Characterization of the Promoter of Human Leukocyte-specific Transcript 1

Abstract

The gene for the human leukocyte-specific transcript 1 (LST1) encodes a small protein that modulates immune responses and cellular morphogenesis. The LST1 transcripts are expressed at high levels in dendritic cells. Because of the complex splicing pattern, use of alternative 5-untranslated exons, and a biologically interesting pattern of expression of LST1 mRNA, we studied the human LST1 gene promoter and regulatory elements. We identified an additional upstream 5-untranslated exon in U937 monocytic cells. Transient transfection studies demonstrated that the combination of regions from ؊1363 to ؊621 with ؊112 to ؊54, relative to the translation start codon, produced the highest level of transcripts from among the various constructs tested, but the pattern of transcripts produced was only a subset of those produced from the endogenous gene. DNase I footprinting analysis and electrophoretic mobility shift assays showed that oligonucleotide probes corresponding to three regions, ؊1171 to ؊1142 (BI), ؊1136 to ؊1111 (BII), and ؊783 to ؊751 (BIV), bound proteins in U937 nuclear extracts. Competition and supershift electrophoretic mobility shift assay did not identify any known transcription factors responsible for BII probe binding. These studies suggest that a novel DNA-binding site and interaction of multiple regulatory elements may be involved in mediating the expression of the various forms of LST1 mRNA.The leukocyte-specific transcript 1 (LST1) 1 gene, the human homologue of the mouse B144 transcript, is located within 15 kilobases upstream of the tumor necrosis factor cluster in the major histocompatibility complex class IV region, a region that contains a number of genes that may play a role in various aspects of stress, inflammation, and immune responses (1-8).The human LST1 mRNA is most actively transcribed in monocytes and dendritic cells, and its mRNA levels can be enhanced by interferon (IFN)-␥, suggesting a role of LST1 in the immune response (9 -12). A recent study showed that this gene product had an inhibitory effect on lymphocyte proliferation (13). Transient transfection studies in our laboratory showed that the LST1 gene product induced extensive thin cytoplasmic extensions in a wide variety of cells in vitro and, in particular, was strongly expressed in dendritic cells in vivo and in vitro. 2Due to alternative splicing, the LST1 gene in human and mouse encodes multiple transcripts, each 800 nucleotides or less in length. Previous studies on the human LST1 gene showed that this gene consisted of at least eight exons and four introns, including four different alternative 5Ј-UT exons (termed 1A, 1B, 1C, and 1D, respectively) and four additional exons spanning 2.7 kilobases. The extensive alternative splicing of LST1 mRNA results in high structural diversity of the putative encoded polypeptides (9). The DNA sequences between these 5Ј-UT exons do not consistently show sequence features resembling promoters.There are certain unusual features of the relationship between the human LST1 mRNA and the gene se...

An Upstream Open Reading Frame Regulates LST1 Expression during Monocyte Differentiation

“…In the present study we focussed on the uORF of exon 1B (uORF4, Figure 1A) for several reasons. First, exon 1B is the most widely used exon 1 variant in LST1 : roughly two thirds of all transcripts include the exon 1B sequence [8]. Second, the stop codon of uORF4 is located only 66 bp upstream of the main ORF and its last codon encodes proline (Figure 1C), a feature that has been shown to delay termination and therefore inhibit binding of other ribosomes to downstream ORFs in close proximity [20], [21].…”

“…The LST1 gene lacks typical TATA promoter sequences and transcription factor binding sites. The region downstream of exon 1B up to exon 1C contains potential binding sites for transcription factors and was identified as the sequence with highest promoter activity in reporter assays [8]. The authors also identified sequences just upstream of exon 1B with protein binding sites but the respective nuclear factors could not be identified.…”

“…Both tasks could be enhanced by the increase in LST1 protein levels. It is noteworthy that we focussed on the effect of the exon 1B and 1C sequences on protein expression mainly because initiation of transcription at exons 1A and 1D is very rare [8] and initiation at exon 1E predominates only in fetal tissue [29]. The presence of several uORFs in exon 1E hints that LST1 expression might be strongly repressed in fetal tissues.…”

“…The expression vectors Exon1B-EGFP, Exon1B-AUGdel-EGFP and Exon1B-frameshift-EGFP include a 23 bp long sequence assigned to intron 1A, this is due to the ambiguity of published exon 1B sequences. The sequence shown here is labelled according to [10], however a second study reports the exon 1B sequence to begin 23 bp upstream [8].…”

“…Depending on exon usage the resulting proteins are either short soluble molecules or transmembrane proteins. LST1 encompasses 9 exons, transcription can be initiated at each of the 5 noncoding exons 1A–E [8], while translation always starts at a specific ATG on exon 2 [9]. LST1 proteins have been postulated to have an immunoregulatory function [10] and to play a role in NFκB regulation [11].…”