We have previously identified a murine leucine zippercontaining transcription factor, GILZ (GILZ: Glucocorticoid Induced Leucine Zipper) able to modulate T lymphocyte activation and apoptosis. 1, 2 In the present study, we describe the isolation and cloning of a human homologue of murine GILZ (mGILZ). In particular, a λgt11 human cDNA library from T lymphocytes was screened using the full length mGILZ cDNA, resulting in the identification of human GILZ (hGILZ). The human cDNA was 1946 bp in total, consisting of a single open reading frame, beginning at nucleotide position 241 and extending to a termination codon at position 643 (Figure 1A). The first ATG of the hGILZ cDNA is surrounded by a minimum Kozak sequence (GAACCATGA) in good agreement with the consensus sequence for initiation of translation in eukaryotes. 3 Besides, a typical polyadenylation signal, appears at the expected distance from the 3'-terminus. The similarity of hGILZ to the nucleotide sequence to mGILZ is 89% in the entire mRNA and 97% in the coding region. The predicted translation product is a leucine zipper of 135 amino acids, highly homologous to mGILZ, with a putative molecular mass of about 15 kd (Figure 1B). In particular, the encoded protein contains a leucine zipper-like structure from leucine-76 to leucine-97, highly conserved among the members of this protein family. Furthermore, a proline rich region (PAR) is present at the 3'-end region. 4 Structure analysis shows that the amino acid sequence in the leucine zipper region is hydrophilic at its C-terminus and hydrophobic at its N-terminus. Consensus casein kinase-II phosphorylation sites are located at threonine-8, at serine-88 (the start of its hydrophilic section), and at serine 114. Besides, a protein kinase-C consensus phosphorylation site at serine-35 and a glycosylation consensus site at asparagine-19 are revealed. We also determined the chromosomal localisation of hGILZ by fluorescence in situ hybridisation (FISH). 5, 6 For that purpose, metaphase spreads were obtained from human PHA-stimulated peripheral blood lymphocytes. Chromosome preparations were hybridised in situ using the entire hGILZ cDNA as a probe. Since the DAPI banding was used to identify the specific chromosome, the assignment between signal from the probe and long arm of chromosome X was obtained (Figure 1C). The detailed position was determined in the diagram based on the summary from 10 photographs (Figure 1Ca and b). Figure 1Ca shows a typical hybridisation of the hGILZ cDNA at the Xq22. 2 region, of the chromatids of both chromosomes (Figure 1Ca and Cc arrow). Interestingly enough, the Xq22 region contains a number of genes associated with various X-linked deficiences including the Bruton agammaglobulinemia, further suggesting a possible role of GILZ in the regulation of immune response. Future studies aimed to identify the chromosomal localisation of mGILZ could furnish indications on the possible role in X-linked deficiencies.

To determine the expression pattern of hGILZ gene, we performed Northern blot experiments with human tissue total RNA. 7 Results of a representative experiment in Figure 1D indicate that hGILZ transcripts can be detected ubiquitously among different organs with the exception of liver (lane 4) and pancreas (lane 6). As shown in the figure, after 24h of autoradiography, hGILZ RNA is clearly detectable in the brain, lung, spleen and skeletal muscle, while expression in heart and kidney is weaker. Evaluation of equal loading was performed by visual inspection of ethidium bromide (data not shown). This finding strongly indicates a widespread distribution of hGILZ and …