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

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

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

Pioneering Activity of the C-Terminal Domain of EBF1 Shapes the Chromatin Landscape for B Cell Programming

Lymphopoiesis requires the activation of lineage-specific genes embedded in naive, inaccessible chromatin or in primed, accessible chromatin. The mechanisms responsible for de novo gain of chromatin accessibility, known as "pioneer" function, remain poorly defined. Here, we showed that the EBF1 C-terminal domain (CTD) is required for the regulation of a specific gene set involved in B cell fate decision and differentiation, independently of activation and repression functions. Using genome-wide analysis of DNaseI hypersensitivity and DNA methylation in multipotent Ebf1(-/-) progenitors and derivative EBF1wt- or EBF1ΔC-expressing cells, we found that the CTD promoted chromatin accessibility and DNA demethylation in previously naive chromatin. The CTD allowed EBF1 to bind at inaccessible genomic regions that offer limited co-occupancy by other transcription factors, whereas the CTD was dispensable for EBF1 binding at regions that are occupied by multiple transcription factors. Thus, the CTD enables EBF1 to confer permissive lineage-specific changes in progenitor chromatin landscape

YY1-mediated enhancer-promoter communication in the immunoglobulin μ locus is regulated by MSL/MOF recruitment

Summary: The rearrangement and expression of the immunoglobulin μ heavy chain (Igh) gene require communication of the intragenic Eμ and 3′ regulatory region (RR) enhancers with the variable (VH) gene promoter. Eμ binding of the transcription factor YY1 has been implicated in enhancer-promoter communication, but the YY1 protein network remains obscure. By analyzing the comprehensive proteome of the 1-kb Eμ wild-type enhancer and that of Eμ lacking the YY1 binding site, we identified the male-specific lethal (MSL)/MOF complex as a component of the YY1 protein network. We found that MSL2 recruitment depends on YY1 and that gene knockout of Msl2 in primary pre-B cells reduces μ gene expression and chromatin looping of Eμ to the 3′ RR enhancer and VH promoter. Moreover, Mof heterozygosity in mice impaired μ expression and early B cell differentiation. Together, these data suggest that the MSL/MOF complex regulates Igh gene expression by augmenting YY1-mediated enhancer-promoter communication

Biallelic inactivation of the NF1 tumour suppressor gene in juvenile myelomonocytic leukaemia: Genetic evidence of driver function and implications for diagnostic workup

: Juvenile myelomonocytic leukaemia (JMML) is characterized by gene variants that deregulate the RAS signalling pathway. Children with neurofibromatosis type 1 (NF-1) carry a defective NF1 allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild-type allele. Here we examined the two-hit concept in leukaemic cells of 25 patients with JMML and NF-1. Ten patients with JMML/NF-1 exhibited a NF1 loss-of-function variant in combination with uniparental disomy of the 17q arm. Five had NF1 microdeletions combined with a pathogenic NF1 variant and nine carried two compound-heterozygous NF1 variants. We also examined 16 patients without clinical signs of NF-1 and no variation in the JMML-associated driver genes PTPN11, KRAS, NRAS or CBL (JMML-5neg) and identified eight patients with NF1 variants. Three patients had microdeletions combined with hemizygous NF1 variants, three had compound-heterozygous NF1 variants and two had heterozygous NF1 variants. In addition, we found a high incidence of secondary ASXL1 and/or SETBP1 variants in both groups. We conclude that the clinical diagnosis of JMML/NF-1 reliably indicates a NF1-driven JMML subtype, and that careful NF1 analysis should be included in the genetic workup of JMML even in the absence of clinical evidence of NF-1

LANGUAGE IN INDIA Strength for Today and Bright Hope for Tomorrow An Economic Analysis of Women Entrepreneurs in Namakkal District, Tamil Nadu

Abstract The paper analyses the economics of women entrepreneurs in Namakkal District, Tamil Nadu. The paper examines the importance and problems of women entrepreneurs in Tamil Nadu and India. In particular, two blocks of Namakkal District is selected for the study where 240 women entrepreneurs are randomly selected, i.e. 120 from Tiruchengode and another 120 from Namakkal block. The profile of the women entrepreneurs are discussed in the first part of the paper and second part examines the economics of women entrepreneurs. That is, nature of business, form of business, sources of funds, sales turnover, net income and return on investment are examined. Of the selected women entrepreneurs, majority of them engage in business

Spontaneous remission and loss of monosomy 7 : a window of opportunity for young children with SAMD9L syndrome

Monosomy 7 is the most common cytogenetic abnormality in pediatric myelodysplastic syndrome (MDS) and associated with a high risk of disease progression. However, in young children, spontaneous loss of monosomy 7 with concomitant hematologic recovery has been described, especially in the presence of germline mutations in SAMD9 and SAMD9L genes. Here, we report on our experience of close surveillance instead of upfront hematopoietic stem cell transplantation (HSCT) in seven patients diagnosed with SAMD9L syndrome and monosomy 7 at a median age of 0.6 years (0.4-2.9). Within 14 months from diagnosis, three children experienced spontaneous hematological remission accompanied by a decrease in monosomy 7 clone size. Subclones with somatic SAMD9L mutations in cis were identified in five patients, three of whom attained hematological remission. Two patients acquired RUNX1 and EZH2 mutations during the observation period, of whom one progressed to MDS with excess of blasts (MDS-EB). Four patients underwent allogeneic HSCT at a median time of 26 months (14-40) from diagnosis for MDS-EB, necrotizing granulomatous lymphadenitis, persistent monosomy 7, and severe neutropenia. At last follow-up, six patients were alive, while one passed away due to transplant-related causes. These data confirm previous observations that monosomy 7 can be transient in young children with SAMD9L syndrome. However, they also indicate that delaying HSCT poses a substantial risk of severe infection and disease progression. Finally, surveillance of patients with SAMD9L syndrome and monosomy 7 is critical to define the evolving genetic landscape and to determine the appropriate timing of HSCT