Sequence-specific binding of proteins from an insulin-secreting cell line (RINm-5F) to the human insulin-gene 5′ region were examined by gel-retardation and methylation-interference analysis. Specific binding of a nuclear factor to sites between nucleotides -210 to -217 and -77 to -84 was detected. The same binding activity was shown at an upstream site (-313 to -320) with low affinity. Studies using mutated binding-site probes delineated a sequence 5′-C(T/C)CTAATG-3′ for high-affinity interactions. This binding activity was also present in another insulin-producing cell line (HIT.T15), but not in extracts from cell lines that did not express the insulin gene (HeLa, HL60). Cross-species comparisons show that this sequence element is highly conserved and may thus play an important role in the cell-specific regulation of insulin-gene transcription.

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