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Summer Research Fellowship Programme of India's Science Academies

EXPLORING THE ROLE OF INO80 AS A REGULATOR OF INTERGENEIC miRNA GENE EXPRESSION IN HUMAN CELLS

MALAVIKA E SANTHOSH

Bharathiar University, Coimbatore. Tamil Nadu.

Guided by:

Prof. Vani Bhrahmachari

Dr. B R Ambedkar Centre for Biomedical Research, Delhi University.

ABSTRACT

INO80, a member of SWI/SNF2 family of ATPases functions as an ATP-dependent chromatin remodeling protein. The human INO80 is functionally very versatile and is involved in replication, DNA repair and stress recovery. Recruitment of INO80 to promoters of target genes is mediated by Yin Yang 1(YY1) protein in humans. But based on the presence of a highly conserved DNA binding domain (DBINO) that recognizes an 11-mer consensus sequence in INO80 subfamily it was predicted that INO80 directly interacts with DNAexclusive of YY1, which was proven in vitro and in vivo . MicroRNAs (miRNA) are small non-coding RNAs that act as regulators, leading either mRNA cleavage or translational repression of their target genes. It is difficult to understand the transcriptional regulation of miRNAs genes, because of difficulty in predicting genuine transcriptional start sites (TSSs) of miRNA genes and different biogenesis pathways involved in the transcription of intergenic and intronic miRNA genes. For this study, multiple alternative TSS for intergenic miRNA was taken from PROmiRNA, a miRNA promoter annotation approach based on deepCAGE data and sequence features. Investigations on the role of INO80 in the expression of intergenic miRNA genes in human cells have been performed in our lab. In silico analysis was used to detect INO80 and YY1 binding motifs 2kb upstream of predicted transcription start sites (TSS) for miRNA genes. Those miRNA genes having exclusive INO80 binding motif were selected for further analysis. Finally, miRNAs whose mRNA targets are already validated and which are known to be endogenously expressed in HeLa cells were prioritized for the experimental validation. The expression of the selected miRNA and their target mRNA was checked using quantitative RT-PCR under 2 experimental conditions: overexpression and knockdown of INO80. This project aims to explore the possible role of INO80 in the transcriptional regulation of intergenic microRNA genes which would subsequently affect their target mRNA in the human cells. In addition to human cells in culture, Drosophila is also used as a model in our lab. I also have learnt basic methods of culturing the Drosophila, recognizing mutants, dissection, DAPI staining, nail polish imprint to study fly ommatidia pattern arrangement, Agarose gel electrophoresis, PCR amplification and optimization, primer designing, restriction digestion, plasmid DNA isolation, genomic DNA isolation from human cells and Drosophila, competent cell preparation and transformation using plasmid vectors.

Keywords: INO80, intergenic miRNA, DBINO, YY1

Abbreviations

INO80human Inositol auxotrophy 80
DNMTsDNA methyltransferases
KMTshistone methyl transferases
HATshistone acetyl transferases
H3Khistone3 lysine
RT PCRreal time PCR
PCRpolymerase chain reactoion
ulmicrolitre
YY1ying yang 1
miRNAmicro RNA
TSStranscription start sites
PMTpost translational modifications
hINO80human INO80

INTRODUCTION /LITERATURE REVIEW

Background/Rationale

Chromatin Remodeling

Eukaryotes are more advanced in structure and function than prokaryotes due to genome complexity and the compartmentalization of their cellular processes into different organelles. The huge eukaryotic genome inside a nucleus is maintained by the DNA organization in a compact chromatin structure, which is brought about by association of DNA with histone proteins. The nucleosome is stabilized by protein-protein interactions within the histone octamer and also by electrostatic and hydrogen bonding between DNA and histones  . This compaction of genomic DNA is important to maintain the genome complexity with several nuclear processes such as DNA replication and transcription.

chromatin.png
     DNA Packaging: Nucleosomes and Chromatin (Annunziato 2008)

    In eukaryotes, the packaging of the DNA into chromatin results in a ground state where transcription is inhibited, preventing the movement of RNA polymerase and the binding of transcription factors. These inhibitory structures therefore require the remodeling of the chromatin to facilitate transcription. A chromatin is dynamically and tightly regulated and many factors and mechanisms participate in this process (​Fig 1​​.2). These include DNA modification, histone post-translational modifications (PTMs) and chromatin remodeling. The compaction of the genetic material into chromatin generally leads to repression of transcription, and genes and their regulatory regions need to be made accessible to transcription factors and chromatin associated proteins in order for gene expression to occur.   

    The major form of DNA modification is methylation of the DNA at the 5-position of cytosine in CpG dinucleotides. The methyl mark is placed by enzymes known as DNA methyltransferases (DNMTs) that transfer a methyl group from S- adenosyl methionine to DNA (for a summary of the functions of DNA methylation and DNMTs (,Jurkowska and Jeltsch 2011). In mammals, DNA methyltransferases can be grouped into de novo DNMTs (DNMT3A and DNMT3B) and maintenance DNMTs (DNMT1).

    met.png
      Summary of the functions of DNA methylation and DNMTs  , Jurkowska and Jeltsch 2011). Mechanisms of chromatin regulation. Actively transcribed genes are found in open euchromatin and are associated with histone acetylation (H3/H4Kac) and tri-methylation of H3 lysine 4 (H3K4me3) at promotors, and tri-methylation of H3 lysine 36 (H3K36me3) over the gene body. Nucleosome positioning at promoters is regulated by ATP-dependent chromatin remodelers (SWI/SNF). Silenced genes are associated with densely packed heterochromatin marked by DNA methylation (5mC) and H3 lysine 9 tri-methylation (H3K9me3) or in silenced polycomb domains marked by tri-methylation of H3 lysine 27 (H3K27me3). Chromatin modifications are deposited by chromatin modifying enzymes such as DNA methyltransferases (DNMTs), histone acetyl transferases (HATs) or histone methyl transferases (KMTs) and removed by de-modifying enzymes such as histone deacetylases (HDACs) or histone demethylases (KDMs). 
      metet.png
        Outcomes of chromatin remodeling (Clapier and Cairns 2009)
        table.PNG
          Known histone modifications

          INO80: A chromatin remodeler

          INO80, was first identified in yeast for inositol biosynthesis. It is a member of SWI/SNF2 family of ATPases remodeling proteins. The unique feature of this subfamily is the presence of a long insertion between IV and V motif of ATPase domain (split ATPase domain). The human INO80 is functionally very versatile and is involved in replication, DNA repair and stress recovery. Recruitment of INO80 to promoters of target genes is mediated by Yin Yang 1(YY1) protein in humans (Y Cai et al., 2007). But based on the presence of a highly conserved DNA binding domain (DBINO) that recognizes a 11-mer consensus sequence in INO80 subfamily, it was predicted that INO80 directly interacts with DNA exclusive of YY1, which was proven in vitro and in vivo (   Bakshi et al 2004 and Mendiratta et al 2016). The INO80 complex was first described in yeast comprising 12 subunits (X. Shen et al 2000). The Multiple modes of regulation of the human INO80 SNF2 ATPase by subunits of the INO80 chromatin-remodeling complex was shown and along with the main catalytic INO80 ATPase subunit, the human INO80 complex shares subunits like AAA+ ATPases, Tip49a/b, actin-related proteins (Arp4, Arp5 and Arp8) and the Ies2 and Ies6 proteins, with its counterpart in yeast (Lu Chen et al 2013). Other additional metazoa-specific factors present in human INO80 complex including Uch37 (deubiquitinating enzyme) and GLI-Kruppel family zinc finger transcription factor YY1 (Yin Yang 1).

          ino80.png
            Modular Assembly of INO80 Complex (Lu Chen et al 2013)

            The presence of DNA binding domain (DBINO) in INO80 led to the hypothesis that INO80 might bind to DNA independently without getting recruited by YY1. By exploring the DNA binding properties of INO80, a conserved consensus 11-mer DNA binding motif 5’-CCCCGTCAGCC-3’ with the core sequence being the 7-mer 5’- GTCAGCC-3’ was identified (Mendiratta et al 2016). This was done using SELEX (Systematic Evolution of Ligands by exponential Enrichment) approach along with further studies revealing the association of INO80 with DNA both in vitro (by electrophoretic mobility assay) and in vivo (by chromatin immunoprecipitation). Sequential ChIP experiment showed the interaction of INO80 with DNA independently of YY1 for a few genes. The human genome was mined for consensus 7-mer sequence upstream of both protein coding and non-coding genes (including microRNA and lncRNA genes). The presence of YY1 binding motif was also looked for and it was found that for many genes INO80 binding motif was present exclusively without the presence of YY1 binding motif. This could show the possibility of independent gene regulation by INO80. Reporter assay by cloning of consensus sequence upstream and downstream of Luciferase gene showed that INO80 causes the downregulation of gene expression (Mendiratta et al 2016). Chromatin Immunoprecipitation (ChIP) revealed that histone markers around INO80 binding motif consisted of repressive markers like H3K27me3 (in concordance with the results of the reporter assay) in majority of the genes analyzed. All of the above findings have established a preliminary basis by which INO80 could be controlling gene regulation. In silico analysis has shown the presence of INO80 binding motif at large numbers of protein coding as well as non-coding genes. Further investigation into the role of INO80 in the regulation of such genes and its consequences might lead us to explore the role of INO80 as a transcriptional regulator. This work is an attempt to confirm the work done by Deepak Pant (Pant 2018, dissertation report) in the lab. The intergenic miRNA list was downloaded from miRStart online database. A list of miRNAs and their multiple alternative TSS were taken from supplementary material of ‘PROmiRNA: a new miRNA promoter recognition method uncovers the complex regulation of intronic miRNAs’ by Marsico et al. (2013). The upstream 2 kb sequences for the promoter coordinates was downloaded from UCSC Genome table (Build: hg19). The upstream sequences were searched for 7-mer INO80 binding motif using a code written in Perl programming language. Validated targets were checked using online databases like miRBase and miRTarBase. MiRNAs having exclusive INO80 binding motif were selected using Venny 2.0.2.

            MicroRNA: Biogenesis and Function

            MicroRNAs (miRNAs) are small non-coding RNAs and act as regulators, leading either mRNA cleavage or translational repression of their target genes. This negative regulatory mechanism is important in controlling diverse biological processes such as carcinogenesis, cellular proliferation, and differentiation. miRNA biogenesis (​Fig 1​.5) starts with the miRNA genes being transcribed to generate long primary transcripts (pri-miRNAs), which are first cropped by RNase-III-type enzyme Drosha to form the hairpin intermediates (pre-miRNAs) in the nucleus. Drosha forms a large (500–650 kDa) complex, known as the Microprocessor complex, together with its essential cofactor DGCR8/Pasha, which contains two dsRNA-binding domains. Pre-miRNA then gets exported to the cytoplasm by exportin-5, which is a member of the Ran-dependent nuclear transport receptor family. Following arrival in the cytoplasm, pre-miRNAs are subjected to the second processing step, which is carried out by Dicer, the cytoplasmic RNase-III-type protein to form mature miRNA (Kim 2014​). Several models have been proposed to describe the cleavage of pre-miRNA into mature miRNA stating the involvement of other proteins like TBRP (HIV Transactivating Response RNA Binding Protein), PACT (Protein Kinase-R Activating Protein) and Ago2 (MacFarlane et al 2010). The result of this cleavage is a linear imperfect double stranded RNA duplex. InmiRNA:miRNA* duplex, one strand mediates silencing (guide strand) and the other one is degraded (passenger strand). The mechanism of Degradation of passenger strand is still unclear. The guide or mature miRNA along with the RISC (RNA Induced Silencing Complex) which consists of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2 bind to the target mRNA to induce gene silencing. Eight such RISC complexes have been found in humans. Silencing by miRNA can be due to two processes: cleavage of mRNA mediated by Ago2 or the more common, inhibition of translation. Cleavage by Ago2 requires nearly perfect base pairing between the seed sequence and 3’-UTR. For translational inhibition, multiple complementary sites are needed with moderate base pairing at each site.

            Since miRNAs play important roles in pathology, their targeting and transcriptional regulation are critical to RNA research. Although miRNA target prediction has remarkable advances in recent years, the transcriptional regulation of it is still inadequate. miRNA genes belong to class II genes, which are transcribed by RNA polymerase II. Genes that code for microRNAs can be found between protein coding genes (intergenic miRNAs) or within introns of protein coding genes (intragenic/intronic). It is implied that intragenic miRNAs and their host genes share the common TSSs and express simultaneously. However, for intergenic miRNAs, their own promoter regions dramatically vary in distance and limit the reliability of predicted results. Moreover, due to the lower expression level of miRNAs, it is more difficult to perform experimental validation and obtain full-length cDNAs of miRNA primary transcripts than coding genes. Therefore, a systematic approach to accurately identify miRNA TSS is necessary to solve these problems.

            mirna.png
              miRNA biogenesis (miRstart database)

              Statement of the Problems

              Preliminary studies have established the role of chromatin remodeling complexes in regulating expression of non-coding RNAs. It is already known that INO80 is involved in shaping long non-coding RNA transcriptome (Alcid & Tsukiyama 2014) Studies have shown correlation between the expression levels of miRNAs with chromatin remodeling proteins. The presence of INO80 binding motif upstream of microRNA genes suggests that INO80 might be playing an important role in shaping up the miRNA expression profiles in cells belonging to different tissues and at different developmental stages, leading to the regulation of their target mRNA.

              Objectives of the Research

              To validate the role of hINO80 in regulating the transcription of miRNAs gene through expression analysis by the induction of overexpression and knockdown of hINO80.

              METHODOLOGY

              Workflow:

              1. Cell Culture

              2. Overexpression/ Downregulation of INO80 in HeLa Cells

              3. RNA Isolation from HeLa cells

              4. cDNA Preparation

              5. PCR reactions for Standardizing Primers 

              6. Quantitative Real Time PCR for expression analysis of selected miRNA gene. 

              Cell Culture Protocol

              HeLa cell line was used in this study. The HeLa cells were cultured in Dulbecco’s Modified Eagle Medium low glucose complete media supplemented with 10% FBS and 1X antibiotic-antimycotic solution and incubated at 37˚C in 5%CO2 in a humidified environment. The cells were maintained at up to 80-90% confluency and then sub cultured. Sub culturing was performed by draining the complete media from the flask, followed by washing the adhered cells with 3 ml of 1X PBS (pH 7.4). The cells were then treated with 1 ml of 0.5% Trypsin-EDTA. Following the treatment, the trypsin-EDTA was drained out and discarded and the flask containing the treated cell is incubated at 37˚C. After 5-10 min, the flask was removed from the incubator and the dislodged cells are resuspended in 1 ml DMEM complete media.

              Transfection Experiment

              For carrying out transfections, 2x105 cells were grown on 6 well plates. To overexpress INO80 in HeLa cells full length hINO80 vector was used. The transfected and empty vector group acted as controls. For downregulation of INO80, siRNA against hINO80 was used. Lipofectamine 2000 reagent was used at 2.5 µl for INO80 siRNA and control (Scrambled) siRNA whereas 3 µl was used for hINO80 and pcDNA empty vector per 6 well plate. The transfection groups were made in the following manner:

              • Untransfected (UT)
              • Transfection with empty (pcDNA) vector
              • INO80 Overexpressed (O/E) by using INO80 full length clone
              • Transfection with control (Scrambled) siRNA (CSiRNA)
              • INO80 knockdown(K/D) using siRNA against INO80

              RNA Isolation

              • Discarded the culture media.
              • Lysed the cells by adding 1ul of Trizol Solutions (TS) reagent to each well. Incubated for 3mins and pipetted until complete lysis.
              • Added 200ul of chloroform/ml of TS.
              • Mixed well and incubated at room temp (RT) for 2-3 mins.
              • Centrifuged at 12000G for 15 mins at 4˚C.
              • Removed aqueous phase and transferred to a new tube.
              • Added 0.5 ml of isopropanol.
              • Stored at RT for 10 mins.
              • Centrifuged at 12000g for 10 mins at 4˚C.
              • Removed supernatant by inverting. Quick spun and excess supernatant was removed by pipette
              • Removed ethanol and air dried the RNA pellet for 30-40 mins.
              • Dissolved the RNA in 25ul RNase free DEPC water.
              • Incubated at 65˚C for 20 mins.
              • Stored at -80˚C.

              cDNA Synthesis:

              • Isolated RNA from two samples was treated with TurboDNasecDNA preparation. The concentration and purity of RNA was determined using nanodrop. 5.5ug RNA/10ul of reaction was treated with DNAase. The mixture was incubated at 37℃ for 40 mins and the DNAse was inactivated at 65℃ in thermocycler. (​Table 1​). Samples were run on 1% gel.
              • Denatured the RNA and random primer hexamer by incubating at 65˚C for 5 minutes in thermocycler and then quick-chilled at -20C for 5mins.
              • Prepared the master mix on ice for cDNA synthesis. Incubated the samples at 25℃ for 5 mins and then at 37℃ for 90 mins. (​Table 2​). The concentration and purity of cDNA was checked using nanodrop.
              • PCR was carried out with gene specific GAPDH primers with amplicon size 163bp to check the reliability of cDNA synthesized (​Table 3​). The PCR products were loaded on 2% gel.
              DNAse treatment  

              Components

              UT(ul)

              pcDNA(ul)

              O/E(ul)

              CSiRNA(ul)

              K/D(ul)

              UT(ul)

              O/E(ul)

              K/D(ul)

              RNA

              4.4

              3

              1.5

              2.4

              3.6

              5.2

              4

              3.5

              10X buffer

              1

              1

              1

              1

              1

              1

              1

              1

              DNAase(1U/ul)

              1.5

              1.5

              1.5

              1.5

              1.5

              1.5

              1.5

              1.5

              Water

              3.1

              4.5

              6

              5.1

              3.9

              3.3

              3.5

              4

              Total volume

              10

              10

              10

              10

              10

              10

              10

              10

              Incubated at 37℃ for 40 mins

              Inactivated DNase at 65℃ for 10 mins

              Random Primer hexamer(0.2ug/ul)

              1

              1

              1

              1

              1

              1

              1

              1

              Incubated at 65℃ for 5 mins

              Quick chilled at -20℃ for 5 mins

              cDNA synthesis  

              Component

              Quantity(ul)

              DNAse treated RNA sample

              10

              5x buffer

              4

              RibolockRNAse inhibitor(20U/ul)

              1

              dNTPs(10mM)

              2

              Revertaid M-mulV reverse transcriptase(200U/ul)

              2

              Incubated at 65℃ for 5 mins

              Incubated at 37℃ for 90 mins

              Stored at -80˚C.

              Polymerase Chain Reaction

              • Standard PCR was done before qPCR with gene specific primers.
              • The following are the sequences of primers, reagents and conditions used in PCR reactions:
              PCR conditions  

              PCR stages

              temperature

              duration

              cycles

              Initial denaturation

              95 °C

              5min

               

              Denaturation

              95 °C

              30 sec

               

              35 cycles

              Annealing

              60 °C

              30sec

              Extension

              72 °C

              30sec

              Final extension

              72 °C

              5 min

               

              PCR reagents  

              PCR Reagents

              volume(µL)

              PCR water (nuclease free)

              11.5

              dNTPs(2mM)

              2

              Buffer(10X)

              2

              Enzyme(5unit/ul)

              0.25

              Forward primer(5picoM/ µL)

              1

              Reverse primer (5picoM/ µL)

              1

              DNA template

              2.5

              Total volume of reaction

              20

              PCR primer sequence  
              miR-193b

              F: 5’-GGTCTCAGAATCGGGGTTTTGA-3’

               

              R: 5’-CCAAAAGCGGGACTTTGAGG-3’

              PCR primer sequence  

              GAPDH

              F: 5’-AGCCTCCCGCTTCGCTCTCT-3’

               

              R: 5’-CGAGGCGCCCAATACGACCA-3

               

              Quantitative Real Time PCR

              RT-PCR reactions were performed to check the levels of selected miRNAs in INO80 overexpressed and INO80 knockdown conditions. The levels of INO80 were also monitored to check whether transfection with INO80 plasmid resulted in increased levels of INO80 mRNA. The mRNA levels of 2 of the targets of our selected miRNAs were also checked. GAPDH was used as an internal control for normalising qPCR results for INO80 and target primers whereas U6 snRNA was used for miRNA primers (​Table 5​). The primer annealing temperatures used for the reactions were 55℃. 

              qPCR internal control primers  

              INO80

              F: 5’-CTGACTCGGCTCAAGTCT-3’

               

              R: 5’-CATGTCTCGCCTCTCCGA-3’

              U6 snRNA

              F: 5’-TCGCTTCGGCAGCACATATA-3’

               

              R: 5’-GGAACGCTTCACGAATTTGC-3’

              GAPDH

              F: 5’-AGCCTCCCGCTTCGCTCTCT-3’

              The reaction mixture was prepared as follows:

              qPCR reagents  

              PCR reagent

              Volume used (ul)

              SYBR green mix

              7.5

              Forward primer

              1

              Reverse primer

              1

              CDNA template(100ng/ul)

              2.5

              water

              2.5

              RESULTS AND DISCUSSION

              In this study we have investigated the role of INO80 protein in the expression of intergenic miRNAs in the human cells. The first part of the project included in silico analysis where we have selected a set of human microRNA genes which could be potential targets for regulation by INO80 and regulatory sequences upstream of intergenic miRNA genes were checked through available in silico tools (Pant 2018).

              In this work, we have focused on hsa-mir-193b which is located on the 16th chromosome. miR-193b is implicated in various kinds of cancer. It is down-regulated in Ewing sarcoma and targets ErbB4 to inhibit anchorage independent growth (Moore et al 2017). It acts as a tumor suppressor and down-regulates proto-oncogenes like Cyclin D1 and ETS1 (Lin SR et al., 2017).

              Experimental Validation of the regulation of miRNA by hINO80 was done by the expression of the precursor transcript for the selected miRNAs in HeLa cells, which was analyzed by qPCR. The expression was compared in the normal cells, cells with over-expression of hINO80 and the hINO80 knock-down cells.

              Capture.PNG
                Expression levels of miR-193b in INO80 O/E and INO80si condition

                In comparison to untransfected Hela controls, the levels of miR-193b showed significant decrease when INO80 was overexpressed. Whereas, the levels showed significant increase upon the siRNA mediated knockdown of INO80. 

                CONCLUSION AND RECOMMENDATIONS

                The miRNAs with upstream INO80 binding motif exclusive of YY1 binding motif had been prioritized in the study. Overexpression of INO80 in Hela cells showed decrease in miR- 193b. This indicated that INO80 is a negative regulator of these miRNA genes. Moreover INO80 down-regulation in HeLa cells showed an increase in the levels of miR-193b when compared to control. This study can be correlated with an earlier study done by Mendiratta et al 2016 ) in which cloning of INO80 binding motif upstream of luciferase gene was done in reporter assay. In that study siRNA against INO80 caused the level of luciferase to increase, which was lower for control. Similarly, our results have also showed the role of INO80’s as a transcriptional repressor.

                Understanding the role of miRNAs in cancer is relevant because differential expression profiles of miRNAs are observed in cancerous tissues when compared to the healthy ones. MiRNAs that are down-regulated in cancer function as tumor suppressors with their targets being proto-oncogenes that are upregulated in cancer. MiR-193b acts like a tumor suppressor and brings down the levels of protooncogenes like Cyclin D1 and ETS1 in human urothelial carcinoma cells   . It is also known to target ErbB4 oncogene in Ewing sarcoma and thereby inhibits anchorage independent growth (Moore et al 2017). Mir-143 has been shown to inhibit cell proliferation and apoptosis in cervical cancer cell lines by targeting HIF-1α   .

                It was shown that on siRNA mediated knockdown of INO80, the progression from G2/M to G1 phase of cell cycle is delayed by Cao L et al. (2015). This was due to the increased levels of cell cycle inhibitor p21 indicating that INO80 negatively regulates p21, which can be linked to cancerous phenotype. A similar approach can be proposed for the transcriptional regulation of miRNA gene expression by INO80.

                By this study, we could interpret that INO80 not only independently regulates the expression of protein coding genes but miRNA genes as well, which had not been shown before and it has enabled us to propose a new mechanism by which INO80 can contribute to cancer progression. This study was done to support and confirm the hypothesiss of the already done work (Pant 2018) where it has been shown that INO80 is a negative regulator of miRNA genes. Clonogenic assay or soft agar assay can be performed in the future to establish a direct correlation between high levels of INO80 and cancer phenotype through a mechanism which involves microRNA down-regulation.

                OTHER TECHNIQUES LEARNT

                • Plasmid DNA isolation from bacterial cells
                • Preparation of competent cells and transformation
                • Genomic DNA isolation from single fly
                • Restriction digestion of plasmid vectors
                • Culturing of Drosophila melanogaster
                • Immunostaining

                Isolation of plasmid DNA from bacterial cells

                Materials required:

                • E coli pUC18 inoculated in LB Medium
                • Solution I-Suspension buffer -Glucose – 50 mM Tris HCl – 25 mM EDTA – 10 mM
                • Solution II -Lysis buffer- NaOH – 0.2N SDS – 1%
                • Solution III - Potassium acetate (5 M) – 60 mL Glacial acetic acid – 11.5 mL Water – 28.5 mL
                • Phenol:Chloroform:IAA (25:24:1)
                • TE buffer Tris HCl – 10 mM EDTA – 1 mM  

                Alkaline lysis solution I

                • 1 M glucose stock solution (50 mL)
                  • Dissolve 9 gram of glucose in 50 mL sterilized de-ion water.
                  • Filter sterilize using membrane millipore (0.20 µM).
                  • Glucose solution is ready to use or store at 4°C cabinet for preservation.
                • 1 M Tris-Cl stock solution (50 mL)
                  • Dissolve 6.057 gram of Tris base in 50 mL sterilized de-ion water
                  • Adjust the pH to the desired value by adding concentrated HCl.
                • 0.5 M EDTA stock solution (100 mL)
                  • Dissolve 14.612 gram of EDTA in 100 mL sterilized de-ion water.
                  • Adjust the pH to 8.0 with NaOH. Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at 15 psi and store at 4°C.

                Alkaline lysis solution II

                • 10 N NaOH stock solution (50 mL) Dissolve 20 gram of NaOH in 50 mL sterilized de-ion water.
                • 1% (w/v) SDS stock solution (30 mL) Dissolve 0.3 gram of SDS in 30 mL sterilized de-ion water. Prepare Solution II afresh and use at room temperature. 

                Alkaline lysis solution III

                • 5 M potassium acetate stock solution (100 mL). Dissolve 49.071 gram of potassium acetate in 100 mL sterilized de-ion water.
                • Store the solution at 4°C and transfer it to an ice bucket just before use.

                Procedure:

                • About 1.5mL of overnight LB broth culture was taken in a microfuge tube.
                • It was centrifuged at 8000rpm for 10 mins at 4°C.
                • The supernatant was discarded and 100 µL ice-cold suspension buffer (solution I) was added. 4) The mixture was then vortexed briefly to break the pellet.
                • To this, 200 µL of freshly prepared lysis buffer was added and mixed gently.
                • Again 150 µL of potassium acetate solution (solution III) was added and mixed.
                • The mixture was centrifuged at 12000 rpm at 4°C for 8 mins.
                • The supernatant was transferred to another microfuge tube.
                • To the contents, equal volume of phenol: chloroform: isoamyl alcohol was added.
                • After inverting the tube several times, the contents were again centrifuged at 12000 rpm for 5 min at 4°C.
                • To the aqueous layer, which was transferred to another tube, equal volume of ice-cold isopropanol was added.
                • It was centrifuged at 12000 rpm for 10 min at 4°C and the supernatant was discarded.
                • To the pellet, 500 µL of 70% ethanol was added and centrifuged at 12000 rpm for 5 mins. Ethanol was discarded and contents were air-dried to facilitate evaporation of ethanol.
                • The DNA pellet was resuspended in 20 µL of sterile water/TE buffer.
                • 5µL of the sample was loaded in 1% Agarose gel and run at 50V for 10 mins. The bands were visualized under UV illumination in the gel documentation system.

                Results: plasmid DNA was isolated and observed using gel documentation system.

                gel1.PNG
                  Plasmid DNA on 1% agarose gel.

                  Preparation of Competent E. coli DH5α and transformation using plasmid vectors (pcDNA3.1)

                  Materials required

                  • LB broth
                  • LB agar plates
                  • CaCl2(0.1M)
                  • Mg Cl2(1M)
                  • Prechilled 50ml polypropylene tubes
                  • Plasmid DNA(PcDNA3.1)
                  • *E. coli DH5alpha Competent cells
                  • Ampicillin(100mg/ml)
                  pcdna.PNG
                    pcDNA map

                     Competent cell preparation:

                    • A single bacterial colony (E. coliDH5α) was picked from a plate that has been incubated for16-22         hours at 37°C.
                    • The colony was transferred into100 mL of Luria broth in a 250 mL flask.
                    • The culture was incubated for 3–4 hours at 37°C with vigorous agitation until its OD600 reaches 0.35-0.4.
                      • Note: To ensure that the culture does not grow to a higher density, measure the OD600 of the culture every 15-20 minutes.
                    • The grown culture was transferred into sterile, disposable, ice-cold 50 mL polypropylene tubes.
                    • Therein the culture was allowed cool to 0° C by storing the tubes on ice for 10 minutes.
                    • The cells were recovered by centrifugation at 4500 rpm for 5 minutes at 4°C.
                    • The medium was decanted from the cell pellets and the tubes were kept in an inverted position on a sterile pad of sterile paper towels for 1 minute to allow the last traces of media to drain out.
                    • Each pellet was re-suspended by swirling or gentle vortexing in 25 mL of ice-cold 0.1M CaCl2 solution and the tubes were kept on ice for 1 hour.
                    • The cells were recovered by centrifugation at 4500 rpm for 5 minutes at 4° C.
                    • Step 7 was repeated.
                    • Each pellet was re-suspended by swirling or gentle vortexing in 30 ml of ice-cold 1M MgCl2 and the tubes were kept on ice for 30-45 minutes.
                    • The cells were recovered by centrifugation at 4500 rpm for 5 minutes at 4° C.
                    • Step 7 was repeated.
                    • About 1 mL of ice-cold 0.1M CaCl2 and 1ml of 60% glycerol (30% working concentration) was added to pellets.
                    • Aliquots of 200 µLwere dispensed and maintained at-70°C. 

                    Bacterial transformation

                    • Thawed aliquots of 200 µL of glycerol stock of competent cells were carefully placed on ice.
                    • About 2 µL of plasmid(pcDNA3.1) solution (concentration1µg/µl) was added to200 µL of competent cells.
                    • The cells were incubated on ice for 30 minutes.
                    • The tubes were quickly transferred to a water bath previously set at 42° C and incubated for 1 minute and then quickly transferred to ice
                    • About 1 mLof Luria broth medium was added to the tube and incubated at 37° C for 30 minute to1 hr.
                    • About 200 µL of culture was spread onto plates containing suitable antibiotic (Ampicillin 1 ug/mL) and the plates were incubated at 37o C for overnight.

                    *E. coli DH5alpha Competent cells are most frequently used E. coli strain for routine cloning applications. he genomic structure of this strain is a singular circular chromosome consisting of 4,686,137 nucleotides, 4359 genes, and 4128 protein encoding genes. This strain also contains plasmids, and has the ability to accept plasmid insertion exceptionally well. In addition to supporting blue/white screening recA1 and endA1 mutations in DH5a™ increase insert stability and improve the quality of plasmid DNA prepared from minipreps. The mutations that the DH5-Alpha strain has are: dlacZ Delta M15 Delta(lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1. lacZ Delta M15 mutation: Allows for blue-white screening for recombinant cells. endA1 mutation: Allows for lower endonuclease degradation which ensures higher plasmid transfer rates. recA1 mutation: reduces homologous recombination for a more stable insert.

                    Isolation of genomic DNA from single Drosophila

                    Materials required

                    • Fruit flies (Drosophila melanogaster)
                    • Eppendorf tubes,pipettes and tips
                    • Hot water bath
                    • Squishing Buffer
                    • Proteinase K
                    • Ice bucket.
                    • Thermocycler and PCR reagents
                    drso.PNG
                      : Fruit fly karyotype (Hales et al 2015)
                      drsos.PNG
                        Drosophila life cycle (Berg Lab. University of Washington)

                         Protocol.

                        • Placed one fly in a 0.25ml tube.Carefully squished the fly for 5-10sec with the help of a pipette tip containing 50ul 0f Squishing Buffer (​Table 1​) without expelling any liquid.
                        • Incubated at 37° C (or room temperature) for 30minutes.
                        • Inactivated the proteinase K by heating at 95° C for 2 minutes.
                        • Held the reaction at 4° C.
                        • Referred ​Table 1​ and 2 to set PCR.
                        • 1.5 % agarose gel electrophoresis was performed.
                        Preparation of Squishing Buffer  
                        ReagentStock concentrationWorking concentrationWorking volume(100ml)

                        Tris-HCl

                        1M

                        10mM

                        1ml

                        EDTA

                        0.5M

                        1mM

                        0.2ml

                        NaCl

                        5M

                        25mM

                        0.5ml

                        Proteinase K

                        10mg/ml

                        200 µg/ml

                         

                        PCR reagents  
                        PCR reagentsTest sampleControl

                        H2O (nuclease free water)

                        12 µl

                        12 µl

                        dNTP (2mM each)

                        2 µl

                        2 µl

                        Buffer (10X)

                        2 µl

                        2 µl

                        Enzyme (5 unit/µl)

                        0.5 µl

                        0.5 µl

                        Primer (5 pmol/µl)

                        2 µl

                        2 µl

                        DNA

                        2 µl

                        -

                        H2O

                        -

                        2 µl

                        Total volume

                        20 µl

                        20 µl

                         PCR conditions  
                        PCR conditionsTemperatureDurationCycles

                        Initial denaturation

                        95 °C

                        10 mis

                         

                        Denaturation

                        95 °C

                        30 sec

                         

                        35 cycles

                        Annealing

                        51 °C

                        30sec

                        Extension

                        72 °C

                        40 sec

                        Final extension

                        72 °C

                        5 min

                         

                         Results:

                        Single fly genomic DNA was isolated and bands with amplicon size 710bp was observed when PCR sample was loaded on 2% agarose gel, using gel documentation system.

                        drso pcr.PNG
                          PCR samples on 2% agarose gel.

                          Restriction digestion of plasmid vector

                          Materials required

                          • EcoR1(10U/ul)
                          • Nde1(20U/ul)
                          • EcoR1/Nde1
                          • Not1(10U/ul)
                          • EcoR1 buffer and “O” buffer
                          • Plasmid Pcas9 DNA (2792.2ng/ul) 

                          Procedure

                          • NEB cutter tool was used to find the restriction enzyme map of the DNA to be digested.
                          • Set up the reaction on ice as in ​Table 4
                          • Incubated the reaction overnight in water bath at 37.
                          • Loaded the sample in 1% agarose gel. The bands were visualized in gel documentation system.
                          Restriction digestion components  

                          component

                          Sample (pcas9) (ul)

                          water(ul)

                          Buffer(ul)

                          Restriction enzyme(ul)

                          pcas9/EcoR1.Nde1

                          2

                          16

                          2

                          1 each

                          pcas9/EcoR1

                          2

                          16

                          2

                          1

                          Pacs9/Nde1

                          2

                          16

                          2

                          1

                          pcas9/Not1

                          2

                          16

                          2

                          1

                          Culturing of Drosophila

                          Preparation of egg laying plates

                          • 3gm of Agar (2.5%) and 3gm of Sucrose (1.5% ) were mixed in 200ml of distilled water.
                          • Boiled for sometimes so that agar is dissolved properly.
                          • The mixture was allowed to cool at room temperature, and then few drops of propionic acid were added.
                          • Appropriate amount of the mixture was poured in small Petri plates and covered slightly so that cover did not develop vapor and stored at 4° C (Figure13).  
                          • Egg laying chamber was prepared and flies were transferred in egg laying chamber for egg laying.
                          • Handling of flies were difficult so they were anesthetized using ether.
                          vials.PNG
                            Drosophila egg laying Agar plate and Maintenance of egg laying chamber. a) egg laying plate. b) yeast paste on egg laying plate. c) preparation of yeast paste. d) egg laying chamber. e)vials containing flyfood.

                             Preparation of fly food

                            • 700ml of fly food was prepared by adding 40gm of Corn flour, 32gm of Jiggery, 4gm of Agar, 12gm ofYeast powder.
                            • The mixture was split into 2 parts of 500ml and 200ml and allowed to boil 2min and three times with proper mixing each time.
                            • The separated mixture was poured together and a final boil of 2min was given.
                            • After cooling of food for 15-20 minutes, 1gm in 3ml ethanol of methy l-4-hydroxybenzoate acid and 3.2ml of propionic acid were added as anti-fungal reagents.
                            • Appropriate amount of food was poured in vials and covered with cheese cloth for 24 hours to avoid contamination. The vials were then covered with cotton plug and stored at 24° C.(Figure24).

                             Drosophila eye examination in neurodegeneration fly models

                            • Embryos were transferred to food vials containing drugs along with the fly food. The embryos were allowed to develop and the eye of adult flies were examined to see the rescue in eye surface and eye pigment degeneration in neurodegeneration fly model.
                            eye.PNG
                              Eye surface degeneration in GMR-GAL4 fly line b) and pigment degeneration in GMR-GAL4:: UAS 127Qflyline. c) nail polish imprint of drosophila eye of neurodegeneration fly model.
                              ins.PNG
                                a), b) BOD maintained at 24 °C for maintaining fly stock. c) fluorescent microscope and GFP scope. 

                                Protocol for Drosophila Larval tissue dissection.

                                Dissection of larval tissue

                                Added a few drops of Phosphate Buffer solution (PBS 1x) onto the slide instead of water to prevent drying of larvae while dissection.

                                Placed the larvae to be dissected.

                                Use two needles to dissect the larvae.

                                Remove cuticle, fat bodies to get salivary glands, CNS (transparent, bilobed structure) along with eye and leg imaginal discs, gut, etc. Fixed the dissected tissue for staining and mounting. 

                                micreo.PNG
                                  Drosophila larval tissue dissection. a) CNS b) salivary gland c) whole gut along with Malpighian tubule.

                                  Protocol for Immunostaining (Drosophila Larval tissue staining)

                                  Fix dissected tissue in fixative for 15 mins.

                                  Wash with PBST three times 10 mins each.

                                  Add 1X blocking solution (blocking solution and PBST in 1:1) and leave for 1 hr.

                                  Remove blocking solution and add primary antibody (anti cut-2B10) with appropriate dilution (1:100 in blocking solution). Incubate at 4℃ in a moist chamber overnight.

                                  Remove the primary antibody and wash the tissue with 1X PBST three times 10 mins each.

                                  Add secondary antibody (anti mouse TRITC) in with appropriate dilution (1:100 in blocking solution) and incubate at room temperature in a moist chamber for 2 hours.

                                  Wash the tissues three times with PBST (10-15 mins each).

                                  Add DAPI with appropriate dilution in PBST (1:2000) and keep for 5 mins.

                                  Wash in PBST(5mins).

                                  Mount the sample in antifade/fluoromount.

                                  Observe under fluorescent microscope. 

                                  Material required

                                  Fixative

                                  • 4% paraformaldehyde in PBS (0.04gm of Paraformaldehyde in 1ml of PBS). Heat in waterbath.

                                  1x PBST (triton-X 100- 0.3%)

                                  • Prepare 1X PBS from 10X PBS (1ml of 10X PBS in 9ml water).
                                  • Add 0.03ml of triton X in 10ml of 1X PBS.

                                  2% BSA solution

                                  • 0.2g of BSA in 10ml of 1X PBS (blocking solution.)
                                  10X PBS Composition 

                                   

                                  Component

                                  Quantity

                                  1

                                  NaCl

                                  40gm

                                  2

                                  KCl

                                  1gm

                                  3

                                  Na2HPo4

                                  7.2gm

                                  4.

                                  KH2Po4

                                  1.2gm

                                  adjust the pH upto 7.4 with NaOH (10N)

                                  Antifade:It is a mounting agent forming a semi-rigid gel with a refractive index ranging from 1.47–1.52. Mounted samples become transparent and can be saved for months with appropriate storage.

                                  Fluoromount-G®: It is a water-soluble compound recommended for slides mounted after a staining procedure having an aqueous final step. Since Fluoromount-G® is water-soluble, the coverslip may be removed by submerging the slide in a PBS solution until the coverslip is loosened. This mounting medium also provides a semi-permanent seal for storage of slide preparation.

                                  aaa.PNG
                                    Fluorescent microscopic (10X) images ofcells CNS of 3rd instarDrosophila larvae.a),c) DAPI and b),d)GFP
                                    sdd.PNG
                                      Fluorescent microscopic images(10x) of Drosophila gut stem cells of 3rd instar larvae a) DAPI b)GFP. Drosophila gut stem cell specific gene(escargot) fused with GFP (esgGal4::UAS GFP).
                                      eed.PNG
                                        Fluorescent microscopic images (40x) of Drosophila gut stem cells of 3rd instar larvae a) DAPI b)GFP c)DAPI-GFP merged. Drosophila gut stem cell specific gene(escargot) fused with GFP (esgGal4::UAS GFP).

                                         ACKNOWLEDGEMENTS

                                        First and foremost, I would like to express my profound gratitude towards Indian academy of Sciences (IAS-INSA-NASI) for giving me this wonderful opportunity to explore science during my summer vacations. I also Thank Authorcafe for providing me an excellent platform to write my report.

                                        Words cannot express my heartiest gratitude towards Prof. Vani Brahmachari for her guidance, encouragement, and support from the very first day I arrived in her lab. It’s my privilege to express my deepest gratitude towards Dr. Akanksha Verma and Dr. Richa Arya for their critical guidance throughout my internship. They had always been with me, with their masterly guidance, valuable suggestions, supervision, and encouragement. I have learnt so many things under their guidance which ultimately helped me in exploring the craze towards science in myself. I express my special thanks to all the lab scholars (Dr. Jayant Maini, Dr. Ankita, Dr. Prachi, Surbhi mam and Kaushik sir), Dr. Susithra, my ACBR friends (Aastha, Richa) who have helped and guided me during the course of my project. Special thanks to my friends Atulya Chandra and Vasudha for all the help, inspiration, and I thank them for being there with me and understanding me so well. I am thankful to ACBR for the inspiring SURP lectures.

                                        I express my sincere gratitude towards my HOD Dr. Vijaya Padma and my mentor Dr. S. Girija for the confidence and faith they have in me.

                                        Words cannot manifest my heartful gratitude towards my friends Pallavi and Manoj who had always been my side from the day first I met them in my university.

                                        I will forever be indebted to my parents, parents-in-laws, my fiancée, sister and sister-in-law for their unconditional love, support, concerns, compromises, and the confidence they always have in me. I hope I will make them proud.

                                        References

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                                        • Shweta Mendiratta, Shipra Bhatia, Shruti Jain, Taniya Kaur, Vani Brahmachari. Interaction of the Chromatin Remodeling Protein hINO80 with DNA. PLOS July 18, 2016.

                                        • Deepak Pant. Investigating the possible role of INO80 in the transcriptional regulation of intergenic microRNA genes in the human genome. Dissertation thesis. 2018.

                                        • Anthony T. Annunziato, Ph.D. (Biology Department, Boston College) DNA Packaging: Nucleosomes and Chromatin. (2008) Nature Education 1(1):26

                                        • Renata Z.Jurkowska , Albert Jeltsch . Review on New concepts in DNA methylation. Cell press.

                                        • Clapier CR, Cairns BR. The biology of chromatin remodeling complexes. Annual Review Biochem. 2009;78:273–304.

                                        • X. Shen, G. Mizuguchi, A. Hamiche, C. Wu. A chromatin remodeling complex involved in transcription and DNA processing. Nature, 406 (2000), pp. 541-54.

                                        • Lu Chen, Ronald C. Conaway, and Joan W. Conaway. Multiple modes of regulation of the human INO80 SNF2 ATPase by subunits of the INO80 chromatin remodeling complex. PNAS. 2013 Dec 17; 110(51): 20497–20502.

                                        • V. Narry Kim. MicroRNA biogenesis: coordinated cropping and dicing. Nature Reviews Molecular Cell Biology Volume 6, pages 376–385 (2005). Volume 39, Issue 7, July 2014, Pages 310-31

                                        • MacFarlane, Leigh-Ann; R. Murphy, Paul. MicroRNA: Biogenesis, Function and Role in Cancer. Current Genomics, Volume 11, Number 7, November 2010, pp. 537-561(25).

                                        • Alcid EA1, Tsukiyama T2. ATP-dependent chromatin remodeling shapes the long noncoding RNA landscape. Genes Dev. 2014 Nov 1; 28(21):2348-60.

                                        • Moore C, Parrish JK, Jedlicka P. MiR-193b, downregulated in Ewing Sarcoma, targets the ErbB4 oncogene to inhibit anchorage-independent growth. PLoS One. 2017 May 18.

                                        • Karen G. Hales, Christopher A. Korey, Amanda M. Larracuente and David M. Roberts. Genetics on the Fly: A Primer on the Drosophila Model System. GENETICS November 1, 2015 vol. 201 no. 3 815-842

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