Isha is a su(Hw) mRNA-binding protein required for gypsy insulator function.

Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here, we utilized RNA-affinity pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.

M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity.

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.

The zinc-finger protein CLAMP promotes gypsy chromatin insulator function in Drosophila.

Chromatin insulators are DNA-protein complexes that establish independent higher-order DNA domains to influence transcription. Insulators are functionally defined by two properties: they can block communication between an enhancer and a promoter, and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. Here, we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is knocked down, gypsy-dependent enhancer-blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap, particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, whereas depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity.

Two way controls of apoptotic regulators consign DmArgonaute-1 a better clasp on it.

Argonaute family proteins are well conserved among all organisms. Its role in mitotic cell cycle progression and apoptotic cell elimination is poorly understood. Earlier we have established the contribution of Ago-1 in cell cycle control related to G2/M cyclin in Drosophila. Here we have extended our study in understanding the relationship of Ago-1 in regulating apoptosis during Drosophila development. Apoptosis play a critical role in controlling organ shape and size during development of multi cellular organism. Multifarious regulatory pathways control apoptosis during development among which highly conserved JNK (c-Jun N-terminal kinase) pathway play a crucial role. Here we have over expressed Ago-1 in Drosophila eye and brain by employing UAS (upstream activation sequence)-GAL4 system under the expression of eye and brain specific driver. Over expression of Ago-1 resulted in reduced number of ommatidia in the eye and produced smaller size brain in adult and larval Drosophila. A drastic reversal of the phenotype towards normal was observed upon introduction of a single copy of the dominant negative mutation of basket (bsk, Drosophila homolog of JNK) indicating an active and physical involvement of the bsk with Ago-1 in inducing developmental apoptotic process. Further study showed that Ago-1 stimulates phosphorylation of JNK through transforming growth factor-ß activated kinase 1- hemipterous (Tak1-hep) axis of JNK pathway. JNK phosphorylation results in up regulation of pro-apoptotic genes head involution defective (hid), grim & reaper (rpr) and induces activation of Drosophila caspases (cysteinyl aspartate proteinases);DRONC (Death regulator Nedd2-like caspase), ICE (alternatively Drice, Death related ICE-like caspase) and DCP1 (Death caspase-1) by inhibiting apoptotic inhibitor protein DIAP1 (Death-associated inhibitor of apoptosis 1). Further, Ago-1 also inhibits miR-14 expression to trigger apoptosis. Our findings propose that Ago-1 acts as a key regulator in controlling cell death, tumor regression and stress response in metazoan providing a constructive bridge between RNAi machinery and cell death.

Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

HDAC inhibitor misprocesses bantam oncomiRNA, but stimulates hid induced apoptotic pathway.

Apoptosis or programmed cell death is critical for embryogenesis and tissue homeostasis. Uncontrolled apoptosis leads to different human disorders including immunodeficiency, autoimmune disorder and cancer. Several small molecules that control apoptosis have been identified. Here, we have shown the functional role of triazole derivative (DCPTN-PT) that acts as a potent HDAC inhibitor and mis-express proto onco microRNA (miRNA) bantam. To further understanding the mechanism of action of the molecule in apoptotic pathway, a series of experiments were also performed in Drosophila, a well known model organism in which the nature of human apoptosis is very analogous. DCPTN-PT mis processes bantam microRNA and alters its down regulatory target hid function and cleavage of Caspase-3 which in turn influence components of the mitochondrial apoptotic pathway in Drosophila. However regulatory microRNAs in other pro-apoptotic genes are not altered. Simultaneously, treatment of same molecule also affects the mitochondrial regulatory pathway in human tumour cell lines suggesting its conservative nature between fly and human. It is reasonable to propose that triazole derivative (DCPTN-PT) controls bantam oncomiRNA and increases hid induced apoptosis and is also able to influence mitochondrial apoptotic pathway.

Bisindole-PBD regulates breast cancer cell proliferation via SIRT-p53 axis.

In a previous study we reported the role of potent bisindole-PBD conjugate as an inclusion in the arsenal of breast cancer therapeutics. In breast cancer cell proliferation, PI3K/AKT/mTOR pathway plays a crucial role by prosurvival mechanism that inhibits programmed cell death. Here, 2 breast cancer cells lines, MCF-7 and MDA-MB-231 were treated with Vorinostat (suberoylanilide hydroxamic acid / SAHA) and bisindole-PBD (5b). We have investigated the effect on PI3K/AKT/mTOR pathway and SIRT expression including epigenetic regulation. There was consistent decrease in the level of PI3K, AKT, mTOR proteins upon treatment of 5b in both MCF-7 and MDA-MB-231 cell lines compared to untreated controls. Treatment with caspase inhibitor (Q-VD-OPH) confirmed that the effect of 5b on PI3K signaling was ahead of apoptosis. Real time PCR and western blot analysis showed profound reduction in the mRNA and protein levels of SIRT1 and SIRT2. Molecular docking studies also supported the interaction of 5b with various amino acids of SIRT2 proteins. Treatment with 5b caused epigenetic changes that include increase of acetylated forms of p53, increase of histone acetylation at p21 promoter as well as decrease in methylation state of p21 gene. Compound 5b thus acts as SIRT inhibitor and cause p53 activation via inhibition of growth factor signaling and activation of p53 dependent apoptotic signaling. This present study focuses bisindole-PBD on epigenetic alteration putting 5b as a promising therapeutic tool in the realm of breast cancer research.

Drosophila oncogene Gas41 is an RNA interference modulator that intersects heterochromatin and the small interfering RNA pathway.

Glioma amplified sequence41 (Gas41) is a highly conserved putative transcription factor that is frequently abundant in human gliomas. Gas41 shows oncogenic activity by promoting cell growth and viability. In the present study, we show that Gas41 is required for proper functioning of RNA interference (RNAi) machinery in the nuclei, although three basic structural domains of RNAi components PAZ, PIWI and dsRNA with respect to binding are absent in the structural sequences. Variations of structural domains are highly conserved among prokaryotes and eukaryotes. Gas41 interacts with cytological RNase III enzyme Dicer1 both biochemically and genetically. However, Drosophila Gas41 functions as chromatin remodeler and interacts with different heterochromatin markers and repeat-induced transgene silencing by modulating position effect variegation. We also show that transcriptional inactive Gas41 mutant interferes with the functional assembly of heterochromatin-associated proteins, dimethylated lysine 9 of histone H3 and heterochromatic protein 1 in developing embryos. A reduction of heterochromatic markers is accompanied by the mini-w promoter sequence in Gas41 mutants. These findings suggest that Drosophila Gas41 guides the repeat associated gene silencing and the Dicer1 interaction, thereby depicting a new role for Gas41. Gas41 is a critical RNAi component. In Drosophila, Gas41 plays a dual role. On the one hand, it appears to participate with Dicer 1 in the RNAi pathway and, alternatively, it also participates in repeat-induced gene silencing by accumulating heterochromatin proteins at the mini-w array promoters. Therefore, it represents an intriguing and apparently paradoxical new finding in RNA technology with respect to the process of heterochromatin gene silencing.

Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle.

Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of 'exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of 'endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of 'endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

Drosophila Argonaute-1 is critical for transcriptional cosuppression and heterochromatin formation.

Argonaute-1 (Ago-1) plays a crucial role in gene regulation and genome stability via biogenesis of small non-coding RNAs. Two "Argonaute" family genes, piwi and Ago-2 in Drosophila are involved in multiple silencing mechanisms in the nucleus, transgene cosuppression, long-distant chromosome interaction, nuclear organization and heterochromatin formation. To investigate whether Ago-1 also plays a similar role, we have generated a series of Ago-1 mutations by excising P element, inserted in the Ago-1 promoter (Ago-1k08121). AGO-1 protein is distributed uniformly in the nucleus and cytosol in early embryos but accumulated predominantly in the cytoplasm during the gastrulation stage. Repeat induced silencing produced by the mini-white (mw) array and transcriptional cosuppression of non-homologous transgenes Adh-w/w-Adh was disrupted by Ago-1 mutation. These effects of Ago-1 are distict from its role in microRNA processing because Dicer-1, a critical enzyme for miRNA biogenesis, has no role on the above silencing. Reduction of AGO-1 protein dislodged the POLYCOMB, EZ (enhancer of zeste) and H3me3K27 binding at the cosuppressed Adh-w transgene insertion sites suggesting its role in Polycomb dependent cosuppression. An overall reduction of methylated histone H3me2K9 and H3me3K27 from the polytene nuclei precisely from the mw promoters was also found that leads to concomitant changes in the chromatin structure. These results suggest a prominent role of Ago-1 in chromatin organization and transgene silencing and demonstrate a critical link between transcriptional transgene cosuppression, heterochromatin formation and chromatin organization. We propose Drosophila Ago-1 as a multifunctional RNAi component that interconnects at least two unrelated events, chromatin organization in the nucleus and microRNA processing in the cytoplasm, which may be extended to the other systems.

The role of MOF in the ionizing radiation response is conserved in Drosophila melanogaster.

In Drosophila, males absent on the first (MOF) acetylates histone H4 at lysine 16 (H4K16ac). This acetylation mark is highly enriched on the male X chromosome and is required for dosage compensation in Drosophila but not utilized for such in mammals. Recently, we and others reported that mammalian MOF, through H4K16ac, has a critical role at multiple stages in the DNA damage response (DDR) and double-strand break repair pathways. The goal of this study was to test whether mof is similarly required for the response to ionizing radiation (IR) in Drosophila. We report that Drosophila mof mutations in males and females, as well as mof knockdown in SL-2 cells, reduce post-irradiation survival. MOF depletion in SL-2 cells also results in an elevated frequency of metaphases with chromosomal aberrations, suggesting that MOF is involved in DDR. Mutation in Drosophila mof also results in a defective mitotic checkpoint, enhanced apoptosis, and a defective p53 response post-irradiation. In addition, IR exposure enhanced H4K16ac levels in Drosophila as it also does in mammals. These results are the first to demonstrate a requirement for MOF in the whole animal IR response and suggest that the role of MOF in the response to IR is conserved between Drosophila and mammals.

Synthesis of aryl-substituted naphthalene-linked pyrrolobenzodiazepine conjugates as potential anticancer agents with apoptosis-inducing ability.

A library of new aryl-substituted naphthalene C8-linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates with various linker architectures were designed, synthesized, and evaluated for their anticancer activity against a panel of 11 human cancer cell lines. All 32 conjugates show anticancer potential, with some of them exhibiting particularly high activity (0.01-0.19 µM). Thermal denaturation studies showed effective DNA binding capacity relative to DC-81. In assays for biological activity relating to cell-cycle distribution, these PBD conjugates induce G0/G1-phase arrest and also cause an increase in the levels of p53 and caspase-9 proteins, followed by apoptotic cell death. One conjugate in particular is the most promising candidate of the series, with the potential to be selected for further studies, either alone or in combination with existing anticancer therapies.

The RNA helicase Rm62 cooperates with SU(VAR)3-9 to re-silence active transcription in Drosophila melanogaster.

Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. We show that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase SU(VAR)3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of SU(VAR)3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range.

Sub-cellular internalization and organ specific oral delivery of PABA nanoparticles by side chain variation.

BACKGROUND: Organic nanomaterials having specific biological properties play important roles in in vivo delivery and clearance from the live cells. To develop orally deliverable nanomaterials for different biological applications, we have synthesized several fluorescently labelled, self-assembled PABA nanoparticles using possible acid side chain combinations and tested against insect and human cell lines and in vivo animal model. Flurophores attached to nanostructures help in rapid in vivo screening and tracking through complex tissues. The sub-cellular internalization mechanism of the conjugates was determined. A set of physio-chemical parameters of engineered nanoskeletons were also defined that is critical for preferred uptake in multiple organs of live Drosophila. RESULTS: The variability of side chains alter size, shape and surface texture of each nanomaterial that lead to differential uptake in human and insect cells and to different internal organs in live Drosophila via energy dependent endocytosis. Our results showed that physical and chemical properties of C-11 and C-16 acid chain are best fitted for delivery to complex organs in Drosophila. However a distinct difference in uptake of same nanoparticle in human and insect cells postulated that different host cell physiology plays a critical role in the uptake mechanism. CONCLUSIONS: The physical and chemical properties of the nanoparticle produced by variation in the acid side chains that modify size and shape of engineered nanostructure and their interplay with host cell physiology might be the major criteria for their differential uptake to different internal organs.

4-N-pyridin-2-yl-benzamide nanotubes compatible with mouse stem cell and oral delivery in Drosophila.

p-aminobenzoic acid (PABA), a structural moiety of many commercial drugs, is self-assembled with linker alkyl side chains to form tubular nanostructures. The tubes exhibited fluorescence either intrinsic or from fluorescent molecules embedded in the wall during self-assembly. Uptake and inter-cellular delivery of the conjugated nanotubes in human cancer cells and in mouse embryonic stem cells were demonstrated by fluorescence imaging and flow cytometry. Biocompatibility, cytotoxicity and clearance were monitored both ex vivo in mouse multipotent embryonic stem cells and in vivo in adult Drosophila. Accumulation of nanotubes had no adverse effects and abnormalities on stem cell morphology and proliferation rate. A distinct distribution of two separate nanotubes in various internal organs of Drosophila interprets that accumulation of nanomaterials might be interdependent on the side chain modifications and physiological settings of cell or tissue types. Unlike carbon nanomaterials, exposure of PABA nanotubes does not produce any hazards including locomotion defects and mortality of adult flies. Despite differential uptake and clearance from multiple live tissues, the use of self-assembled nanotubes can add new dimensions and scope to the development of dual-purpose oral carriers for the fulfilment of many biological promises.