Eukaryotic organisms contain families of DNA-binding transcription factors comprised of structurally related proteins that are encoded by different genes. Individual members of the family are often co-expressed in the same cell, and in many cases they can associate with each other to generate heteromeric transcription factors. In addition, transcription factor families can exhibit cross-regulation, in which one family member affects the expression and/or function of another family member. In general, individual transcription factors within the family have both distinct and overlapping biological functions.
An important transcription factor family in mammalian cells includes the p53 tumor suppressor and two other proteins, p63 and p73, that are strikingly similarity to each other and less similar to p53 
. p63 and p73 share ~85% amino acid identity in their DNA binding domain, and they show strong structural and sequence similarity in their activation, oligomerization, and isoform-specific, C-terminal domains. p53 binds its target sites as a tetramer 
, and it is presumed that this is the case for p63 and p73. p63 and p73 exist as stable tetramers, and they interact efficiently to form heterotetramers 
, although the DNA-binding activity of the heterotetramers has not been tested directly. Neither p63 nor p73 can form heterotetramers with p53, because p53 lacks a critical second helix in the tetramerization domain that is present in p63 and p73 
. The various family members can co-exist in the same cell, and they exhibit cross-regulation 
. In addition, p63 and p73 can transcriptionally regulate genes involved in DNA repair 
Despite the very high degree of similarity between p63 and p73, mouse knockout models reveal distinct and non-redundant physiological roles. p63-deficiency is associated with severe defects in epithelial development 
and DNA damage responses in the female germline 
. In contrast, p73 is implicated in various biological pathways including neurogenesis, inflammation, sensory pathways, and osteoblastic differentiation 
as well as genomic stability and tumor suppression 
. The molecular basis for these distinct physiological roles is unknown.
There are multiple explanations, not mutually exclusive, for how two highly related members of the same protein family can have distinct biological functions. First, differences in tissue- and cell type-specific expression patterns can underlie distinct biological functions, even if the proteins are functionally equivalent. Differences in expression patterns might involve some or all of the structurally distinct isoforms that arise via alternative splicing, promoter usage, or 3′ end formation. Second, the two proteins can have distinct target specificities in vivo, either due to subtle differences in their DNA-binding domains and/or to differences in cooperative interactions with other DNA-binding proteins. Third, the two proteins can have functionally distinct domains that differentially mediate transcriptional activation or repression, interactions with co-activators or co-repressors, or interactions with other regulatory proteins. In cases where the proteins themselves are functionally distinct, the differences could be intrinsic to the protein sequence and/or reflect differences in phosphorylation or other post-translational modifications.
The in vivo
binding behavior of highly related transcription factors in the same cells has rarely been examined in a global, unbiased manner. In the case of the ETS family of transcription factors, analysis of in vivo
binding using genome-wide promoter microarrays revealed redundant and specific occupancy by individual members of the family 
. Comparison of Stat5a and Stat5b, demonstrated that these highly homologous factors bind the same sites in vivo
, albeit with different kinetics that may underlie differences in Stat5 biology 
. A comparison of E2F family members in normal and tumor cells revealed very similar DNA-binding profiles in some cell types but not others 
In previous work, we used tiled microarrays covering the human genome to identify ~5800 target sites for p63 in ME180, a cervical carcinoma cell line 
. Here, we generate a DNA-binding profile of p73 in the same cells, thereby permitting a comparison of its in vivo
target specificity to that of p63. We show that the p73 and p63 binding profiles are indistinguishable, with a similar p73
p63 binding ratio at essentially all genomic loci. Furthermore, we show that p63 and p73 co-occupy DNA target sites in vivo
, suggesting that p63 and p73 bind primarily as heteromeric complexes. The observation that p63 and p73 directly associate with the same set of genomic targets suggests that their distinct biological functions are due to cell-type specific expression and/or protein domains that involve functions other than intrinsic or cooperative DNA binding to target sites.