T cell-specific expression of the human TNF-alpha gene involves a functional and highly conserved chromatin signature in intron 3

Using a phylogenetic approach, we identified highly conserved sequences within intron 3 of the human TNF-alpha gene. These sequences form cell type-specific DNase I hypersensitivity sites and display cell type-specific DNA-protein contacts in in vivo genomic footprints. Consistent with these results, intron 3 confers specific activity upon a TNF-alpha reporter gene in Jurkat T cells, but not THP-1 monocytic cells. Thus, using a combinatorial approach of phylogenetic analysis, DNase I hypersensitivity analysis, in vivo footprinting, and transfection analysis, we demonstrate that intronic regulatory elements are involved in the cell type-specific regulation of TNF-alpha gene expression.     

Analysis of cis-acting elements present in the CD20/B1 antigen promoter.

A genomic clone spanning a large portion of the 5' untranscribed region of the CD20 gene was isolated. Deletion analysis of subcloned fragments identified several regulatory elements. A major positive cis-acting element was localized between base pairs -290/-186. A second positive regulatory element was localized between -454/-280 and negative regulatory elements were present in the region between bp -828/-454. The sequence -280/-186 conferred B cell-specific expression on a heterologous, TATA box containing c-fos promoter. Electrophoretic mobility shift assays with overlapping oligonucleotide probes spanning -280/-186 revealed that a 25-bp probe (-225/-201) bound a nuclear protein present in B cell lines expressing the CD20/B1 antigen but not in Jurkat (T cell), U937 (promonocytic), U251 (glioma), or HeLa cells. To confirm the functional significance of this sequence, a trimer of this region was subcloned into the c-fos promoter containing CAT plasmid. Expression was observed only in BJA-B and HS-Sultan cells but not in CD20/B1- cell lines. This sequence element is also important in phorbol ester-induced CD20 expression in the pre-B cell line BP-697. These results partially characterize several regulatory elements present in the CD20 promoter that are likely important in the B cell-specific expression of the CD20 gene.     

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.     

Multiple regulatory elements determine adrenocortical expression of steroid 21-hydroxylase

Steroid 21-hydroxylase (21-OHase) is specifically expressed at high levels in the adrenal cortex, where it is required for the synthesis of mineralocorticoids and glucocorticoids. In this study, we have investigated the regulatory elements in the 21-OHase promoter region which contribute to the expression of this gene in Y1 adrenocortical cells. Eight potential regulatory elements in the 5'-flanking region of the 21-OHase gene were identified by DNase I footprinting and gel mobility shift experiments. Some of these footprints were produced by nuclear extracts from many cell lines, whereas other interactions were seen only when using nuclear extracts from Y1 adrenocortical and MA-10 Leydig tumor cells. Mutation of most of the elements markedly decreased the expression of a 21-OHase gene transfected into Y1 cells, thus documenting their functional importance for expression. Moreover, oligonucleotides containing the sequences of two related elements at -65 and -210, which share the heptamer AGGTCAG, increased the activity of a heterologous promoter in a Y1 cell-specific manner. Collectively, these results demonstrate that expression of 21-OHase in Y1 adrenocortical cells requires interactions among multiple cis-acting elements and regulatory proteins.