Signaling by Decapentaplegic (Dpp), a member of the TGFβ superfamily of signaling molecules similar to vertebrate BMP2 and BMP4, has been implicated in many developmental processes in Drosophila melanogaster . Notably, Dpp acts as a long‐range morphogen during imaginal disc growth and patterning. Genetic approaches led to the identification of a number of gene products that constitute the core signaling pathway. In addition to the ligand‐activated heteromeric receptor complex and the signal‐transducing intracellular Smad proteins, Dpp signaling requires two nuclear proteins, Schnurri (Shn) and Brinker (Brk), to prime cells for Dpp responsiveness. A complex interplay between the nuclear factors involved in Dpp signaling appears to control the transcriptional readout of the Dpp morphogen gradient. It remains to be seen whether similar molecular mechanisms operate in the nucleus in vertebrate systems.

P63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The ΔNp63α isoform is thought to play a ‘master’ role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing ΔNp63α; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.

The CCAAT box is a prototypical promoter element, almost invariably found between –60 and –100 upstream of the major transcription start site. It is bound and activated by the histone fold trimer NF-Y. We performed chromatin immunoprecipitation (ChIP) on chip experiments on two different CpG islands arrays using chromatin from hepatic HepG2 and pre-B cell leukemia NALM-6 cell lines, with different protocols of probe preparation and labeling. We analyzed and classified 239 known or predicted targets; we validated several by conventional ChIPs with anti-YB and anti-YC antibodies, in vitro EMSAs, and ChIP scanning. The importance of NF-Y binding for gene expression was verified by the use of a dominant negative NF-YA mutant. All but four genes are new NF-Y targets, falling into different functional categories. This analysis reinforces the notion that NF-Y is an important regulator of cell growth, and novel unexpected findings emerged from this unbiased approach. (i) A remarkable proportion of NF-Y targets, 40%, are complex transcriptional units composed of divergent, convergent, and tandem promoters. (ii) 40–50% of NF-Y sites are not in core promoters but are in introns or at distant 3′ or 5′ locations. The abundance of “unorthodox” CCAAT positions highlights an unexpected complexity of the NF-Y-mediated transcriptional network.

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether ‘phenotypic’ disease progression is really associated with ‘molecular’ progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.