Identification of a Hoxc8-regulated transcriptional network in mouse embryo fibroblast cells

The transcription factor, Hoxc8, is a member of the homeobox gene family that is vital for growth and differentiation. Previously, we identified 34 genes whose expression levels were changed at least 2-fold by forced expression of Hoxc8 in C57BL/6J mouse embryo fibroblast cells using a mouse 16,463-gene oligonucleotide microarray. In the present study, we used the combined power of microarray profiling, global Hoxc8 DNA-binding site analysis, and high-throughput chromatin immunoprecipitation assays to identify direct and biologically relevant targets of Hoxc8 in vivo. Here we show that 19 of the 34 responsive genes contain Hoxc8 consensus DNA-binding sequence(s) in their regulatory regions. Chromatin immunoprecipitation analysis indicated that Hoxc8-DNA interaction was detected in five of the 19 candidate genes. All of these five target genes have been implicated in oncogenesis, cell adhesion, proliferation, and apoptosis. Overall, the genes described here should aid in the understanding of global regulatory networks of Hox genes and to provide valuable insight into the molecular basis of Hoxc8 in development and carcinogenesis

Improved in situ beta-galactosidase staining for histological analysis of transgenic mice

The present study describes a novel method for the histochemical demonstration of beta-galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) with 5-bromo-indolyl-beta-o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial beta-galactosidase (lacZ). After beta-galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for beta-galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of beta-galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter