TRANSCRIPTION FACTOR ZF5 REGULATES EXPRESSION OF MAMMALIAN GENE CONTAINING GCC-TRIPLET REPEATS IN
5'-REGULATORY REGION IN HUMAN HEPATOMA HepG2 CELLS
S. V. Orlov,1, 2 K. B. Kuteikin-Teplyakov,3 A. V. Grishin,2
E. B. Dizhe,1 E. B. Prokhorchuk,4 A. P. Perevozchikov 2, 5
1 Institute of Experimental Medicine RAMS, St. Petersburg; 2 Department of Embryology,
St. Petersburg State University, 3 Ruhr University, Department of Neurobiochemistry, Bohum, Germany, and
4 Institute of Biology of Gene RAS, Moscow;
5 e-mail: app@iem.sp.ru
Some nuclear proteins of human HeLa and HepG2 cells are capable of binding to GCC-triplet repeats -
(GCC)n>3 in 5'-regulatory regions of a number of mammalian genes - GCC-elements. According to our
previous data, nucleotide sequence (GCC)4 in promoter of mouse ribosomal protein L32 gene (rpL32)
between 17 and 6 bp upstream of transcription start site interacts to nuclear proteins from HepG2 cells, and may be
considered as a GCC-element. We suggest that one of those proteins, with molecular weight about 52 kDa, which may
interact with rpL32 GCC-element, is a known conservative mammalian transcription factor ZF5. DNA-binding
domain of ZF5 contains a few Kruppel-like Zn-fingers (Cys2His2-type) interacting with the
GC-rich nucleotide sequences in 5'-regulatory regions of a number of mammalian genes. Our results (obtained by
EMSA) showed that recombinant GST-ZF5 fused protein containing ZF5 DNA-binding domain specifically binds a few
GS-rich sequences: (GCC)9-triplet repeats, 5'-GCGCGC-3' (known ZF5 consensus binding site) and (more
preferable) the fragment (-24 … +11 bp) of rpL32 promoter. The high affinity of ZF5 DNA-domain binding with
the latter may be explained by the presence in this fragment of two overlapped subsequences, each being capable of
binding to ZF5: (GCC)4 and 5'-GCGCGC-3'. Zf5 cDNA was cloned from HepG2 cells by RT-PCR method,
and then used for construction of the gene expression vector. It has been shown that Zf5 cDNA expression
vector specifically down-regulates (in luciferase assays) the activity of rpL32 promoter (-155 … +159) including
the above mentioned GN-rich subsequences by cotransfection of HepG2 cells. Therefore, our results enable us to
consider GCC-elements as a novel class of ZF5 targets in 5'-regulatory regions of mammalian genes.
Key words: nuclear proteins of cultured human tumor cells, GCC-triplet repeats, ZF5 transcription
factor; GS-rich nucleotide sequences in 5'-regulatory regions of mammalian genes
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