BACKGROUND
p <dig> is a transcription factor -tf-homologous to the tumour suppressor p <dig> and to p <dig>  <cit> .
this class of proteins activate and repress genes as a result of binding to promoters and enhancer regions.
six isoforms can be found as a result of different transcription initiation sites and alternative splicing.
p <dig> proteins contain -ta- or lack -Δn- a transcriptional activation domain at the n-terminal, and the sterile alpha motif -sam- domain, presumably a protein-protein interaction module.
the resulting proteins have dissimilar transcriptional properties and, as a result, different biological behaviour.
the paramount importance of p <dig> in development has been broadly illustrated by genetic experiments in different organisms.
mice lacking p <dig> die soon after birth with severe defects in limb, craniofacial and skin development  <cit> .
the major isoform present in keratinocytes and other epithelia -Δnp63α- is essential for ectodermal development in zebrafish  <cit> .
in humans, several syndromes showing abnormalities in limbs, skin and annexes are caused by mutations in the p <dig> gene  <cit> .
in general, therefore, p <dig> is essential for the biology of multilayered epithelia.
several tfs have an established role in keratinocytes programs  <cit> , but few of them have so far been linked to p <dig> regulation.
this topic is quite relevant since renewal of stem cells and ongoing active terminal differentiation most likely requires the progressive fine tuning of transctriptional programs, unlikely to be masterminded by a single tf.
c/ebps are a family of six b-zip tfs that activate and repress transcription under different differentiation and growth arrest conditions  <cit> .
c/ebpα and c/ebpβ are required for differentiation of adipocytes  <cit> .
inactivation of c/ebpε in ko mice leads to lack of natural killer cells  <cit> .
c/ebpδ ko has a mammary phenotype, with an alteration in the involution of the mammary glands upon lactation  <cit> .
adipocyte differentiation is also impaired in cells that lack c/ebpδ when cultured in vitro, similarly to c/ebpβ  <cit> .
by inactivating p <dig> in hacat and primary cells, it was possible to investigate the roles of p <dig> in keratinocytes, notably the importance for differentiation and cell adhesion programmes  <cit> .
at the same time, however, p <dig> plays a major role in maintaining the proliferative potential of stem cells of the multilayered epithelia  <cit> .
through the use of rnai inactivation coupled to gene expression profiling, as well as chip on chip experiments, several labs have recently identified hundreds of p <dig> targets  <cit> .
specifically, c/ebpδ emerged in the rnai profiling of human hacat cells and primary keratinocytes as a target of p <dig>  <cit> .
this prompted us to validate it and characterize the p63-c/ebpδ connections by rt-pcr, chips and immunofluorescence in human keratinocytes.
RESULTS
validation of c/ebpδ as a target of p63
we performed rnai inactivation of p <dig> in human hacat cells followed by gene expression profiling with the affymetrix platform  <cit> ; one of the genes that was specifically increased under these conditions was c/ebpδ.
this gene was similarly found to be affected in primary keratinocytes in ref.
<cit> .
we confirmed this data by performing rt-pcr to examine the mrna levels of c/ebpδ, as well as two other members of the family.
upon p <dig> inactivation, a strong increase was specifically obeserved for c/ebpδ, but not for c/ebpα, while c/ebpβ showed a modest increase .
β-actin, an invariant mrna, was used to normalize samples.
Δnp <dig> was decreased, as expected .
the pattern was also seen after rnai inactivation of p <dig> in primary human keratinocytes .
in this setting, we also checked the protein levels of c/ebpδ by immunofluorescence: fig.
1b shows that under normal growth conditions, kcs are weakly but uniformly stained with anti-p <dig> and anti-c/ebpδ antibodies; removal of p <dig> by rnai transfections  lead to a substantial increase in the c/ebpδ staining .
to ascertain whether c/ebpδ repression is a primary event, we perfomed chip assays, using multiple anti-p <dig> and control  antibodies.
the anti-p <dig> antibody we produced and purified recognize all isoforms of p <dig> .
scanning the human c/ebpδ upstream region, we identified a cluster of potential p53/p <dig> binding sites around  <dig> kb upstream from the transcriptional start site.
these sites are in a cluster that is conserved with the mouse sequence .
chip analysis with hacat and primary kc chromatin showed strong positivity with the two p <dig> antibodies .
because hacat cells contain two p <dig> alleles mutated in the dna-binding domain  <cit> , hence generating proteins presumably incapable of dna-binding, only in primary cells did we include an antibody against p53: p <dig> was as positive as p <dig>  an indication that both proteins are bound to the c/ebpδ promoter .
as a control, we amplified a region of the human α-globin gene, which was devoid of any binding with any of the binding analyzed .
we conclude that c/ebpδ is under direct regulation of p <dig> in human keratinocytes.
c/ebpδ is regulated upon keratinocytes differentiation
the role of c/ebpδ in skin differentiation is unknown.
as a first step to shed light on this, we wished to know whether it is regulated during differentiation models of human keratinocytes.
hacat cells can be differentiated upon withdrawal of serum and calcium addition: the cells stop growing, alter their morphology, and express several markers of suprabasal keratinocytes.
we checked the levels of c/ebps by rt-pcr analysis at two times following the differentiation stimulus: early - <dig> hours- and late,  <dig> days.
the results are shown in fig.
2a, upper panels: the keratin  <dig> -ck1- marker was increased, a very modest increase of c/ebpδ, and no change of c/ebpβ or c/ebpα was scored; the invariant histone-like nf-yb was used as an internal control.
an increase in c/ebpδ protein level was seen in western blots  p <dig> was checked and showed a concomitant reduction.
protein levels were normalized with nf-yb and laminb.
immunofluorescence analysis confirmed that c/ebpδ is increased after differentiation, in contrast to c/ebpα and c/ebpβ .
ck <dig> was used to monitor differentiation .
similar experiments with kcs showed a greater increase of c/ebpδ at the mrna level in rt-pcr  and at the protein level in immunofluorescence .
mrna levels of ck <dig> decreased after differentiation, as expected .
we conclude from these data that c/ebpδ is subject to regulation during differentiation of hacat cells and primary human keratinocytes, suggesting that it might play a role in the process.
c/ebpδ is differentially expressed in human skin
differential expression in keratinocytes cultured in vitro suggests that c/ebpδ might be differentially expressed in the skin.
to verify whether this is the case, we evaluated human skin sections of healthy individuals by immunofluorescence confocal microscopy, double staining with p <dig> and c/ebpδ antibodies.
fig.
<dig> shows representative immunofluorescence images from two sections.
consistent with previous reports, p <dig> is strictly confined to nuclei of epidermis and abundant in the basal layer, with expression progressively fading in spinous cells and absent in the granular and corneum strata.
c/ebpδ staining is absent in the derma and confined to keratinocytes: it is nuclear and maximal in the granular layer and overlappping with p <dig> in the spinous layer .
interestingly, c/ebpδ also shows co-staining in some, but not all, cells of the basal compartment of interfollicular skin .
parallel staining with secondary anti-mouse and anti-rabbit antibodies were essentially negative, ensuring that the signals observed above are specific.
in conclusion, c/ebpδ is keratinocyte-restricted and clearly regulated in human skin, reinforcing the idea that it is involved in regulating genes during differentiation of keratinocytes.
furthermore, the expression patterns suggest that p <dig> and c/ebpδ may influence their reciprocal levels by transcriptionally regulating each other.
regulation of Δnp <dig> by c/ebpδ
to study the role of c/ebpδ on p <dig> expression, we overexpressed c/ebpδ in primary keratinocytes.
rt-pcr analysis of p <dig> isoforms is shown in fig.
4a .
Δnp63α, the only detectable isoform in mock transfected cells  <cit> , increases at  <dig> hours; surprisingly, Δnp63β and Δnp63γ were strongly induced at the same time point.
none of the ta isoforms were initially detectable, but they became apparent after  <dig> hours.
in parallel, we inactivated c/ebpδ by sirna interference in the same cellular setting, using three different oligonucleotides: fig.
4a  shows that using two of these sirnas the endogenous c/ebpδ drops to low levels in rt-pcr analysis; oligo  <dig> was then used for further analysis of the p <dig> isoforms upon c/ebpδ removal.
Δnp63α indeed decreased dramatically, while none of the other isoforms was expressed, confirming the role of c/ebpδ in Δn regulation.
these data indicate that the Δn -and ta- p <dig> promoters are potentially under control of c/ebpδ in keratinocytes.
to test this, we searched for c/ebp sites by using different algorithms in the human Δnp <dig> promoter, and found a consensus of two juxtaposed boxes at - <dig>  in a region that shows considerable homology in the mouse gene .
this led us to verify the activation of the Δnp <dig> promoter using luciferase constructs of different lengths, from - <dig> to - <dig> relative to the transcriptional start site.
as the promoter is known to be dependent from nf-y, a ubiquitous activator  <cit> , we included it in our cotransfections assays as a positive control.
these experiments were performed in u2os, which provide a background that is free of the possible dominant self-regulating activity of p <dig> on its own promoter  <cit> .
we found that nf-y and c/ebpδ singularly activated the - <dig> contruct .
remarkably, addition of the two tfs together yielded a clear cooperative effect.
however, shorter constructs  did not show any significant increase upon c/ebpδ overexpression, while still retaining some nf-y inducibility, most likely due to core promoter ccaat boxes detailed in refs.
<cit>  and  <cit> .
the decrease in nf-y inducibility with respect of the - <dig> construct is probably due to a functional ccaat box binding nf-y in this region .
the simplest explanation for these results is that the - <dig> c/ebp sites are vital for induction.
to confirm that the activation observed is a primary phenomenon, we performed scanning chips analysis on different regions of the c/ebpδ promoter in human kcs; fig.
4d shows that c/ebpδ and nf-y are found in the upstream region, in at least three different positions.
note that predicted and actual sites do not always ovelap .
taken together, these data indicate that  c/ebpδ binds and activates the Δnp <dig> promoter in keratinocytes,  point at the - <dig> binding sites as important for regulation,  confirm that nf-y is a bona fide activator of the Δnp <dig> promoter.
consequences of c/ebpδ overexpression/inactivation on expression of p <dig> targets
the finding that c/ebpδ regulates p <dig> prompted us to ascertain whether it would affect the expression of some of p <dig> targets recently identified, in overexpression and rnai inactivation experiments in human primary keratinocytes .
we also analyzed by rt-pcr markers of differentiation, such as ck <dig> and ck <dig>  and desmocollin   <dig> and  <dig>  important for keratinocyte biology and presumptive c/ebpδ targets  <cit> .
c/ebpδ overexpression lead to variations in all genes tested, with the exception of ck <dig>  pcna, a marker of cell proliferation and itself a p <dig> target  <cit>  decreased modestly after  <dig> hours, suggesting a slowdown, but not a stop in proliferation.
dsc <dig>  but not dsc <dig>  was activated by c/ebpδ overexpression.
essentially all p <dig> targets identified in our recent screenings varied in expression, some decreasing, others increasing.
interestingly, some of these genes showed a response at  <dig> hours, others -egf-r, c-jun, e- and t-cadherin- a delayed one.
rt-pcr analysis of keratinocytes in which c/ebpδ was inactivated showed an equally dramatic change in gene expression.
in most cases, there is a perfect match with overexpression: genes that are activated,  <dig> or  <dig> hours after c/ebpδ transfections are down-regulated by rnai, consistent with a positive role of c/ebpδ in their regulation.
the exception is esr, whose undetectable levels could not be further decreased by c/ebpδ inactivation.
the genes that are repressed in overexpression are increased by rnai, except egf-r and desmocollin  <dig>  in which a dominant role of p <dig> can be envisaged.
in summary, the c/ebpδ rnai and overexpression experiments are consistent with this tf being important for the expression of many p <dig> targets.
p <dig> targets are bound by c/ebpδ in vivo
to verify whether the effect of c/ebpδ on expression of the p <dig> targets analyzed above is a primary event, we performed chip analysis with an anti-c/ebpδ antibody in primary kcs and in hacat cells.
the regions analyzed were the same that showed p <dig> binding .
essentially all targets are bound by c/ebpδ, both in hacat and in primary kcs, the only exception being zeb <dig>  except c-jun and etar, these regions contain one or more c/ebp consensus sequences, as defined by analyzing the common sites derived by the use of the consite, rvista and custom algorithms used in fig.
4; some are in core promoters, others in upstream locations.
the pattern of p <dig> is somewhat similar, with one target -etar- absent in primary kcs.
nf-y sites are found in expected locations, except for e-cadherin, which was shown to be a positive promoter for nf-y.
note that nf-y binding on c/ebpδ in primary keratinocytes differs in fig.
<dig>  and  <dig> .
individual variations might account for these discrepancies, as we have also noticed differences in positivity in p <dig> among keratinocytes derived from individual donors with respect of some of the targets .
these exceptions notwithstanding, we conclude that the majority of p <dig> targets are bound by c/ebpδ in vivo in primary and immortalized keratinocytes.
c/ebpδ-p <dig> targets are regulated during differentiation
the reciprocal roles of p <dig> and c/ebpδ for keratinocyte differentiation suggests that coregulated genes would be differentially regulated during this process.
we checked their expression levels by rt-pcr analysis in primary keratinocytes induced to differentiate .
the process was monitored as in fig.
<dig> with ck <dig> and ck <dig> -the former increasing, the latter decreasing- and normalized with gapdh and nf-yb rnas.
all genes showed remarkable changes.
in particular, the coregulated junb and c-jun varied, while jund did not.
in keeping with the increase in c/ebpδ levels after differentiation, activated genes -tgfβ-rii, t-cad, junb- showed a positive variation, irrespective of p <dig> expression .
other genes behaved dissimilarly with respect to c/ebpδ: e-cadherin and egf-r, which are repressed, increased considerably upon differentiation.
we conclude that all coregulated targets tested change during differentiation, but this complex process, as in the case of p <dig>  cannot be fully recapitulated through variations generated by a single tf.
discussion
the concerted modulation of specific gene expression programs is controlled by transcription factors.
in specific pathways of response to external stimuli, one, or a few, tfs play a dominant role in this response.
matters are far more complex in multi-cellular systems involving terminal differentiation and the specification of exquisite features.
what emerged is the concept of "master" regulators, whose role is to confer cell identity and to drive gene regulation programs accordingly.
in the muscle system, for example, some of these masters -myod, myogenin, mef2- were identified and their gene regulation programs studied: they apparently show a preference for fellow tfs genes.
a robust hierarchical cascade of specific programs is adjusted in such multisteps and multi-functions programs  <cit> .
interestingly, this network is not restricted to tfs exquisitely expressed in muscle, since ubiquitous, or near ubiquitous tfs are an integral part of the network  <cit> .
similarly, the recent identification of hundreds of p <dig> targets in various cell types  highlighted links with tfs that are known, or likely, to play an important role in skin biology  <cit> .
the direct cross-talk between p <dig> and c/ebpδ, originally emerging from p <dig> rnai screenings  <cit>  is confirmed by chip and c/ebpδ rnai experiments.
the decrease in Δnp63α by inactivation of c/ebpδ strongly argues in favour of an activating role of c/ebpδ on Δnp <dig> transcription, confirmed by overexpression; reporter constructs experiments and chips point at important upstream sites at - <dig>  kb.
interestingly, the synergistic activation observed with nf-y is a further argument in favour of the physiologic role of this particular member of the c/ebp family in Δn p <dig> regulation: nf-y, itself a poor activator, acts primarily in conjunction with a neighbouring tf .
in the mhc class ii promoters, for example, only the relevant rfx- <dig>  and not other rfxs, is capable to synergize with nf-y  <cit> .
an intriguing finding is the differential regulation of p <dig> splicing isoforms in keratinocytes upon c/ebpδ overexpression.
normally, these cells express exclusively Δnp63α and it is currently unclear whether ta isoforms are induced upon differentiation in vitro, since different results were reported .
overexpression of c/ebpδ led to the appearance of Δnβ and γ, and -later- of the ta isoforms.
thus, c/ebpδ acts not only to support the activity of the Δn promoter in general, but, directly or indirectly, to impinge on an isoform switch that modifies the p <dig> portfolio.
Δnp63β and γ induction was also observed upon activation and differentiation of cells of the corneal limbus  <cit> , and a role of ta isofoms in differentiation has been proposed  <cit> .
the switch could have a far reaching effect on expression of p <dig> targes, particularly in the upper epidermal layers, since the Δn and ta isoforms have often quite dissimilar transcriptional effects.
how modification of the splicing is accomplished is unclear, at the moment; it has become recently evident, however, that regulation of transcription and rna processing are highly coordinated events and that specific transcription factors -and cofactors- are known to play a role in pre-mrna processing  <cit> .
interestingly, one of the cofactors involved is carm <dig>  <cit> , an arginine methyltransferase which modifies other factors promoting exon-skipping  <cit> .
carm <dig> is activated by Δnp63α  <cit>  and it was shown to be involved in other differentiation systems  <cit> .
thus, it is possible that p <dig> regulators, such as c/ebpδ, impact on factors loaded on the Δn promoter and travel along the gene with polii complexes to adjust splicing events.
our results indicate that many, perhaps most of the p <dig> targets are coregulated by c/ebpδ.
an intersection between c/ebps and p <dig> family members was noticed before.
typical p53-induced pro-apoptotic targets are not activated in the absence of c/ebpδ in ko mammary cells and anti-apoptotic genes are not repressed  <cit> .
the two factors are apparently engaged in a positive feed-back loop, with many genes commonly controlled: cyclin d <dig>  bfl <dig>  sgp <dig>  gas <dig>  bak and igfbp <dig>  in addition, expression of c/ebpδ is induced upon vitamin d treatment in prostate cells, leading to a dramatic arrest in clonal expansion  <cit> , presumably through vdr, another p <dig> target  <cit> .
given the pivotal role of p <dig> in prostate development  <cit> , the interplay between p <dig> and c/ebpδ could be both direct and indirect, through vdr.
note that the overlap between p <dig> and p <dig> targets in the skin is large, but not absolute  <cit> .
hacat cells are an epithelial line derived from the back of adult human skin that spontaneusly underwent immortalization in vitro  <cit> ; they exhibit normal differentiation and have provided a valuable tool for studying regulation of keratinization in human cells.
the interpretation of data concerning p <dig> in hacat is often complicated by the presence of two mutated p <dig> alleles  <cit> , which are known to impinge on the p <dig> pathways.
this is apparently ot the case for c/ebpδ, but for other p <dig> targets we are analyzing, there are dramatic differences in regulation between hacat and kcs .
in light of the overlap between the targets of the two tfs, this implies that a mutated p <dig> might subvert the tumor suppressive and pro-differentiation role of c/ebpδ.
based on current knowledge derived mostly from rnai experiments, a tentative view of the influence of p <dig> and c/ebpδ is presented in fig.
<dig>  the picture is complicated by the effect of p <dig> inactivation through c/ebpδ rnai and by the differential role of tap <dig>  <cit>  activated at late time points.
with two exceptions, egf-r and dsc <dig>  all genes show a consistent behaviour in overexpression and rnai.
genes that are repressed by p <dig>  etar, aim <dig>  t-cadherin, junb, and should be up-regulated by p <dig> decrease, are rather decreased, an indication that they are mostly rely on c/ebpδ.
some of these -aim <dig>  t-cadherin, junb- limit growth.
a role of c/ebpδ as a tumor suppressor was established by the finding that knock-out mefs show genomic instability, impaired contact inhibition and reduced serum dependance  <cit> .
for the p63-repressed c-jun, up-regulation by c/ebpδ could either be because of removal of p <dig> or repression by c/ebpδ, as indicated by overexpression at late time points.
as for genes activated by Δnp <dig>  such as egf-r, tgfβ-rii and e-cadherin, the latters could be under joint control; egf-r, which drops after c/ebpδ and p <dig> rnai, could decrease after c/ebpδ overexpression at  <dig> hours through the increase of tap <dig> isoforms, shown to be strong repressors of the egf-r promoter.
this would be consistent with a growth control and pro-differentiative role of both tap <dig> and c/ebpδ.
similarly, removal of the repressive Δnp <dig>  by c/ebpδ rnai is consistent with an increase in pcna mrna and with a small, late decrease in overexpression.
in conclusion, it is clear that coregulation of p63/p <dig> and c/ebpδ is part of a larger program that controls cell-cycle progression and differentiation.
to reconstruct the network, it will now be necessary to obtain unbiased information about the panel of c/ebpδ targets in keratinocytes, through the use of rnai and chip on chip technology.
c/ebpδ belongs to a family of tfs important for several differentiation pathways, including adipocytes, liver and haematopoietic lineages  <cit> .
specifically, it has been associated to the early phases of adipocyte differentiation, together with c/ebpβ.
it is induced upon several environmental changes that lead to growth arrest  <cit>  and serum withdrawal in mammary cells and lung epithelia cells  <cit> .
overexpression leads to growth arrest in mammary and prostate epithelial cell lines  <cit> .
the growth arrest features are common to other c/ebps .
our findings that c/ebpδ is up-regulated during cell-cycle exit and differentiation of immortalized hacat and primary keratinocytes, and the decrease in expression of pcna, a proliferative marker, are in agreement with these results.
nothing is known about c/ebpδ expression during epidermal development of mouse skin.
despite careful examination, the available antibodies are unable to stain mouse cells in immunofluorescence analysis .
in humans, little variation of expression of c/ebpδ was detected in the different phases of maturation of skin annexes, the hair follicle and the sebaceous gland, unlike other members of the family  <cit> .
at the opposite, we observe here that c/ebpδ staining is not uniform in human interfollicular skin, suggesting that the reciprocal interplay with p <dig> is complex.
co-expression is seen in selected basal cells, which is in line with the activating role of c/ebpδ on the Δn promoter; in other cells of the same layer, strongly positive for p <dig>  c/ebpδ fades: this is also consistent with the negative role of p <dig> on c/ebpδ expression.
there is a coexpression and a balance in the spinous layer, and in the upper granular layer, with cells exiting from a proliferation status and terminally differentiating, c/ebpδ is prominent.
in transfection assays, desmocollin  <dig>  which is expressed in basal cells, is transactivated by c/ebpδ and c/ebpβ, the suprabasal desmocollin  <dig> by c/ebpδ and c/ebpα  <cit> .
we confirm that both these genes require c/ebpδ expression, as determined by rnai experiments.
thus our results are consistent with c/ebpδ playing a dual role in skin differentiation, both in very early and late stages.
in the former, it could limit, through its growth suppressive properties the growth potential of early progenitors.
in the later stages, it may coordinate cell-cycle exit and induce differentiation markers.
our observations could now be extended in other systems, notably in the mammary gland and in prostate, in which both tfs have been singularly been shown to play key roles.
one caveat to the c/ebpδ role in skin is the fact that ko mice have no apparent alterations.
however, this might be due to redundancy with c/ebpβ: it was reported that c/ebpβ is expressed in the cytoplasm of basal keratinocytes and becomes nuclear in the spinous layer  <cit> .
c/ebpβ ko mice have skin hyperplasia with downplay of keratin  <dig> and  <dig> expression  <cit>  and c/ebpβ is important for keratinocyte survival  <cit> .
of particular significance are the data recently reported by the group of vinson on a transgenic model expressing a dominant negative c/ebp -termed a-c/ebp- in basal keratinocytes: these mice have hyperplasia of the basal epidermis and increased apoptosis of the upper layers  <cit> .
p <dig> and pro-apoptotic markers are induced and c/ebpβ dramatically reduced; as a-c/ebp is not c/ebpβ-specific, c/ebpδ might contribute to the observed phenotype.
given the well known complexity within this family, through homo- and heterodimerizations, production of dominant negatives through the use of internal aug within c/ebpβ, much work lies ahead to establish their role in the different layers of epidermis.
CONCLUSIONS
we identified a direct and mutual link between p <dig> and a member of the c/ebp family of transcription factors, c/ebpδ.
the following relevant findings are the reported:  c/ebpδ expression is confined to keratinocytes and it is up-regulated in cells committed to differentiation; it is increased in in vitro differentiation systems, both of hacat and primary keratinocytes.
c/ebpδ activates transcription of the Δnp <dig> promoter, and it appears to be part of the mechanisms that control p <dig> splicing.
it is involved in regulation of other p <dig> targets regulated during differentiation, as verified by rnai, overexpression and chip assays.
we therefore suggest that the mutual link between these tfs is important for the correct differentiation of keratinocytes.

 p is a transcription factor -tf-homologous to the tumour suppressor p and to p. this class of proteins activate and repress genes as a result of binding to promoters and enhancer regions. six isoforms can be found as a result of different transcription initiation sites and alternative splicing. p proteins contain -ta- or lack -Δn- a transcriptional activation domain at the n-terminal, and the sterile alpha motif -sam- domain, presumably a protein-protein interaction module. the resulting proteins have dissimilar transcriptional properties and, as a result, different biological behaviour. the paramount importance of p in development has been broadly illustrated by genetic experiments in different organisms. mice lacking p die soon after birth with severe defects in limb, craniofacial and skin development. the major isoform present in keratinocytes and other epithelia -Δnp63α- is essential for ectodermal development in zebrafish. in humans, several syndromes showing abnormalities in limbs, skin and annexes are caused by mutations in the p gene. in general, therefore, p is essential for the biology of multilayered epithelia. several tfs have an established role in keratinocytes programs, but few of them have so far been linked to p regulation. this topic is quite relevant since renewal of stem cells and ongoing active terminal differentiation most likely requires the progressive fine tuning of transctriptional programs, unlikely to be masterminded by a single tf. c/ebps are a family of six b-zip tfs that activate and repress transcription under different differentiation and growth arrest conditions. c/ebpα and c/ebpβ are required for differentiation of adipocytes. inactivation of c/ebpε in ko mice leads to lack of natural killer cells. c/ebpδ ko has a mammary phenotype, with an alteration in the involution of the mammary glands upon lactation. adipocyte differentiation is also impaired in cells that lack c/ebpδ when cultured in vitro, similarly to c/ebpβ. by inactivating p in hacat and primary cells, it was possible to investigate the roles of p in keratinocytes, notably the importance for differentiation and cell adhesion programmes. at the same time, however, p plays a major role in maintaining the proliferative potential of stem cells of the multilayered epithelia. through the use of rnai inactivation coupled to gene expression profiling, as well as chip on chip experiments, several labs have recently identified hundreds of p targets. specifically, c/ebpδ emerged in the rnai profiling of human hacat cells and primary keratinocytes as a target of p. this prompted us to validate it and characterize the p63-c/ebpδ connections by rt-pcr, chips and immunofluorescence in human keratinocytes.  validation of c/ebpδ as a target of p63 we performed rnai inactivation of p in human hacat cells followed by gene expression profiling with the affymetrix platform; one of the genes that was specifically increased under these conditions was c/ebpδ. this gene was similarly found to be affected in primary keratinocytes in ref.. we confirmed this data by performing rt-pcr to examine the mrna levels of c/ebpδ, as well as two other members of the family. upon p inactivation, a strong increase was specifically obeserved for c/ebpδ, but not for c/ebpα, while c/ebpβ showed a modest increase. β-actin, an invariant mrna, was used to normalize samples. Δnp was decreased, as expected. the pattern was also seen after rnai inactivation of p in primary human keratinocytes. in this setting, we also checked the protein levels of c/ebpδ by immunofluorescence: fig. 1b shows that under normal growth conditions, kcs are weakly but uniformly stained with anti-p and anti-c/ebpδ antibodies; removal of p by rnai transfections lead to a substantial increase in the c/ebpδ staining. to ascertain whether c/ebpδ repression is a primary event, we perfomed chip assays, using multiple anti-p and control antibodies. the anti-p antibody we produced and purified recognize all isoforms of p. scanning the human c/ebpδ upstream region, we identified a cluster of potential p53/p binding sites around kb upstream from the transcriptional start site. these sites are in a cluster that is conserved with the mouse sequence. chip analysis with hacat and primary kc chromatin showed strong positivity with the two p antibodies. because hacat cells contain two p alleles mutated in the dna-binding domain, hence generating proteins presumably incapable of dna-binding, only in primary cells did we include an antibody against p53: p was as positive as p an indication that both proteins are bound to the c/ebpδ promoter. as a control, we amplified a region of the human α-globin gene, which was devoid of any binding with any of the binding analyzed. we conclude that c/ebpδ is under direct regulation of p in human keratinocytes. c/ebpδ is regulated upon keratinocytes differentiation the role of c/ebpδ in skin differentiation is unknown. as a first step to shed light on this, we wished to know whether it is regulated during differentiation models of human keratinocytes. hacat cells can be differentiated upon withdrawal of serum and calcium addition: the cells stop growing, alter their morphology, and express several markers of suprabasal keratinocytes. we checked the levels of c/ebps by rt-pcr analysis at two times following the differentiation stimulus: early - hours- and late, days. the results are shown in fig. 2a, upper panels: the keratin -ck1- marker was increased, a very modest increase of c/ebpδ, and no change of c/ebpβ or c/ebpα was scored; the invariant histone-like nf-yb was used as an internal control. an increase in c/ebpδ protein level was seen in western blots p was checked and showed a concomitant reduction. protein levels were normalized with nf-yb and laminb. immunofluorescence analysis confirmed that c/ebpδ is increased after differentiation, in contrast to c/ebpα and c/ebpβ. ck was used to monitor differentiation. similar experiments with kcs showed a greater increase of c/ebpδ at the mrna level in rt-pcr and at the protein level in immunofluorescence. mrna levels of ck decreased after differentiation, as expected. we conclude from these data that c/ebpδ is subject to regulation during differentiation of hacat cells and primary human keratinocytes, suggesting that it might play a role in the process. c/ebpδ is differentially expressed in human skin differential expression in keratinocytes cultured in vitro suggests that c/ebpδ might be differentially expressed in the skin. to verify whether this is the case, we evaluated human skin sections of healthy individuals by immunofluorescence confocal microscopy, double staining with p and c/ebpδ antibodies. fig. shows representative immunofluorescence images from two sections. consistent with previous reports, p is strictly confined to nuclei of epidermis and abundant in the basal layer, with expression progressively fading in spinous cells and absent in the granular and corneum strata. c/ebpδ staining is absent in the derma and confined to keratinocytes: it is nuclear and maximal in the granular layer and overlappping with p in the spinous layer. interestingly, c/ebpδ also shows co-staining in some, but not all, cells of the basal compartment of interfollicular skin. parallel staining with secondary anti-mouse and anti-rabbit antibodies were essentially negative, ensuring that the signals observed above are specific. in conclusion, c/ebpδ is keratinocyte-restricted and clearly regulated in human skin, reinforcing the idea that it is involved in regulating genes during differentiation of keratinocytes. furthermore, the expression patterns suggest that p and c/ebpδ may influence their reciprocal levels by transcriptionally regulating each other. regulation of Δnp by c/ebpδ to study the role of c/ebpδ on p expression, we overexpressed c/ebpδ in primary keratinocytes. rt-pcr analysis of p isoforms is shown in fig. 4a. Δnp63α, the only detectable isoform in mock transfected cells, increases at hours; surprisingly, Δnp63β and Δnp63γ were strongly induced at the same time point. none of the ta isoforms were initially detectable, but they became apparent after hours. in parallel, we inactivated c/ebpδ by sirna interference in the same cellular setting, using three different oligonucleotides: fig. 4a shows that using two of these sirnas the endogenous c/ebpδ drops to low levels in rt-pcr analysis; oligo was then used for further analysis of the p isoforms upon c/ebpδ removal. Δnp63α indeed decreased dramatically, while none of the other isoforms was expressed, confirming the role of c/ebpδ in Δn regulation. these data indicate that the Δn -and ta- p promoters are potentially under control of c/ebpδ in keratinocytes. to test this, we searched for c/ebp sites by using different algorithms in the human Δnp promoter, and found a consensus of two juxtaposed boxes at - in a region that shows considerable homology in the mouse gene. this led us to verify the activation of the Δnp promoter using luciferase constructs of different lengths, from - to - relative to the transcriptional start site. as the promoter is known to be dependent from nf-y, a ubiquitous activator, we included it in our cotransfections assays as a positive control. these experiments were performed in u2os, which provide a background that is free of the possible dominant self-regulating activity of p on its own promoter. we found that nf-y and c/ebpδ singularly activated the - contruct. remarkably, addition of the two tfs together yielded a clear cooperative effect. however, shorter constructs did not show any significant increase upon c/ebpδ overexpression, while still retaining some nf-y inducibility, most likely due to core promoter ccaat boxes detailed in refs. and. the decrease in nf-y inducibility with respect of the - construct is probably due to a functional ccaat box binding nf-y in this region. the simplest explanation for these results is that the - c/ebp sites are vital for induction. to confirm that the activation observed is a primary phenomenon, we performed scanning chips analysis on different regions of the c/ebpδ promoter in human kcs; fig. 4d shows that c/ebpδ and nf-y are found in the upstream region, in at least three different positions. note that predicted and actual sites do not always ovelap. taken together, these data indicate that c/ebpδ binds and activates the Δnp promoter in keratinocytes, point at the - binding sites as important for regulation, confirm that nf-y is a bona fide activator of the Δnp promoter. consequences of c/ebpδ overexpression/inactivation on expression of p targets the finding that c/ebpδ regulates p prompted us to ascertain whether it would affect the expression of some of p targets recently identified, in overexpression and rnai inactivation experiments in human primary keratinocytes. we also analyzed by rt-pcr markers of differentiation, such as ck and ck and desmocollin and important for keratinocyte biology and presumptive c/ebpδ targets. c/ebpδ overexpression lead to variations in all genes tested, with the exception of ck pcna, a marker of cell proliferation and itself a p target decreased modestly after hours, suggesting a slowdown, but not a stop in proliferation. dsc but not dsc was activated by c/ebpδ overexpression. essentially all p targets identified in our recent screenings varied in expression, some decreasing, others increasing. interestingly, some of these genes showed a response at hours, others -egf-r, c-jun, e- and t-cadherin- a delayed one. rt-pcr analysis of keratinocytes in which c/ebpδ was inactivated showed an equally dramatic change in gene expression. in most cases, there is a perfect match with overexpression: genes that are activated, or hours after c/ebpδ transfections are down-regulated by rnai, consistent with a positive role of c/ebpδ in their regulation. the exception is esr, whose undetectable levels could not be further decreased by c/ebpδ inactivation. the genes that are repressed in overexpression are increased by rnai, except egf-r and desmocollin in which a dominant role of p can be envisaged. in summary, the c/ebpδ rnai and overexpression experiments are consistent with this tf being important for the expression of many p targets. p targets are bound by c/ebpδ in vivo to verify whether the effect of c/ebpδ on expression of the p targets analyzed above is a primary event, we performed chip analysis with an anti-c/ebpδ antibody in primary kcs and in hacat cells. the regions analyzed were the same that showed p binding. essentially all targets are bound by c/ebpδ, both in hacat and in primary kcs, the only exception being zeb except c-jun and etar, these regions contain one or more c/ebp consensus sequences, as defined by analyzing the common sites derived by the use of the consite, rvista and custom algorithms used in fig. 4; some are in core promoters, others in upstream locations. the pattern of p is somewhat similar, with one target -etar- absent in primary kcs. nf-y sites are found in expected locations, except for e-cadherin, which was shown to be a positive promoter for nf-y. note that nf-y binding on c/ebpδ in primary keratinocytes differs in fig. and. individual variations might account for these discrepancies, as we have also noticed differences in positivity in p among keratinocytes derived from individual donors with respect of some of the targets. these exceptions notwithstanding, we conclude that the majority of p targets are bound by c/ebpδ in vivo in primary and immortalized keratinocytes. c/ebpδ-p targets are regulated during differentiation the reciprocal roles of p and c/ebpδ for keratinocyte differentiation suggests that coregulated genes would be differentially regulated during this process. we checked their expression levels by rt-pcr analysis in primary keratinocytes induced to differentiate. the process was monitored as in fig. with ck and ck -the former increasing, the latter decreasing- and normalized with gapdh and nf-yb rnas. all genes showed remarkable changes. in particular, the coregulated junb and c-jun varied, while jund did not. in keeping with the increase in c/ebpδ levels after differentiation, activated genes -tgfβ-rii, t-cad, junb- showed a positive variation, irrespective of p expression. other genes behaved dissimilarly with respect to c/ebpδ: e-cadherin and egf-r, which are repressed, increased considerably upon differentiation. we conclude that all coregulated targets tested change during differentiation, but this complex process, as in the case of p cannot be fully recapitulated through variations generated by a single tf. discussion the concerted modulation of specific gene expression programs is controlled by transcription factors. in specific pathways of response to external stimuli, one, or a few, tfs play a dominant role in this response. matters are far more complex in multi-cellular systems involving terminal differentiation and the specification of exquisite features. what emerged is the concept of "master" regulators, whose role is to confer cell identity and to drive gene regulation programs accordingly. in the muscle system, for example, some of these masters -myod, myogenin, mef2- were identified and their gene regulation programs studied: they apparently show a preference for fellow tfs genes. a robust hierarchical cascade of specific programs is adjusted in such multisteps and multi-functions programs. interestingly, this network is not restricted to tfs exquisitely expressed in muscle, since ubiquitous, or near ubiquitous tfs are an integral part of the network. similarly, the recent identification of hundreds of p targets in various cell types highlighted links with tfs that are known, or likely, to play an important role in skin biology. the direct cross-talk between p and c/ebpδ, originally emerging from p rnai screenings is confirmed by chip and c/ebpδ rnai experiments. the decrease in Δnp63α by inactivation of c/ebpδ strongly argues in favour of an activating role of c/ebpδ on Δnp transcription, confirmed by overexpression; reporter constructs experiments and chips point at important upstream sites at - kb. interestingly, the synergistic activation observed with nf-y is a further argument in favour of the physiologic role of this particular member of the c/ebp family in Δn p regulation: nf-y, itself a poor activator, acts primarily in conjunction with a neighbouring tf. in the mhc class ii promoters, for example, only the relevant rfx- and not other rfxs, is capable to synergize with nf-y. an intriguing finding is the differential regulation of p splicing isoforms in keratinocytes upon c/ebpδ overexpression. normally, these cells express exclusively Δnp63α and it is currently unclear whether ta isoforms are induced upon differentiation in vitro, since different results were reported. overexpression of c/ebpδ led to the appearance of Δnβ and γ, and -later- of the ta isoforms. thus, c/ebpδ acts not only to support the activity of the Δn promoter in general, but, directly or indirectly, to impinge on an isoform switch that modifies the p portfolio. Δnp63β and γ induction was also observed upon activation and differentiation of cells of the corneal limbus, and a role of ta isofoms in differentiation has been proposed. the switch could have a far reaching effect on expression of p targes, particularly in the upper epidermal layers, since the Δn and ta isoforms have often quite dissimilar transcriptional effects. how modification of the splicing is accomplished is unclear, at the moment; it has become recently evident, however, that regulation of transcription and rna processing are highly coordinated events and that specific transcription factors -and cofactors- are known to play a role in pre-mrna processing. interestingly, one of the cofactors involved is carm, an arginine methyltransferase which modifies other factors promoting exon-skipping. carm is activated by Δnp63α and it was shown to be involved in other differentiation systems. thus, it is possible that p regulators, such as c/ebpδ, impact on factors loaded on the Δn promoter and travel along the gene with polii complexes to adjust splicing events. our results indicate that many, perhaps most of the p targets are coregulated by c/ebpδ. an intersection between c/ebps and p family members was noticed before. typical p53-induced pro-apoptotic targets are not activated in the absence of c/ebpδ in ko mammary cells and anti-apoptotic genes are not repressed. the two factors are apparently engaged in a positive feed-back loop, with many genes commonly controlled: cyclin d bfl sgp gas bak and igfbp in addition, expression of c/ebpδ is induced upon vitamin d treatment in prostate cells, leading to a dramatic arrest in clonal expansion, presumably through vdr, another p target. given the pivotal role of p in prostate development, the interplay between p and c/ebpδ could be both direct and indirect, through vdr. note that the overlap between p and p targets in the skin is large, but not absolute. hacat cells are an epithelial line derived from the back of adult human skin that spontaneusly underwent immortalization in vitro; they exhibit normal differentiation and have provided a valuable tool for studying regulation of keratinization in human cells. the interpretation of data concerning p in hacat is often complicated by the presence of two mutated p alleles, which are known to impinge on the p pathways. this is apparently ot the case for c/ebpδ, but for other p targets we are analyzing, there are dramatic differences in regulation between hacat and kcs. in light of the overlap between the targets of the two tfs, this implies that a mutated p might subvert the tumor suppressive and pro-differentiation role of c/ebpδ. based on current knowledge derived mostly from rnai experiments, a tentative view of the influence of p and c/ebpδ is presented in fig. the picture is complicated by the effect of p inactivation through c/ebpδ rnai and by the differential role of tap activated at late time points. with two exceptions, egf-r and dsc all genes show a consistent behaviour in overexpression and rnai. genes that are repressed by p etar, aim t-cadherin, junb, and should be up-regulated by p decrease, are rather decreased, an indication that they are mostly rely on c/ebpδ. some of these -aim t-cadherin, junb- limit growth. a role of c/ebpδ as a tumor suppressor was established by the finding that knock-out mefs show genomic instability, impaired contact inhibition and reduced serum dependance. for the p63-repressed c-jun, up-regulation by c/ebpδ could either be because of removal of p or repression by c/ebpδ, as indicated by overexpression at late time points. as for genes activated by Δnp such as egf-r, tgfβ-rii and e-cadherin, the latters could be under joint control; egf-r, which drops after c/ebpδ and p rnai, could decrease after c/ebpδ overexpression at hours through the increase of tap isoforms, shown to be strong repressors of the egf-r promoter. this would be consistent with a growth control and pro-differentiative role of both tap and c/ebpδ. similarly, removal of the repressive Δnp by c/ebpδ rnai is consistent with an increase in pcna mrna and with a small, late decrease in overexpression. in conclusion, it is clear that coregulation of p63/p and c/ebpδ is part of a larger program that controls cell-cycle progression and differentiation. to reconstruct the network, it will now be necessary to obtain unbiased information about the panel of c/ebpδ targets in keratinocytes, through the use of rnai and chip on chip technology. c/ebpδ belongs to a family of tfs important for several differentiation pathways, including adipocytes, liver and haematopoietic lineages. specifically, it has been associated to the early phases of adipocyte differentiation, together with c/ebpβ. it is induced upon several environmental changes that lead to growth arrest and serum withdrawal in mammary cells and lung epithelia cells. overexpression leads to growth arrest in mammary and prostate epithelial cell lines. the growth arrest features are common to other c/ebps. our findings that c/ebpδ is up-regulated during cell-cycle exit and differentiation of immortalized hacat and primary keratinocytes, and the decrease in expression of pcna, a proliferative marker, are in agreement with these results. nothing is known about c/ebpδ expression during epidermal development of mouse skin. despite careful examination, the available antibodies are unable to stain mouse cells in immunofluorescence analysis. in humans, little variation of expression of c/ebpδ was detected in the different phases of maturation of skin annexes, the hair follicle and the sebaceous gland, unlike other members of the family. at the opposite, we observe here that c/ebpδ staining is not uniform in human interfollicular skin, suggesting that the reciprocal interplay with p is complex. co-expression is seen in selected basal cells, which is in line with the activating role of c/ebpδ on the Δn promoter; in other cells of the same layer, strongly positive for p c/ebpδ fades: this is also consistent with the negative role of p on c/ebpδ expression. there is a coexpression and a balance in the spinous layer, and in the upper granular layer, with cells exiting from a proliferation status and terminally differentiating, c/ebpδ is prominent. in transfection assays, desmocollin which is expressed in basal cells, is transactivated by c/ebpδ and c/ebpβ, the suprabasal desmocollin by c/ebpδ and c/ebpα. we confirm that both these genes require c/ebpδ expression, as determined by rnai experiments. thus our results are consistent with c/ebpδ playing a dual role in skin differentiation, both in very early and late stages. in the former, it could limit, through its growth suppressive properties the growth potential of early progenitors. in the later stages, it may coordinate cell-cycle exit and induce differentiation markers. our observations could now be extended in other systems, notably in the mammary gland and in prostate, in which both tfs have been singularly been shown to play key roles. one caveat to the c/ebpδ role in skin is the fact that ko mice have no apparent alterations. however, this might be due to redundancy with c/ebpβ: it was reported that c/ebpβ is expressed in the cytoplasm of basal keratinocytes and becomes nuclear in the spinous layer. c/ebpβ ko mice have skin hyperplasia with downplay of keratin and expression and c/ebpβ is important for keratinocyte survival. of particular significance are the data recently reported by the group of vinson on a transgenic model expressing a dominant negative c/ebp -termed a-c/ebp- in basal keratinocytes: these mice have hyperplasia of the basal epidermis and increased apoptosis of the upper layers. p and pro-apoptotic markers are induced and c/ebpβ dramatically reduced; as a-c/ebp is not c/ebpβ-specific, c/ebpδ might contribute to the observed phenotype. given the well known complexity within this family, through homo- and heterodimerizations, production of dominant negatives through the use of internal aug within c/ebpβ, much work lies ahead to establish their role in the different layers of epidermis.  we identified a direct and mutual link between p and a member of the c/ebp family of transcription factors, c/ebpδ. the following relevant findings are the reported: c/ebpδ expression is confined to keratinocytes and it is up-regulated in cells committed to differentiation; it is increased in in vitro differentiation systems, both of hacat and primary keratinocytes. c/ebpδ activates transcription of the Δnp promoter, and it appears to be part of the mechanisms that control p splicing. it is involved in regulation of other p targets regulated during differentiation, as verified by rnai, overexpression and chip assays. we therefore suggest that the mutual link between these tfs is important for the correct differentiation of keratinocytes. 
c/ebpδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of hacat and primary keratinocytes. c/ebpδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p we validated c/ebpδ as a primary target of Δnp63α by rt-pcr and chip location analysis in hacat and primary cells. overexpression of c/ebpδ leads to alteration in the normal profile of p isoforms, with the emergence of Δnp63β and γ, and of the ta isoforms, with different kinetics. finally, c/ebpδ is found on the p targets in vivo by chip analysis, indicating that coregulation is direct. our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets. genetic experiments have clarified that p is a key transcription factor governing the establishment and maintenance of multilayered epithelia.
 genetic experiments have clarified that p is a key transcription factor governing the establishment and maintenance of multilayered epithelia. key to our understanding of p strategy is the identification of target genes. we perfomed an rnai screening in keratinocytes for p followed by profiling analysis.  c/ebpδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p we validated c/ebpδ as a primary target of Δnp63α by rt-pcr and chip location analysis in hacat and primary cells. c/ebpδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of hacat and primary keratinocytes. it is bound to and activates the Δnp promoter. overexpression of c/ebpδ leads to alteration in the normal profile of p isoforms, with the emergence of Δnp63β and γ, and of the ta isoforms, with different kinetics. in addition, there are changes in the expression of most p targets. inactivation of c/ebpδ leads to gene expression modifications, in part due to the concomitant repression of Δnp63α. finally, c/ebpδ is found on the p targets in vivo by chip analysis, indicating that coregulation is direct.  our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets. 