Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity.The highly conserved heterochromatin protein 1 (HP1) class of chromobox genes (HP1) encode structural adapters whose probable role is to assemble a variety of macromolecular complexes in chromatin (30). The possible functions of these complexes are wide-ranging and include roles in transcriptional repression (12,36,54,55), transgene silencing (17, 26), chromosome segregation (14, 31), recruitment of silent genes to heterochromatin (7, 54), localization of heterochromatin to the nuclear periphery (67), and sex chromosome inactivation during mammalian spermatogenesis (44).The swi6 gene in Schizosaccharomyces pombe is a nonessential gene that is required for the recombination-suppression and silencing which encompasses the mat2-K-mat3 region (33). Cloning of the gene showed that swi6 is a member of the HP1 class of chromobox genes (38), suggesting that the recombination-suppression and silencing are due to the packaging of the mat2-K-mat3 region into a heterochromatin-like complex that renders the region inaccessible to the transcriptional and recombination machinery (38,63). Other trans-acting factors that are required for repression at the silent loci include rik1, clr1, clr2, clr3, clr4, and clr6 (34, 64). rik1, clr1, and clr4 are thought to encode structural components of the heterochromatin-like complex, while clr3 and clr6 share considerable homology with histone deacetylases (20). Along with the silent mating-type loci, swi6p is also involved in silencing at the fission yeast centromeres and telomeres (14, 47) and plays a role in chromosome segregation at anaphase (14).HP1 proteins are characterized by the possession of both a classical chromodomain (CD) and a chromo shadow domain (CSD) (2) linked by a variable intervening region (IVR) or "hinge" (16). In addition, a stretch of acidic amino acids immediately precedes the CD of HP1 proteins (see Fig. 1A). The solution structure of the CD from the murine HP1-like heterochromatin-associated protein, M31 (also known as mH...
We have raised a monoclonal antibody (MoAb), MAC 402, to the mouse M33 protein, a mammalian homologue of the Drosophila Polycomb protein. MAC 402 recognises a band of 59.7 kDa by Western blot analysis and gives rise to two types of staining pattern within interphase nuclei. Surprisingly, at metaphase, M33 protein is enriched within centromeric heterochromatin. This pattern is also seen for the product of the bmi-1 gene, which is a homologue of the Drosophila Polycomb-group (Pc-G) gene Posterior sex combs. We speculate as to the reasons for this relocalisation of mammalian Pc-G gene products to the centromere at metaphase and its implication for the cell-to-cell inheritance of the states of gene repression that are conferred by this highly conserved group of genes.
The rat sperm surface antigen, 2B1, that has been proposed to play a key role in sperm adhesion to the zona pellucida, has been cloned and its entire cDNA sequenced. Northern blot analysis indicates that 2B1 is encoded by a 2.2-kb RNA transcript that is abundantly expressed in the testis. The deduced protein sequence contains 512 amino-acid residues with a strong candidate signal sequence and C-terminal transmembrane domain. Data base searches reveal a high degree of sequence similarity to guinea pig, rabbit, monkey, and human PH20 sperm surface antigens, and a lower degree of similarity to honey bee and whiteface hornet venom hyaluronidases. Rat 2B1 antigen also possesses hyaluronidase activity, suggesting that it is a bifunctional protein with putative roles in the dispersion of cumulus oophorus cells as well as zona adhesion. However, while it would appear that 2B1 is the rat homologue of the guinea pig PH20 antigen, they differ in a number of important biochemical respects (including their mode of attachment to the sperm membrane and distribution between soluble and membrane-bound fractions), as well as in their localization on the sperm membrane. Expression of regions of the 2B1 protein in recombinant bacterial cells has allowed a preliminary mapping of the 2B1 epitope, and has provided more definitive information on the endoproteolytic processing of 2B1 during epididymal transit.
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