Characterization of BCE-1, a Transcriptional Enhancer Regulated by Prolactin and Extracellular Matrix and Modulated by the State of Histone Acetylation

Abstract

We have previously described a 160-bp enhancer (BCE-1) in the bovine ␤-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-␤ and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin-and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-␤ but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.It is now well established that the processes of development and differentiation depend on a cell's ability to correctly perceive its microenvironment (reviewed in references 1 and 43). A key component of this environment is the extracellular matrix (ECM). The ECM is an organized network of glycoproteins, proteoglycans, and glycosaminoglycans, components important for cell morphology as well as for signal transduction via cell surface integrins and ultimately for tissue-specific gene expression (reviewed in reference 43).The mammary gland appears to be particularly well suited for the study of ECM-induced differentiation and gene expression. In the adult animal, the gland develops after puberty and functionally differentiates in response to pregnancy. The mechanisms involved in these developmental processes are complex and guided by various hormones (54), growth factors (53), and the ECM (3). Milk protein expression is initiated at mid-pregnancy and correlates with the synthesis and deposition of a specialized laminin-rich ECM during alveolar development. Expression of these milk proteins can be used as markers for the differentiated state of the gland. In the last decade, a number of model systems using mammary epithelial cells to study ECM-dependent gene regulation have been developed. These range from primar...

Inhibition of MMTV transcription by HDAC inhibitors occurs independent of changes in chromatin remodeling and increased histone acetylation

“…Previous studies examining regulation of the MMTV promoter by HDAC inhibitors found that, depending on a variety of conditions, butyrate or TSA could activate or repress the MMTV promoter in organized chromatin (Bresnick et al, 1990;Bartsch et al, 1996;Lambert and Nordeen 1998;Myers et al, 1998;Wilson et al, 2002). Repression was observed in seven distinct cell lines with two exceptions.…”

“…This promoter, when assembled into organized chromatin, can be repressed by the HDAC inhibitors trichostatin A (TSA) or sodium butyrate at concentrations which cause the accumulation of acetylated histones (Bresnick et al, 1990;Bartsch et al, 1996;Lambert and Nordeen 1998;Myers et al, 1998;Wilson et al, 2002). We have investigated the mechanism by which TSA represses the MMTV promoter in organized chromatin.…”

Extracellular matrix and integrin signalling: the shape of things to come

“…We hypothesized previously that the structural continuity that exists between the ECM, integrins, the cytoskeleton and the nuclear matrix may regulate the access of transcription factors to their target genes, perhaps through modulation of histones and chromatin structure [5,13]. Consistent with this notion, in mammary epithelial cells, where β1 integrin ligation, tyrosine kinase signalling and morphological changes induced by basement membrane are all required for transcriptional activation of the milk protein β-casein, access of transcription factors to the β-casein gene depends ultimately upon chromatin organization [95]. Therefore it is likely not only that cell shape permits the interaction of upstream effectors, such as Ras and Raf, with downstream mediators including ERKs, but that cell shape also influences the interaction of these downstream mediators with their growth-or differentiation-specific target genes at the level of nuclear structure ( Figure 3).…”

The contribution of adhesion signaling to lactogenesis

“…The extracellular matrix (ECM) induces a complex interaction between bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones at the β-casein enhancer and promoter regions (Myers et al 1998). There is evidence suggesting that the ECM maintains a high level of histone H4 acetylation upstream of the αs1-casein gene, especially at the level of a distal prolactin and ECM-sensitive enhancer region (Jolivet et al 2005).…”