Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway.

CFTR expression is tightly controlled by a complex network of ubiquitous and tissue-specific cis-elements and trans-factors. To better understand mechanisms that regulate transcription of CFTR, we examined transcription factors that specifically bind a CFTR CArG-like motif we have previously shown to modulate CFTR expression. Gel mobility shift assays and chromatin immunoprecipitation analyses demonstrated the CFTR CArG-like motif binds serum response factor both in vitro and in vivo. Transient co-transfections with various SRF expression vector, including dominant-negative forms and small interfering RNA, demonstrated that SRF significantly increases CFTR transcriptional activity in bronchial epithelial cells. Mutagenesis studies suggested that in addition to SRF other co-factors, such as Yin Yang 1 (YY1) previously shown to bind the CFTR promoter, are potentially involved in the CFTR regulation. Here, we show that functional interplay between SRF and YY1 might provide interesting perspectives to further characterize the underlying molecular mechanism of the basal CFTR transcriptional activity. Furthermore, the identification of multiple CArG binding sites in highly conserved CFTR untranslated regions, which form specific SRF complexes, provides direct evidence for a considerable role of SRF in the CFTR transcriptional regulation into specialized epithelial lung cells.

Engrailed and polyhomeotic maintain posterior cell identity through cubitus-interruptus regulation.

In Drosophila, the subdivision into compartments requires the expression of engrailed (en) and hedgehog (hh) in the posterior cells and of cubitus-interruptus (ci) in the anterior cells. Whereas posterior cells express hh, only anterior cells are competent to respond to the hh signal, because of the presence of ci expression in these cells. We show here that engrailed and polyhomeotic (ph), a member of the Polycomb Group (PcG) genes, act concomitantly to maintain the repression of ci in posterior compartments during development. Using chromatin immunoprecipitation (ChIP), we identified a 1 kb genomic fragment located 4 kb upstream of the ci coding region that is responsible for the regulation of ci. This genomic fragment is bound in vivo by both Polyhomeotic and Engrailed. In particular, we show that Engrailed is responsible for the establishment of ci repression early during embryonic development and is also required, along with Polyhomeotic, to maintain the repression of ci throughout development.

Chimeric human CstF-77/Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3' end processing in Drosophila.

The Suppressor of forked [Su(f)] protein is the Drosophila homologue of CstF-77, a subunit of human cleavage stimulation factor (CstF) that is required for the first step of the mRNA 3' end processing reaction in vitro. We have addressed directly the role of su(f) in the mRNA 3' end processing reaction in vivo. We show that su(f) is required for the cleavage of pre-mRNA during mRNA 3' end formation. Analysis of the functional complementation between Su(f) and CstF-77 shows that most of the Drosophila protein (85%) can be exchanged for the human protein to produce chimeric CstF-77/Su(f) proteins that rescue lethality and cleavage defect during mRNA 3' end formation in su(f) mutants. Interestingly, we show that a domain in human CstF-77 is limiting for the rescue and that this domain is not able to reproduce protein interactions with the CstF subunits of Drosophila. We also show that chimeric CstF-77/Su(f) proteins that rescue lethality of su(f) mutants cannot restore utilization of a regulated poly(A) site in Drosophila. Taken together, these results demonstrate that CstF-77 and Su(f) have the same function in mRNA 3' end formation in vivo, but that these two proteins are not interchangeable for regulation of poly(A) site utilization.