Cell-type specific and differential regulation of the human metallothionein genes. Correlation with DNA methylation and chromatin structure.

The expression of three human metallothionein genes, MT-IIA, MT-IF, and MT-IG was studied in the human hepatoblastoma (HepG2), the hepatocarcinoma (Hep3B2), the embryonic kidney (Hek 293), and the lymphoblastoid-derived (Wi-L2) cell lines. The pattern of expression of each specific MT gene in response to various heavy metals was different among the four cell lines studied indicating differential regulation of MT gene expression. The MT-IF or MT-IG and the MT-IIA genes were regulated in a cell-type specific manner in response to heavy metals and dexamethasone, respectively. DNA methylation was shown to be correlated to cell-type specific regulation of MT gene expression since 5-azacytidine treatment resulted in the expression of the MT-IF and MT-IG genes in response to cadmium and zinc in Wi-L2 cells, of the MT-IIA gene in response to dexamethasone in Wi-L2 cells, and of the MT-IG in response to zinc and copper in Hek 293 cells. Furthermore, transfection studies indicated that all the trans-acting factors necessary for the expression of these genes were present and functional in Wi-L2 and Hek 293 cells. The differential level of expression of the MT-IF and MT-IG genes in response to heavy metals in the Hek 293 cell line was shown to be correlated to their chromatin structure.     

Similarities and differences in the function of regulatory elements at the 5' end of the human apolipoprotein B gene in cultured hepatoma (HepG2) and colon carcinoma (CaCo-2) cells.

The arrangement of regulatory elements along the apolipoprotein B promoter region (positions -898 to +1) has been examined in transient transfection experiments performed in HepG2 and Hep3B (hepatic) and CaCo-2 (intestinal) cell lines, all of which express the apoB gene, and also in Chinese hamster ovary cells, which do not express the gene. The overall distribution of positive and negative regulatory segments was very similar in the two hepatoma cell lines (HepG2 and Hep3B) but different from that observed in the colon carcinoma cells (CaCo-2). Thus, whereas 260 base pairs of 5'-flanking sequence were sufficient for maximal expression of the promoter in HepG2 cells, only 139 nucleotides were required for maximal expression in CaCo-2 cells. Promoter activity in Chinese hamster ovary cells was exhibited by short constructs, with maximal activity for the -85 construct. DNase I footprinting of the apolipoprotein B promoter region using hepatic and intestinal extracts revealed multiple sites of interaction between the DNA and nuclear proteins. Gel retention experiments using the region from -262 to -88 (the region of greatest contrast between HepG2 and CaCo-2 cells) revealed interesting variations in the relative abundance of various nuclear proteins between the two cell types. A major functional difference between HepG2 and CaCo-2 cells was localized to the region between -111 and -88, which harbors the sequence TGTTTGCT, a motif present in the promoter region of several liver-specific genes. The molecular basis for the functional differences between these two cell types may be attributable to a difference in the relative abundance of three proteins that bind to sequences between -111 and -88.     

Functional analysis of the promoter of a sea urchin metallothionein gene.

The 5'-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides +31 to -309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injection into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5'-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.