dispensable for the pro-survival function of sprunt-1 a morpholino antisense oligonucleotide was designed to target the translational start site in the spcbfβ mrna. introduction of this maso into embryos produces a distinctive phenotype that is milder than that obtained with sprunt- masos. while spcbfβ morphants display morphological defects, most notably a failure of skeletogenesis and a poorly differentiated ectoderm, unlike sprunt- morphants, they gastrulate. the spcbfβ morphants also have a secondary axis and appear to form bilateral clusters of skeletogenic mesenchyme cells, which normally occurs in response to signals from the oral ectoderm, suggesting that the ectodermal and skeletogenic differentiation defects are not caused by a failure of regional specification, but rather of subsequent cell differentiation. that the spcbfβ maso effectively depletes embryos of spcbfβ is shown by immunoblot analysis of endogenous protein. the gastrulation defective phenotype associated with sprunt- deficiency is largely a secondary effect of extensive apoptosis that occurs throughout the post-blastula stage embryo. since spcbfβ morphants gastrulate, we hypothesized that they do not undergo the extensive apoptosis characteristic of sprunt- morphants. this is indeed the case: unlike embryos injected with the sprunt- maso, those injected with the spcbfβ maso display negligible tunel signal. this suggests that spcbfβ is dispensable for sprunt- function in promoting cell survival. sprunt- derived from exogenous mrna rescues cell survival in sprunt- morphants. if spcbfβ is dispensable for the survival function of sprunt- then a sprunt- protein that has been mutated so that it cannot interact with spcbfβ should retain the ability to rescue cell survival in sprunt- morphants. to test this, we constructed a sprunt- mutant protein wherein an arginine is substituted for a glycine residue that is essential for the interaction between the runt domain and cbfβ. electrophoretic mobility shift analysis verifies that the runt domain from sprunt-1-g115r retains the ability to bind dna as a monomer, but does not heterodimerize with spcbfβ. importantly, sprunt-1-g115r rescues cell survival in sprunt- morphants, indicating that heterodimerization with spcbfβ is not required for the anti-apoptotic function of sprunt- sppkc is a spcbfβ-independent runx target the apoptotic phenotype in sprunt- deficient embryos is specifically caused by a deficit in the expression of sppkc a direct regulatory target of sprunt- that encodes a conventional protein kinase c. the fact that spcbfβ-deficient embryos do not display an apoptotic phenotype suggests that loss of spcbfβ function does not adversely affect the expression of sppkc to test this we used reverse transcription-coupled pcr to measure the relative levels of sppkc transcripts in either sprunt- morphants or spcbfβ morphants with respect to controls. whereas sppkc is ~7-fold under-expressed in sprunt-1-deficient gastrula stage embryos, its expression at that stage is not significantly affected by knockdown of spcbfβ. in contrast, expression of the aboral ectoderm differentiation marker cyiiia is diminished to an equivalent extent by knockdown of either sprunt- or spcbfβ, as are expression of two other putative sprunt- targets, spcyclind and spdri. these results indicate that at gastrula stage, sprunt-1-mediated activation of cyiiia, spcyclind and spdri requires heterodimerization with spcbfβ, whereas sprunt-1-mediated activation of sppkc does not. interestingly, cyiiia expression is not as strongly affected by sprunt- depletion as is sppkc expression; this could relate to the fact that the splice-blocking maso does not completely deplete sprunt- protein, some of which is provided maternally. these data suggest that sppkc activity is particularly sensitive to sprunt- protein levels, which might occur if the architectural context of the sppkc cis-regulatory system confers selectivity for zygotically-expressed sprunt- and/or sprunt- isoforms that cannot heterodimerize with spcbfβ. while these results indicate that spcbfβ is not required for the transcriptional activation of sppkc they do not rule out the possibility that spcbfβ nonetheless heterodimerizes with sprunt- on its target sequences in the sppkc promoter. to address this issue, we performed chromatin immunoprecipitation of chromatin prepared from late gastrula stage embryos using antibodies to either sprunt- or spcbfβ. consistent with the expression data indicating that cyiiia activation requires the heterodimer, sequences from the cyiiia promoter are recovered by chip using both antibodies, as are sequences from spcyclind. in contrast, whereas sequences from the sppkc promoter are recovered by chip using the sprunt- antibody, they are not recovered by the spcbfβ antibody. we conclude that in the gastrula stage embryo, spcbfβ does not exist in a heterodimeric complex with sprunt- within the context of the sppkc promoter.  our data are consistent with the proposition that cbfβ is a facultative runx partner that participates in the regulation of a subset of runx target genes. it will be important to analyze the sppkc cis-regulatory system to learn the contextual rules that facilitate cbfβ-independent runx function, and to determine the biological rationale for runx-mediated transcriptional regulation that does not involve cbfβ. one intriguing possibility is that genes that need to be rapidly responsive to physiological signals require runx-dna binding complexes that are less stable and/or have shorter half lives. addressing this issue will require identification and comparison of the cis-regulatory systems from additional cfbβ-dependent and -independent runx target genes. 
spcbfβ is entirely of zygotic provenance and its expression is similar that of sprunt- accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. spcbfβ is a facultative runx partner that appears to be required specifically for cell differentiation. we report the initial characterization of spcbfβ, the heterodimeric partner of sprunt- from the sea urchin stronylocentrotus purpuratus. we showed previously that morpholino antisense-mediated knockdown of sprunt- leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the runx target gene sppkc in contrast, we show here that knockdown of spcbfβ does not negatively impact cell survival or sppkc expression, although it does lead to differentiation defects similar to those associated with sprunt- deficiency. moreover, sprunt- containing a single amino acid substitution that abolishes its ability to interact with spcbfβ retains the ability to rescue cell survival in sprunt- morphant embryos. runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein core binding factor β. spcbfβ is remarkably similar to its mammalian homologues, and like them it enhances the dna binding of the runt domain. cbfβ allosterically enhances runx dna binding but does not bind dna itself.
 runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein core binding factor β. cbfβ allosterically enhances runx dna binding but does not bind dna itself. we report the initial characterization of spcbfβ, the heterodimeric partner of sprunt- from the sea urchin stronylocentrotus purpuratus.  spcbfβ is remarkably similar to its mammalian homologues, and like them it enhances the dna binding of the runt domain. spcbfβ is entirely of zygotic provenance and its expression is similar that of sprunt- accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. unlike sprunt- however, spcbfβ is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. we showed previously that morpholino antisense-mediated knockdown of sprunt- leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the runx target gene sppkc in contrast, we show here that knockdown of spcbfβ does not negatively impact cell survival or sppkc expression, although it does lead to differentiation defects similar to those associated with sprunt- deficiency. moreover, sprunt- containing a single amino acid substitution that abolishes its ability to interact with spcbfβ retains the ability to rescue cell survival in sprunt- morphant embryos. chromatin immunoprecipitation shows that while the cyiiia promoter engages both proteins, the sppkc promoter only engages sprunt-  spcbfβ is a facultative runx partner that appears to be required specifically for cell differentiation. 