In vivo modulation of HMGA2 expression.

While the biochemical role of the HMGA proteins has largely been elucidated in tissue culture, the majority of the insight as to their physiological functions in the processes of proliferation and development has been established in animal models of overexpression (transgenic) and null mice (knockouts). An emphasis has been placed on the HMGA2 studies which have defined its critical role in mesenchymal proliferation and differentiation.

Hmga1 is required for normal sperm development.

The Hmgi protein family of chromosomal architectural factors is extensively studied for its roles in embryogenesis and its association with benign mesenchymal tumors. Although the biochemical function of Hmga1 has been studied in vitro, to provide in vivo insight into its biological function, a targeted disruption of Hmga1 was initiated. Chimeric founder mice were derived from embryonic stem (ES) cells harboring a targeted mutation in a single Hmga1 allele. These 14 different chimeric founders produced 494 black progeny. Since none of these 494 progeny were agouti, none of them were derived from ES cells. Control injections of the wild-type ES cell lines resulted in ES cell derived agouti mice, indicating that the ES cells were totipotent. Therefore, our results indicate that one intact Hmga1 allele was not sufficient for germ-line transmission of the ES cells. Seven chimeric founder mice that were examined histologically demonstrated aberrant regions in their reproductive organs. Aberrant regions of seminiferous tubules were reduced in diameter, demonstrated vacuolated Sertoli cells, and had an absolute deficiency of sperm. While the Hmga1(+/-) ES cells were shown to contribute to the formation of the epididymides, they did not significantly contribute to the testes of chimeric founder mice. No sperm isolated from any of the Hmga1(+/-) chimeric mice were shown to arise from the ES cells, as none of them contained the targeted disruption of the Hmga1 gene. Our results suggest that both alleles of Hmga1 are required for normal sperm production in the mouse.

HMGA2 is expressed in an allele-specific manner in human lipomas.

The architectural transcription factor HMGA2 is almost exclusively expressed in undifferentiated mesenchymal cells. Interestingly, it has been mapped to the translocation site in a variety of human mesenchymal tumors that reveal a terminally differentiated phenotype. The expression of chimeric HMGA2 transcripts encoding three DNA-binding domains fused to novel transcriptional regulatory domains was previously described in lipomas. In this study with lipoma ST91-198, we report the expression of truncated HMGA2 transcripts that gained no functional domains. The highly polymorphic region in the 5' untranslated region (UTR) of HMGA2 was used to determine the allele-specific expression of HMGA2 in lipomas. Microsatellite PCR revealed a monoallelic expression pattern, and only the translocated allele was expressed when the DNA-binding domains of the rearranged allele were fused with transcription activation domains. Surprisingly, a diallelic expression pattern of HMGA2 was observed in lipoma ST91-198, and the wild-type allele was also expressed. In conjunction with studies involving rearrangements of HMGA genes in other benign mesenchymal tumors, our results support a model in which the expression of the wild-type HMGA allele is critical for the pathogenesis of mesenchymal tumors and in which rearrangements of HMGA do not lead to a gain of function in the chimeric HMGA protein.

Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway.

Survivin is an inhibitor of apoptosis protein, which is over-expressed in most tumors. Aberrant expression of survivin and loss of wild-type p53 in many tumors prompted us to investigate a possible link between these two events. Here we show that wild-type p53 represses survivin expression at both mRNA and protein levels. Transient transfection analyses revealed that the expression of wild-type p53, but not mutant p53, was associated with strong repression of the survivin promoter in various cell types. The over-expression of exogenous survivin protein rescues cells from p53-induced apoptosis in a dose-dependent manner, suggesting that loss of survivin mediates, at least, in part the p53-dependent apoptotic pathway. In spite of the presence of two putative p53-binding sites in the survivin promoter, deletion and mutation analyses suggested that neither site is required for transcriptional repression of survivin expression. This was confirmed by chromatin immunoprecipitation assays. Further analyses suggested that the modification of chromatin within the survivin promoter could be a molecular explanation for silencing of survivin gene transcription by p53.

Hmgi-c-independent Activation of MuRantes in Vivo.

The architectural factor HMGI-C is of considerable interest for its recognized roles in mammalian development and tumorigenesis. As a result, the identification of downstream target genes of HMGI-C is the present focus of active research. In vitro evidence from macrophage cell lines has previously suggested that Hmgi-c is necessary for the inducible activation of MuRantes expression. To attempt to verify this hypothesis, an in vivo analysis was performed that took advantage of the existence of the Hmgi-c null mouse strain. The ability of cells and tissues extracted from Hmgi-c null mice to express the inflammatory chemokine MuRantes was investigated. The investigation examined MuRantes expression in primary embryonic fibroblasts and fresh peritoneal macrophages after Newcastle disease virus induction and whole organs after lipopolysaccharide induction. Each of these systems clearly demonstrates that Hmgi-c is not required for the activation of MuRantes expression.