Cell-type specificity of regulatory elements identified by linker scanning mutagenesis in the promoter of the chicken lysozyme gene

The chicken lysozyme gene is constitutively expressed in macrophages, in oviduct cells its expression is controlled by steroid hormones, and in fibroblasts the gene is not expressed. A fusion gene consisting of promoter sequences of the lysozyme gene from –208 to +15 in front of the chloramphenicol acetyltransferase (CAT) coding region was more than 50 times less active in non-expressing cells as compared to expressing cells. In order to identify the element(s) responsible for this cell-type specificity 31 different linker scanning mutations were generated within this promoter fragment and analyzed by transient transfections in the three types of chicken cells mentioned above. Three mutation sensitive regions located around position –25, –100 and between –158 and –208 were detected in each cell type, however, several LS mutations displayed clear cell-type specific differences in their phenotypic effects. Interestingly, a few LS mutations led to an increase in promoter activity in fibroblasts suggesting that the corresponding wildtype sequences represent binding sites for negatively acting transcription factors

Extinction of gene expression in somatic cell hybrids. a reflection of important regulatory mechanisms?

Extinction in somatic cell hybrids is a multifactorial process that leads to loss of cell-type-specific gene expression. The underlying mechanisms are thought to mirror, at least in part, the repertoire of regulatory mechanisms controlling mammalian cell differentiation

Extinction of tyrosine aminotransferase gene activity in somatic cell hybrids involves modification and loss of several essential transcription factors

Extinction is defined as the loss of cell type-specific gene expression that occurs in somatic cell hybrids derived by fusion of cells with dissimilar phenotypes. To explore the basis of this dominant-negative regulation, we have studied the activities of the control elements of the liver-specific gene encoding tyrosine aminotransferase (TAT) in hepatoma/fibroblast hybrid crosses. We show that extinction in complete somatic cell hybrids is accompanied by the loss of activity of all known cell type-specific control elements of the TAT gene. This inactivity is the result of first, lack of expression of genes coding for the transcriptional activators HNF4 and HNF3[~ and HNF33,, which bind to essential elements of the enhancers; and second, loss of in vivo binding and activity of ubiquitous factors to these enhancers, including CREB, which is the target for repression by the tissue-specific extinguisher locus TSE1. Complete extinction of TAT gene activity is therefore a multifactorial process affecting all three enhancers controlling liver-specific and hormone-inducible expression. It results from lack of activation, rather than active repression, and involves both post-translational modification and loss of essential transcriptional activators

A new method for constructing linker scanning mutants

A new procedure for the construction of linker scanning mutants is described. A plasmid containing the target DNA is randomly linearized and slightly shortened by a novel combination of established methods. After partial apurination with formic acid a specific nick or small gap is introduced at the apurinic site by exonuclease III, followed by nuclease S1 cleavage of the strand opposite the nick/gap. Synthetic linkers are ligated to the ends and plasmids having the linker inserted in the target DNA are enriched. Putative linker scanning mutants are identified by their topoisomer patterns after relaxation with topoisomerase I. This technique allows the distinction of plasmids differing in length by a single basepair. We have used this rapid and efficient strategy to generate a set of 32 linker scanning mutants covering the chicken lysozyme promoter from –208 to +1

Tissue-specific DNase I-hypersensitive sites in the 5´flanking sequences of the trytophane oxygenase and tyrosine aminotransferase genes

The genes for tryptophan oxygenase (TO) and tyrosine aminotransferase (TAT) are expressed in a tissue- and development-specific manner and are regulated by glucocorticoids (TO and TAT) and glucagon or its intracellular mediator cAMP (TAT) in rat liver. We have analyzed the chromatin structure of these genes in the vicinity of the 5' ends with regard to DNaseI hypersensitivity and have found DNaseI hypersensitive sites upstream of each of the promoters. Mapping of this region reveals three closely spaced cleavage sites near the TO promoter and a doublet of sites near the TAT promoter. In both genes additional cleavage sites are found further upstream. All hypersensitive sites of both genes are absent in kidney nuclei and therefore appear to be specific for the tissue expressing the genes. A correlation of expression and modified chromatin structure was also observed in a hepatoma cell line expressing TAT but not TO: hypersensitive sites are present in TAT but not in TO chromatin. Upon glucocorticoid induction an additional hypersensitive site is detected approximately 2 kb upstream of the TAT promoter in liver and hepatoma cells

Role of cyclic AMP in the control of cell-specific gene expression

Genes have to be expressed in specific cell types at appropriate times of development dependent on external signals. cAMP signaling occurs in all cells, thus raising the question of how this signal transduction pattern is integrated into mechanisms determining cell-specific gene expression. We have analyzed expression of the tyrosine aminotransferase gene as a model to study the basis of this cell type specificity of hormone induction. We found that cell-type-specific expression is generated by combined action of cAMP signal-dependent and liver cell-specific transcription factors. The interdependence of the cAMP response element and an element determining liver cell specificity enables a gene to respond to an ubiquitous signal in a cell-specific manner