Functional diversification of yeast telomere associated protein, Rif1, in higher eukaryotes.

BACKGROUND: Telomeres are nucleoprotein complexes at the end of linear eukaryotic chromosomes which maintain the genome integrity by regulating telomere length, preventing recombination and end to end fusion events. Multiple proteins associate with telomeres and function in concert to carry out these functions. Rap1 interacting factor 1 (Rif1), was identified as a protein involved in telomere length regulation in yeast. Rif1 is conserved upto mammals but its function has diversified from telomere length regulation to maintenance of genome integrity. RESULTS: We have carried out detailed bioinformatic analyses and identified Rif1 homologues in 92 organisms from yeast to human. We identified Rif1 homologues in Drosophila melanogaster, even though fly telomeres are maintained by a telomerase independent pathway. Our analysis shows that Drosophila Rif1 (dRif1) sequence is phylogenetically closer to the one of vertebrates than yeast and has identified a few Rif1 specific motifs conserved through evolution. This includes a Rif1 family specific conserved region within the HEAT repeat domain and a motif involved in protein phosphatase1 docking. We show that dRif1 is nuclear localized with a prominent heterochromatin association and unlike human Rif1, it does not respond to DNA damage by localizing to damaged sites. To test the evolutionary conservation of dRif1 function, we expressed the dRif1 protein in yeast and HeLa cells. In yeast, dRif1 did not perturb yeast Rif1 (yRif1) functions; and in HeLa cells it did not colocalize with DNA damage foci. CONCLUSIONS: Telomeres are maintained by retrotransposons in all Drosophila species and consequently, telomerase and many of the telomere associated protein homologues are absent, including Rap1, which is the binding partner of Rif1. We found that a homologue of yRif1 protein is present in fly and dRif1 has evolutionarily conserved motifs. Functional studies show that dRif1 responds differently to DNA damage, implying that dRif1 may have a different function and this may be conserved in other organisms as well.

Nuclear matrix proteome analysis of Drosophila melanogaster.

The nucleus is a highly structured organelle and contains many functional compartments. Although the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat, although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we found structural proteins, chaperones, DNA/RNA-binding proteins, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. Comparison of the two-dimensional profile of NuMat proteome from different developmental stages of Drosophila embryos showed that less than half of the NuMat proteome is constant, and the rest of the proteins are stage-specific dynamic components. These NuMat dynamics suggest a possible functional link between NuMat and embryonic development. Finally, we also showed that a subset of NuMat proteins remains associated with the mitotic chromosomes, implicating their role in mitosis and possibly the epigenetic cellular memory. NuMat proteome analysis provides tools and opens up ways to understand nuclear organization and function.

Repeat performance: how do genome packaging and regulation depend on simple sequence repeats?

Non-coding DNA has consistently increased during evolution of higher eukaryotes. Since the number of genes has remained relatively static during the evolution of complex organisms, it is believed that increased degree of sophisticated regulation of genes has contributed to the increased complexity. A higher proportion of non-coding DNA, including repeats, is likely to provide more complex regulatory potential. Here, we propose that repeats play a regulatory role by contributing to the packaging of the genome during cellular differentiation. Repeats, and in particular the simple sequence repeats, are proposed to serve as landmarks that can target regulatory mechanisms to a large number of genomic sites with the help of very few factors and regulate the linked loci in a coordinated manner. Repeats may, therefore, function as common target sites for regulatory mechanisms involved in the packaging and dynamic compartmentalization of the chromatin into active and inactive regions during cellular differentiation.

Novel motifs distinguish multiple homologues of Polycomb in vertebrates: expansion and diversification of the epigenetic toolkit.

BACKGROUND: Polycomb group (PcG) proteins maintain expression pattern of genes set early during development. Although originally isolated as regulators of homeotic genes, PcG members play a key role in epigenetic mechanism that maintains the expression state of a large number of genes. Polycomb (PC) is conserved during evolution and while invertebrates have one PC gene, vertebrates have five or more homologues. It remains unclear if different vertebrate PC homologues have distinct or overlapping functions. We have identified and compared the sequence of PC homologues in various organisms to analyze similarities and differences that shaped the evolutionary history of this key regulatory protein. RESULTS: All PC homologues have an N-terminal chromodomain and a C-terminal Polycomb Repressor box. We searched the protein and genome sequence database of various organisms for these signatures and identified approximately 100 PC homologues. Comparative analysis of these sequences led to the identification of a novel insect specific motif and several novel and signature motifs in the vertebrate homologue: two in CBX2 (Cx2.1 and Cx2.2), four in CBX4 (Cx4.1, Cx4.2, Cx4.3 and Cx4.4), three in CBX6 (Cx6.1, Cx6.2 and Cx6.3) and one in CBX8 (Cx8.1). Additionally, adjacent to the chromodomain, all the vertebrate homologues have a DNA binding motif - AT-Hook in case of CBX2, which was known earlier, and 'AT-Hook Like' motif, from this study, in other PC homologues. CONCLUSION: Our analysis shows that PC is an ancient gene dating back to pre bilaterian origin that has not only been conserved but has also expanded during the evolution of complexity. Unique motifs acquired by each homologue have been maintained for more than 500 millions years indicating their functional relevance in boosting the epigenetic 'tool kit'. We report the presence of a DNA interaction motif adjacent to chromodomain in all vertebrate PC homologues and suggest a three-way 'PC-histoneH3-DNA' interaction that can restrict nucleosome dynamics. The signature motifs of PC homologues and insect specific motif identified in this study pave the way to understand the molecular basis of epigenetic mechanisms.

CD137L- and RANKL-mediated reverse signals inhibit osteoclastogenesis and T lymphocyte proliferation.

Members of the tumor necrosis factor-related family of ligands and receptors appear to be critical regulators of osteoclastogenesis and various cellular responses in T cells. In the present study, we have investigated CD137L and RANKL (receptor activator of nuclear factor (NF)-kappaB ligand)-induced biological responses in osteoclasts and T cells, respectively. Osteoclast-like cells were generated from murine bone marrow in the presence of RANKL and monocyte-macrophage colony-stimulating factor (M-CSF). RAW264.7 cells (murine monocytic cell line) constitutively express CD137L. Ligation of CD137L with anti-CD137L mAb (TKS-1) inhibits RANKL-induced osteoclast formation in a dose-dependent manner. Bone marrow cells expressed CD137L only when induced by treatment with M-CSF. In bone marrow cells, cross-linking of CD137L with anti-CD137L mAb (TKS-1) inhibits M-CSF/RANKL-evoked formation of multi-nucleated osteoclasts. Further we examined RANKL-mediated regulation of T cell proliferation. Both mouse CD4(+) and CD8(+) T cells expressed RANKL following their activation by anti-CD3 Ab and anti-CD137 Ab. Ligation of RANKL with OPG-Fc, the decoy receptor for RANKL, inhibited both mouse CD4(+) and CD8(+) T cell proliferation. From the above results, we suggest that the cellular responses in cell-to-cell interactions between T cells and osteoclasts are regulated through reciprocal regulations of CD137/CD137L and RANK/RANKL interactions.