Negative regulatory role of Sp1 in metal responsive element-mediated transcriptional activation

Transcription of mammalian metallothionein (MT) genes is activated by heavy metals via multiple copies of a cis-acting DNA element, the metal-responsi ve element (MRE), Our previous studies have shown that certain MREs of the human MT-IIA gene (MREb, MREc, MREd, and MREf) are less active than the oth ers (MREa, MREe, and MREg). Gel shift analysis of HeLa cell nuclear protein s revealed that whereas the active MREs strongly bind the transcription fac tor MTF-1 essential for metal regulation, the less active MREs bind another distinct protein, MREb-BF. This protein recognizes the GC-rich region of M REb rather than the MRE core required for MTF-1 binding. All the MREs recog nized by MREb-BF contain the CGCCC and/or CACCC motif, suggesting that the MREb-BF-MRE complex contains Sp1 or related proteins. Supershift analysis u sing antibodies against Sp1 family proteins as well as gel shift analysis u sing the recombinant Sp1 demonstrated that Sp1 represents the majority of M REb-BF activity. An MREb mutant with reduced affinity to Sp1 mediated zinc- inducible transcription much more actively than the wild-type MREb, Further more, when placed in the native promoter, this mutant MREb raised the overa ll promoter activity. These results strongly suggest that Sp1 acts as a neg ative regulator of transcription mediated by specific MREs.