Differential Regulation of Human T-Plastin Gene in Leukocytes and Non-Leukocytes: Identification of the Promoter, Enhancer, and CpG Island

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Abstract
Plastins (fimbrins) are a family of actin-bundling proteins conserved from yeast to humans. In humans, three tissue-specific plastin isoforms have been identified. The T isoform (T-plastin) is unique in that it is expressed in all tissues except leukocytes. To investigate how the T-plastin gene is differentially regulated in leukocytes and non-leukocytes, we isolated a genomic clone that included 9 kb of the upstream flanking region, 0.1 kb of the first exon, and 5.9 kb of the first intron. From this clone, we obtained a continuous sequence of 5535 bp, including 3138 bp of the upstream flanking region, the first exon, and 2286 bp of the first intron. A cluster of four transcription initiation sites was located by S1 mapping. A region spanning these sites and extending 1.4 kb into the first intron had the characteristics of a CpG island. Three CG-containing restriction sites within this island were analyzed and found all or variably methylated in four T-plastin-negative leukemia cell lines. In contrast, the same sites were not methylated in three T-plastin-expressing cell lines or in a sample of normal blood lymphocytes. A basal promoter was located 250 bp upstream from the transciption initiation sites. It comprised a CCAAT box, an Sp1 motif, and four AP2 motifs. No TATA or Inr sequence was found. The basal promoter exhibited weak activity when assayed in fibrosarcoma cells. Stronger promoter activities were found in the presence of the SV40 enhancer or a T-plastin enhancer located some 500 bp from the basal promoter. In T-plastin-negative leukemia cells, the T-plastin basal promoter could be activated by the SV40 enhancer but not by the T-plastin enhancer. DNA footprinting identified the T-plastin enhancer as two inverted symmetric octamers (AGATAACCTC and GAGGTCAGCT) separated by 17 nucleotides.
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