Till date

Spatial dynamics of chromosome territory (CT) during DNA damage response (DDR)

We had shown earlier that certain gene-rich CTs relocate to newer locations and revert back to native positions in mammalian fibroblasts during DDR. In the recent work, we uncovered the role of gamma-H2Ax signalling, nuclear myosin recruitment in CT relocations. Apical kinases such as ATM, ATR, DNAPK and sensor proteins such as PARP-1 are essential for promoting nuclear myosin recruitment to chromatin following DNA damage. Interestingly, abrogation of gamma-H2Ax signalling leads to failure of such nuclear myosin recruitment to chromatin, either of which is essential for mediating CT relocations. Our current model describing the underlying mechanism of changes in CT dynamics points out to the functional coupling between DDR kinases/sensors, leading to gamma-H2Ax signalling in turn facilitating nuclear myosin recruitment to chromatin, all of which act concertedly in bringing about DDR response leading to dynamic changes in CT positions.

Mugdha Kulashrestha, Ishita Mehta (DAE-UM-CBS), Pradeep Kumar & BJRao

Till date

Replication restart critically depends on ATR signaling in mammalian nuclei

Hydroxyurea (HU) treated cells exhibit classic responses of replication stress. Short treatment (2h) of HU leads ssDNA breaks whereas long treatment (24h) leads to dsDNA breaks causing two distinct types (early versus late) of replication stress (RS) responses, where ATR, ATM respectively act to facilitate restart biology. Using specific chemical inhibitors that target ATR kinase, we studied the role of ATR kinase in early replication restart biology. We asked whether inhibiting ATR prior to or after HU treatment gives rise to distinct mechanistic functional insights of ATR. Active ATR is essential for rapid spike of gamma-H2Ax during HU mediated RS. If ATR is inhibited following HU mediated RS, heightened gamma-H2Ax response persists, unabated, which is independent of ATM. Interestingly, ATR inhibition prior to HU treatment enables bypass of S-phase checkpoint, whereas the same following HU treatment leads to G2/M checkpoint avoidance, suggesting different roles of ATR in different time phase of HU mediated RS. We also show that prior inhibition of ATR to HU treatment results in less of replication fork collapse than later inhibition of ATR to HU treatment. Our studies, for the first time, therefore indicate, two temporally separable functional phases of ATR action during early RS.

Debadrita Bhattacharya & BJRao

Till date

Cellular and tissue homeostasis defects in Drosophila carrying ATM or Rad51 null mutations

Whole animal null mutants of ATM or Rad51 exhibit several tissue specific developmental defects, both leading to cell-death as well as accompanying adaptive compensatory cellular proliferations. We established that abrogation of Rad51 or atm in Drosophila leads to larval as well as pupal lethality. Interestingly, ATM mutation leads to about 80% of the cells in the wing imaginal disc tissue undergoing apoptosis due to endogenous damages. As expected, the damaged cells failed to express gammaH2av foci as ATM was inactive, but interestingly, PH3 levels rise high in the tissue around dying cells. The cells which escape apoptosis stain very high for P53, an anti-apoptotic marker. Upon staining for wingless, a mitogenic signal, in ATM mutant and comparing it with controls, we found that the boundaries of wingless expression were disrupted and high zoom imaging showed some ectopic expression of wingless marker. In contrast, Rad51 mutants seem to exhibit p53 and rpr independent cellular compensatory proliferation and validate the role of JNK pathway required for tissue homeostasis. We also found out Rad51 protein localizes to centrosomes in cell cycle dependent manner in early embryo of Drosophila. Future direction is geared towards obtaining more detailed mechanistic description of how ATM and Rad51 proteins lead to such contrasting cellular effects.

Chaitali Khan, Sonia Ray, Naini Chakraborty, Champakali Ayyub & BJRao

Till date

Computational analyses of chromosomal organization during eukaryotic evolution

Eukaryotic cells show largely either Rabl (tethered to nuclear membrane) or radial (non-randomly packed) chromosome territory (CT) organization in the interphase nuclei. The evolutionary basis of these two organizational designs has been elusive. In the current study, we computationally modeled genomes from lower to higher eukaryotic nuclei. We discovered that Rabl nuclei, largely associated with lower eukaryotes, exhibit uniformly high gene-density, relatively small chromosomes whereas, in contrast, higher eukaryotes contain radial chromosomes, which are much larger and contain highly variable gene-density. We simulated a minimal model of interactive chromosomes by assessing the relationship between two novel computational constructs, namely, effective protein coding gene density (EPGD) and effective chromosome length (ECL). These two parameters were computed for all pairs of chromosomes in a eukaryotic organism, and compared the same across several lower to higher eukaryotes. The relationship between these two computational constructs uncovered important differences between Rabl versus radial chromosomal organization: Rabl chromosome pairs showed a linear regression between these two parameters, whereas the radial nuclei showed non-linear clustered pattern of regression. A hierarchical decomposition of the pattern, in radial nuclei, uncovered spatially segregated pattern of radial chromosomes, which was consistent with experimentally observed pattern. Interestingly, the relationship between EPGD versus ECL also revealed a hybrid pattern between Rabl versus radial in a few eukaryota such as Aves and Reptilia. We are now testing an Insilco experiment to assess how large number of micro-chromosomes in such hybrid-patterns might drive the transition from a classical Rabl to radial chromosomal organization during eukaryotic evolution.

Sarosh Fatakia, Ishita Mehta (DAE-MU-CBS) & BJRao

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Mechanism of nuclear PARP-1 action in mammalian cells

In this project, we are studying Human foreskin fibroblast cells (SV-40 transformed): GMU6 (PARP +ve cell lines); GM-Sip (Stable PARP-1 knockout cell lines); and GMRSip (PARP-1 replenished cell lines). All of these cell lines are grown under hygromycin selection. We are testing the mechanistic basis of how PARP-1 knockout cells adapt to survive the loss of such crucial cellular regulator as PARP-1, the most abundant NAD+ requiring nuclear enzyme. Immunostaining showed that nucleolin punctas in GM-Sip cells (PARP-/-) increase significantly as compared to that of GM-U6 cells (PARP control) and the puncta numbers revert back to normal level when PARP-1 restored in the mutant cells (GM-Rsip). We confirmed the same, by an immunoblot analyses for nucleolin protein and Q-PCR for nucleolin RNA. As expected of nucleolin upregulation, an anti-apoptotic regulator, we detected reduced apoptotic response during H2O2 stress induced oxidative stress in PARP -/- cells compared to the control cells. However, PARP-/- cells did exhibit delayed DNA repair as revealed by higher steady-state level of ss-DNA strand breaks. When we analyzed P-Chk1, it was found that PARP-/- cells seem to avoid check-point control by down regulating the checkpoint kinase. We are now assessing detailed mechanistic angles to understand the nature of adaptive state PARP-/- cells have reached during genotoxic stress responses.

Soumajit Saha, Ishita Mehta (DAE-UM-CBS), Dr. Girish Shah (CHUL, Quebec, Canada) & BJRao

Till date

Circadian regulation of carbon concentrating mechanism (CCM) and photorespiration (PR) genes expression in Chlamydomonas reinhardtii cells

We showed earlier that CCM gene expression is not only under the control of inorganic carbon supply to the cells during light, but also the dark exposure conditions. Several important CCM genes show transcriptional upregulation specifically in dark, but get the proteins expressed and localized to pyrenoid, the final site of CCM in chloroplast, only in light. We are now testing the co-regulation by dark and light cycles of a set of PR genes and compare the same with CCM gene expression. It appears that PR and CCM genes are tightly co-regulated in the same set of conditions, suggesting that such set of genes are perhaps subject to the same master-regulatory control at transcriptional level.

Srikanth Tirumani & BJRao