The HMG1 genes

Abstract

The High Mobility Group 1 (HMGl) protein is a ubiquitous and abundant nuclear protein found in higher eukaryotes. The amino acid and nucleic acid sequences of HMGl are highly conserved between different organisms. Although no definite function has yet been assigned to it, HMGl has been suggested to be involved in many processes, e.g. DNA replication, recombination, packaging and gene regulation. In order to have a better knowledge of the HMGl genes, the present study was carried out at three different levels: i.e. transcriptional regulation of the HMGl gene in the human, the HMGl gene structure and organization in the chicken genome, and the evolution of the HMGl gene in the actively speciating Israel mole rats. Though the HMGl gene has been studied by researchers for almost two decades, little is known about its transcriptional regulation. In this study, the 5'-upstream region of the human HMGl gene was cloned by using a PCR-based gene walking approach. This region consists of a 4-kbp 5'-upstream fragment together with the exon 1 and the 2-kbp intron 1 of the human HMGl gene. By primer extension, a major transcriptional start site was found at 57bp upstream of the exon 1-intron 1 boundary. The position of the human HMGl core promoter was mapped and it lacks the canonical TATA box. Furthermore, a strong silencer element immediately upstream of the core promoter and an enhancer in intron 1 have also been identified. The silencer element can effectively repress HMGl expression down to less than one sixth of the activity of the core promoter. The intron 1 enhancer can effectively enhance activity of the human HMGl promoter as well as that of the heterologous SV40 promoter by at least 4-fold. The region of intron 1 enhancer is hypersensitive to DNase I digestion as demonstrated in the DNase I hypersensitivity experiment. Both the silencer and the intron 1 enhancer specifically interact with MCF-7 nuclear proteins as revealed by EMSA. Results from this study suggest that under normal conditions, HMGl expression is probably under tight control exerted by the 5'-upstream silencer. Elevated expression of HMGl may be achieved by activation of the intron 1 enhancer. At the genome level of the present study, the chicken HMGl gene was cloned and analyzed. HMGl retropseudogenes are commonly found in mammalian genomes, with more than 50 copies per genome. However, in the chicken genome, there is no HMGl retropseudogene. This finding suggests that the HMGl retropseudogenes arose in mammals after their divergence away from the birds. Moreover, the functional HMGl gene in chicken, like the other mammalian functional HMGl genes, exists as a single copy gene in the genome. The chicken HMGl gene is highly homologous to the human and the mouse HMGl genes, with similar gene structure and the same exon-intron boundaries. However, in contrast to other avian genes that have shorter introns, the chicken HMG1 gene has introns that are twice as long as their mammalian homologues. A study of the HMG1 gene in the actively speciating Israel mole rats has shedded new light on the fate of highly conserved genes during speciation. The four chromosomal species of Israel mole rats, Spalax galili (2n=52), Spalax golani (2n=54), Spalax carmeli (2n=58) and Spalax judadei (2n=60) formerly collectively known as Spalax ehrenbergi, are known to be undergoing active speciation. The HMG1 genes from these four mole rat species were cloned and analyzed. The translated amino acid sequence from each of them shows three to four changes from the mouse HMG1 protein. These changes are all located in the HMG-box A and the acidic tail. The amino acid sequence of the HMG-box B is absolutely conserved. The DNA sequences of HMG1 genes are very conserved among these four Spalax species, with 98% to 99% identities, but the ratios of non-synonymous to synonymous substitutions (Ka/Ks) are unexpectedly high. Further analysis has revealed that the Ka/Ks ratio differs a lot between the different HMG1 domains. The HMG-box A and the acidic tail have much higher Ka/Ks ratios than the HMG-box B. Compared to the introns, the HMG1 coding region also shows an elevated substitution rate. It appears that in the actively speciating Israel mole rats, there is a relief of the evolutionary constraint on the highly conserved HMG1 protein. Such a relief might be a general feature, allowing organisms to diverge.