Towards construction and validation of an ends-in recombination system in Escherichia coli

Homologous recombination is the primary DNA repair pathway in bacteria and it is immensely important in repairing DNA double strand breaks. Components of the homologous recombination pathway have been well conserved throughout evolution as an essential part of cell survival. Homologous recombination plays an important role in cellular processes like DNA repair as well as exchange of genetic information through chromosomal crossover. During homologous recombination, DNA strand exchange leads to formation of a heteroduplex joint between the invading and displaced DNA strands. This hetereoduplex joint is called a Holliday Junction. Resolution of the Holliday Junction proceeds via one of two pathways. In the presence of RuvC and/or RecG, Holliday Junction resolution proceeds via a “cut and paste” pathway where the invading DNA strand replaces a region of homologous DNA on the target DNA. In the absence of RuvC and RecG, Holliday Junction resolution takes place via a “copy and paste” pathway during which DNA synthesis needs to be primed at Holliday Junction intermediates formed during strand invasion. In an effort to separate this myriad of different requirements, I have attempted to develop a novel “ends-in” recombination assay system using E. coli as a model organism. This ends-in system would allow recombinant molecule formation by DNA synthesis of approximately 200 to 2000 bp size interval between the two converging ends of an invading linear dsDNA substrate oriented just like the greek letter Ù, but with the arms pointing inwards. In this study, a number of linear dsDNA assay templates were constructed and analyzed. All the constructs had two “arms” of homology to the chromosome pointing inwards i.e. in the ends-in orientation. Using this ends-in system, it was demonstrated that the presence of chi (Crossover Hotspot Initiator) sites was an important requirement for ends-in recombination in wild type E. coli cells. Our studies also showed that ends-in homologous recombination did not occur if chi sites were placed at or very near to the ends of the incoming linear dsDNA molecule, suggesting that the chi site recognition is efficient only if the incoming dsDNA has chi sites internal to the ends. Moreover, it was shown that neither RuvC nor RecG were required for successful recombinant product formation using the ends-in assay. This finding reinforces previous observations that suggest the idea that Holliday Junctions can be resolved independent of both RuvC and RecG

Coupling Protein Catabolism to Lifespan and Reproduction in Caenorhabditis elegans

There is an undisputable link between aging and reproduction and it has both puzzled and fascinated biologists for decades. Evolutionary biologists suggest that the rate of aging depends on the complex tug-of-war between maintenance of the soma and maintenance of the germ cells. The former is essential for longevity while the latter is essential for transmitting genetic information from parents to progeny. Any perturbation in the fertility and fecundity of C. elegans influences lifespan and vice-versa. Interestingly, germline deficient animals have increased resistance to environmental and proteotoxic stress. All eukaryotic cells reproduce for a finite amount of time before irreversibly ceasing reproduction, a phenomenon called reproductive senescence. Recent research has been aimed at trying to elucidate the genetic factors that regulate reproductive and post-reproductive lifespan. As part of this ongoing process, the initial part of my work aims to characterize the increased reproductive lifespan of a C. elegans mutant that is deficient for the gene rer-1. I further demonstrate that rer-1 mutants show a higher level of autophagy which is responsible for the enhanced reproductive lifespan of these mutants. Aging is thought to be a stochastic process, and cessation of reproduction is one of the biological hallmarks of aging. Although both reproduction and aging are well studied processes, there is very little mechanistic understanding of how these processes are connected and coordinated. The latter part of this study aims to answer some of these questions about the cross-talk between the germ cells and somatic tissue in C. elegans using a panel of sterile hermaphrodites impaired for specific stages of reproduction. My findings show that cessation of fertilization triggers a signalling cascade from the germ cells to the soma and that this signalling is brought about by steroid hormones, presumably synthesized by the somatic gonad. In actively reproducing worms, the forkhead transcription factor DAF-16 drives expression of vha genes which encode a multi-subunit proton pump that is responsible for maintaining lysosomal acidity. Nuclear exclusion of DAF-16 in post-reproductive worms co-ordinately reduces the expression of vha genes resulting in lysosomal alkalinization that culminates in acute loss of fitness and contributes to organismal senescence. My data shows a plausible mechanistic pathway by which lysosomal acidity is regulated via gonad to soma signalling in young (reproducing) animals

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis. Rhabdomyosarcomas are tumours blocked in myogenic differentiation, which despite the expression of master muscle regulatory factors, including MYOD, are unable to differentiate. Here, the authors show that SNAI2 is upregulated by MYOD through super enhancers, binds to MYOD target enhancers, and arrests differentiation

A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan.

Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exiting mitosis. Yet pathways that normally limit the number of cell divisions remain poorly characterized. Here we describe a screen of a collection of 3762 single gene mutants in the yeast Saccharomyces cerevisiae, accounting for 2/3 of annotated yeast ORFs, to search for mutants that undergo an atypically high number of cell divisions. Many of the potential longevity genes map to cellular processes not previously implicated in mitotic senescence, suggesting that regulatory mechanisms governing mitotic exit may be broader than currently anticipated. We focused on an ER-Golgi gene cluster isolated in this screen to determine how these ubiquitous organelles integrate into mitotic longevity. We report that a chronic increase in ER protein load signals an expansion in the assembly of autophagosomes in an Ire1-independent manner, accelerates trafficking of high molecular weight protein aggregates from the cytoplasm to the vacuoles, and leads to a profound enhancement of daughter cell production. We demonstrate that this catabolic network is evolutionarily conserved, as it also extends reproductive lifespan in the nematode Caenorhabditis elegans. Our data provide evidence that catabolism of protein aggregates, a natural byproduct of high protein synthesis and turn over in dividing cells, is among the drivers of mitotic longevity in eukaryotes

Rapid Nuclear Exclusion of Hcm1 in Aging Saccharomyces cerevisiae Leads to Vacuolar Alkalization and Replicative Senescence

The yeast, Saccharomyces cerevisiae, like other higher eukaryotes, undergo a finite number of cell divisions before exiting the cell cycle due to the effects of aging. Here, we show that yeast aging begins with the nuclear exclusion of Hcm1 in young cells, resulting in loss of acidic vacuoles. Autophagy is required for healthy aging in yeast, with proteins targeted for turnover by autophagy directed to the vacuole. Consistent with this, vacuolar acidity is necessary for vacuolar function and yeast longevity. Using yeast genetics and immunofluorescence microscopy, we confirm that vacuolar acidity plays a critical role in cell health and lifespan, and is potentially maintained by a series of Forkhead Box (Fox) transcription factors. An interconnected transcriptional network involving the Fox proteins (Fkh1, Fkh2 and Hcm1) are required for transcription of v-ATPase subunits and vacuolar acidity. As cells age, Hcm1 is rapidly excluded from the nucleus in young cells, blocking the expression of Hcm1 targets (Fkh1 and Fkh2), leading to loss of v-ATPase gene expression, reduced vacuolar acidification, increased α-syn-GFP vacuolar accumulation, and finally, diminished replicative lifespan (RLS). Loss of vacuolar acidity occurs about the same time as Hcm1 nuclear exclusion and is conserved; we have recently demonstrated that lysosomal alkalization similarly contributes to aging in C. elegans following a transition from progeny producing to post-reproductive life. Our data points to a molecular mechanism regulating vacuolar acidity that signals the end of RLS when acidification is lost

The RNA Demethylase ALKBH5 Maintains Endoplasmic Reticulum Homeostasis by Regulating UPR, Autophagy, and Mitochondrial Function

Eukaryotic cells maintain cellular fitness by employing well-coordinated and evolutionarily conserved processes that negotiate stress induced by internal or external environments. These processes include the unfolded protein response, autophagy, endoplasmic reticulum-associated degradation (ERAD) of unfolded proteins and altered mitochondrial functions that together constitute the ER stress response. Here, we show that the RNA demethylase ALKBH5 regulates the crosstalk among these processes to maintain normal ER function. We demonstrate that ALKBH5 regulates ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain the calcium flux between the ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently, mitochondrial biogenesis. Thus, these findings reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness

A genome scale screen for isolating mutants with extended mitotic lifespan in the yeast <i>S</i>. <i>cerevisiae</i>.

<p><b>A.</b> The screen work flow. The design rationale is detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s001" target="_blank">S1 Fig</a>. <b>B.</b> Cy5:Cy3 signal ratios of mutants maintained in a dividing state for 16 days. Ranked values were log₂ normalized and projected. Of the starting collection of 3762, 52 mutants that maintained negative log₂ Cy5:Cy3 signal ratios at both day 6 and day 16 and displayed signal ratios <-2.3 at day 16 were classified as potentially long-lived (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s003" target="_blank">S3 Fig</a>). <b>C.</b> Broad functional clustering of the putative longevity genes isolated in this screen using GO Ontology. Genes that function in protein modification and trafficking across the ER-Golgi network are outlined.</p

Enhanced clearance of protein aggregates in yeast and worm <i>rer1</i> mutants.

<p><b>A.</b> Live cell imaging of α-syn-GFP after a 4 hr induction in 0.2% galactose. TM (0.02 μg/ml) was added to the cultures after an initial induction in galactose and cells were imaged 2 hours later. In a marked contrast to untreated cells, the GFP foci were largely vacuolar in <i>rer1Δ</i> or wild type cells treated with TM (arrows). <b>B.</b> GFP immunoblot in whole cell lysates prepared from cells in (<b>A)</b>. The intravacuolar proteolysis of α-syn-GFP generates the transiently stable GFP moiety detected as a discrete fragment in SDS-PAGE. <b>C.</b> Fluorescent images of transgenic hermaphrodites stably expressing α-syn-GFP from a body wall muscle promoter (<i>Punc-</i>_<i>54</i>::<i>α-syn</i>::<i>GFP</i>). Arrows denote a subset of the α-syn-GFP foci. Worms co-expressing heat shock protein torsinA (<i>tor-2</i>) in the same cells (<i>P</i>_{<i>unc-54</i>}::<i>tor-2</i>) served as a positive experimental control. The average number of α-syn-GFP foci per worm scored from digitized Z-stacked images ± s.e.m are plotted in <b>D</b> (n >20).</p

ER stress extends reproductive lifespan in yeast and worms.

<p><b>A.</b> Mitotic lifespan of wild type yeast grown on YPD supplemented with the indicated concentrations of TM. Mean mitotic lifespans for WT (18.5) and cells treated with TM at 0.005 (24.5) and 0.02 (27.5) μg/ml were statistically significant (p-values<0.01). <b>B.</b> Reproductive lifespan in worms with or without dietary supplementation with TM. <b>C.</b> Live cell imaging of GFP-Rer1 in wild type yeast and <i>ire1Δ</i> mutants treated with DMSO or after a 2 hr exposure to 0.02 μg/ml TM. (Box) In tandem with inducing canonical UPR, ER stress increases trafficking from ER to Golgi, reflected in the Golgi redistribution of GFP-Rer1 in cells treated with TM. Conversely, deleting <i>RER1</i> increases ER load denoted by the high basal UPR in <i>rer1Δ</i> mutants. <b>D</b>. Mitotic lifespan of WT (16.5 days) and isogenic <i>rer1∆</i> (26.5), <i>ire1∆</i> (16.5) and <i>rer1∆ ire1∆</i> (24.5) mutants at 30°C. Differences between WT mean mitotic lifespan were significant for <i>rer1∆</i> and <i>rer1∆ ire1∆</i> mutants (p-values<0.001).</p