Interactome of vertebrate GAF/ThPOK reveals its diverse functions in gene regulation and DNA repair

GAGA associated factor (GAF) is a sequence-specific DNA binding transcription factor that is evolutionarilyconserved from flies to humans. Emerging evidence shows a context-dependent function of vertebrate GAF(vGAF, a.k.a. ThPOK) in multiple processes like gene activation, repression, and enhancer-blocking. Wehypothesize that context-dependent interaction of vGAF with a diverse set of proteins forms the basis for themultifunctional nature of vGAF. To this end, we deciphered the protein–protein interactome of vGAF andshow that vGAF interacts with chromatin remodelers, RNA metabolic machinery, transcriptional activators/repressors, and components of DNA repair machinery. We further validated the biological significance of ourprotein–protein interaction data with functional studies and established a novel role of vGAF in DNA repairand cell-survival after UV-induced DNA damage. One of the major risk factors for skin cutaneous melanoma isprolonged exposure of UV and subsequent DNA damage. vGAF is highly expressed in normal skin tissue.Interestingly, our analysis of high-throughput RNA-sequencing data shows that vGAF is heavily downregulated across all major stages of skin cutaneous melanoma suggesting its potential as a diagnostic biomarker.Taken together, our study provides a plausible explanation for the diverse gene regulatory functions of vGAFand unravels its novel role in DNA repair.

Genomic organization of Polycomb Response Elements and its functional implication in Drosophila and other insects

The epigenetic memory is an essential aspect of multicellular organisms to maintain several cell types and their geneexpression pattern. This complex process uses a number of protein factors and specific DNA elements within thedevelopmental cues to achieve this. The protein factors involved in the process are the Polycomb group (PcG)members, and, accordingly, the DNA sequences that interact with these proteins are called Polycomb ResponseElements (PREs). Since the PcG proteins are highly conserved among higher eukaryotes, including insects, andfunction at thousands of sites in the genomes, it is expected that PREs may also be present across the genome. However,the studies on PREs in insect species, other thanDrosophila, is currently lacking.We took a bioinformatics approach todevelop an inclusive PRE prediction tool, ‘PRE Mapper’, to address this need. By applying this tool on the Drosophilamelanogaster genome, we predicted[20,000 PREs. When compared with the available PRE prediction methods, thistool shows far better performance by correctly identifying the in vivo binding sites of PcG proteins, identified bygenome-scale ChIP experiments. Further analysis of the predicted PREs shows their cohabitation with chromatindomain boundary elements at several places in the Drosophila genome, possibly defining a composite epigeneticmodule. We analysed 10 insect genomes in this context and find several conserved features in PREs across the insectspecies with some variations in their occurrence frequency. These analyses leading to the identification of PRE in insectgenomes contribute to our understanding of epigenetic mechanisms in these organisms.

Finding clues to the riddle of sex determination in zebrafish.

How sex is determined has been one of the most intriguing puzzles in biology since antiquity. Although a fundamental process in most metazoans, there seems to be myriad of ways in which sex can be determined – from genetic to environmental sex determination. This variation is limited mainly to upstream triggers with the core of sex determination pathway being conserved. Zebrafish has gained prominence as a vertebrate model system to study development and disease. However, very little is known about its primary sex determination mechanism. Here we review our current understanding of the sex determination in zebrafish. Zebrafish lack identifiable heteromorphic sex chromosomes and sex is determined by multiple genes, with some influence from the environment. Recently, chromosome 4 has been identified as sex chromosome along with few sex-linked loci on chromosomes 5 and 16. The identities of candidate sex-linked genes, however, have remained elusive. Sex in zebrafish is also influenced by the number of meiotic oocytes in the juvenile ovary, which appear to instruct retention of the ovarian fate. The mechanism and identity of this instructive signal remain unknown. We hypothesize that sex in zebrafish is a culmination of combinatorial effects of the genome, germ cells and the environment with inputs from epigenetic factors translating the biological meaning of this interaction.

Dynamics of nuclear matrix proteome during embryonic development in Drosophila melanogaster.

Embryonic development is a complex and dynamic process that involves spatiotemporal expression of genes in a highly coordinated manner. Multiple levels of nuclear architecture maintain the fidelity of gene expression programme. One of the components of nuclear architecture, which is believed to play an important role in regulation of gene expression, is the nuclear matrix (NuMat). Many studies over the past few years have tried to analyse the components of this non-chromatin scaffolding of the nucleus and have provided evidences of its structural and functional complexity. However, the relationship of NuMat with the process of embryonic development still remains poorly understood. Here, we report a comparative analysis of the NuMat proteomes of early and late stage Drosophila melanogaster embryos and show that 65% of the NuMat proteome is dynamic during development. Our study establishes links between the dynamics of nuclear architecture and embryonic development and provides tools to further understand the process such as cellular differentiation in the context of higher-order nuclear organization.

Tone up your chromatin and stay young.

There are several observations that are difficult to explain using classical Mendelian inheritance. These include position effect variegation, transvection, telomere position effect and imprinting. These phenomena are now known to be based on changes in chromatin structure and epigenetic modifications that can be transmitted to daughter cells. It is, therefore, possible that abnormal chromatin packaging can lead to abnormal cellular processes that ultimately disturb the cell’s sustenance. For example, de-repression of telomeric heterochromatin can lead to cellular senescence (Kennedy et al. 1997; Moazed 2001). This indicates that during aging the ability to compensate for cellular damage fails to meet the need for repair or replenishment of the maintenance factors (Steinkraus et al. 2008). Non-dividing cells maintain their pool to repair internal damage by optimizing the supply of chromatin components. Actively dividing cells need massive synthesis of such components. Eukaryotic cells cannot undergo cell division indefinitely and, therefore, have an inherent lifespan, called the replicative lifespan. After a certain number of replications, the mother cell accumulates aging-related damage, which ultimately causes it to cease further divisions. In multicellular organisms, each tissue has a characteristic replicative capacity (Cavalier-Smith 1978). Much of the information about aging has come from the yeast Saccharomyces cerevisiae (Steinkraus et al. 2008). Several key factors affecting aging are conserved from yeast to worms to mammals (Kaeberlein et al. 1999; Wood et al. 2004; Kenyon 2010). These include calorie restriction, radical oxygen species (ROS)-dependent signal transduction, sirtuin-mediated anti-aging functions and the target of rapamycin (TOR) pathway. Budding yeasts try to prevent the passing of cellular components with aging-related damage such as damaged proteins, or extra-chromosomal DNA fragments, to the daughter cells during cell division (Kaeberlein et al. 1999). As a cell undergoes many divisions, it becomes increasingly difficult for the cell to prevent passage of this load to the daughter cells. So the accumulation of damaged components can itself act as a trigger for replicative senescence (Steinkraus et al. 2008). Consequently, factors that reduce this load assume the role of safeguarding against the aging process. Many of the factors that prevent aging can alter gene expression via chromatin modifications. Therefore, chromatin modifiers such as Sir2, the Polycomb group (PcG) of proteins (Jacobs et al. 1999), histone deacetylases and histone chaperones (Chen et al. 2008; Dang et al. 2009) are being related to aging processes. Can key structural components of chromatin such as the histone proteins be directly involved in replicative aging? A recent report demonstrates a direct connection between the levels of histones and the lifespan of budding yeast (Feser et al. 2010). It was shown that aging cells have low histone protein levels, resulting in loose packaging of the genome and hence inappropriate gene regulation.

Promoter search and strength of a promoter: two important means for regulation of gene expression in Escherichia coli.

Search for a promoter element by RNA polymerase from the extremely large DNA base sequence is thought to be the slowest and rate-determining for the regulation of transcription process. Few direct experiments we described here which have tried to follow the mechanistic implications of this promoter search. However, once the promoter is located, transcription complex, constituting mainly the RNA polymerase molecule and few transcription factors has to unidirectionally clear the promoter and elongate the RNA chain through a series of steps which altogether define the initiation of transcription process. Thus, it appears that the promoter sequence acts as a trap for RNA polymerase associated with a large binding constant, although to clear the promoter and to elongate the transcript such energy barrier has to be overcome. Topological state of the DNA, particularly in the neighbourhood of the promoter plays an important role in the energetics of the whole process.