Cloning and knock-out of the mouse gene coding for the high mobility group 2 protein (HMG2).

High Mobility Group proteins 1 and 2 are highly conserved nuclear proteins ubiquitously expressed in higher eukaryotic cells. HMG1 and HMG2 each contain two similar DNA binding domains, called HMG-boxes, and an acidic tail. HMG1 and HMG2 are able to facilitate the binding to DNA of several transcription factors (Steroid Hormone Receptors, HOX and OCT proteins) and they are involved in V(D)J recombination. hmg1 -1- mice, produced in our lab by conventional knock-out, are bom and die within the first day of life. The cause of death is a severe hypoglycaemia: the level of glucose is low in the blood of mutant mice, while abundant glycogen is still present in their liver. Glucocorticoid-dependent gene expression is impaired in -/- mice. Surprisingly, HMG1 is not essential for the life of the cell, as it might be suggested by the abundance of the protein and its evolutionary conservation. We then suggested that another protein can substitute HMG1 in its function in the cell. The best candidate is HMG2 because of its remarkable similarity with HMG1. Therefore, I decided to investigate the function of HMG2 by gene targeting, and I planned the work so as to investigate similarities and differences between HMG1 and HMG2. The mouse hmg2 gene was cloned, characterised and mapped on the centromeric region of chromosome 8. Further, functional analysis were done. By transient transfection assays, I demonstrated that HMG2 is very similar to HMG1 in the transcriptional activation mediated by HOX proteins. Using the same assay, I demonstrated that differences in the length of the acidic tail of HMG1 and HMG2 determine no functional differences. Moreover, I showed that HMG2, as HMG1, is not stably associated to chromatin. These data, together with previously published data, suggest that HMG2 and HMG1 play very similar functions in the cell. Nevertheless, a difference between the two proteins was found in their expression pattern. HMG2 is absent in adult liver and brain, while is very abundant in adult testis, spleen, and thymus. On the contrary, HMG1 is ubiquitously expressed. In situ hybridisation revealed that in the testis HMG2 has a specific distribution: it is absent in spermatogonia and in late spermatids and spermatozoa, while is very abundant in spermatocytes and round spermatids. The peculiar protein distribution during spermatogenesis correlates with the phenotype we found in hmg2 -/- mice. hmg2 -/- mice had no obvious phenotypic differences from wild type. No immunological, homeotic, and chromatin defects were found. However, -/- male mice are partially sterile. After six months of continuous breeding studies some -/- mice gave no litters, some others gave litters of reduced size while others gave normal litters. Histological analysis of the testis in completely sterile mice revealed the presence of a significant number of dysmorphogenic seminiferous tubules, similar to that described in knock-out mice for estrogen receptor and in mice overexpressing ABP (Androgen Binding Protein). Degenerated tubules were also found in fertile -/- mice, but the number was drastically reduced. Moreover, electron microscope analysis revealed severe defects in elongating spermatids. Spermatozoa are produced, but are mostly immobile. Nevertheless, they are able to fertilise eggs. The testes of many knock-outs were found in the inguinal ring instead of in the scrotum. The correct position of testes is regulated by androgens. The correct function of androgen receptor was tested in mouse embryonic fibroblasts, but no large difference in the response to testosterone was found in this kind of cells. Thus, HMG1 and HMG2 play a very similar role in the cell and it is highly probable that they can substitute each other to a large extent. HMG1 expression is high and ubiquitous, whereas HMG2 expression is limited to a subset of tissues and organs. In the testes, the absence of HMG2 leads to a reduction in the number of viable spermatozoa, suggesting that during male meiosis a high level ofHMGs is required.