NiH_Presentation
- 1. Genetic interactors of BRCA2 in BRCA2 deficient tumorigenesis Shrenik Jain
- 2. Breast Cancer as a whole Breast Cancer accounts for 22.9% of all cancers diagnosed worldwide1 Generally affects women more than men in a 100:1 ratio2 It is estimated that 232,340 women will be diagnosed with and 39,620 women will die of cancer of the breast in 20132 Breast Cancer can be caused by a variety of environmental and hereditary factors 2 Statistics from: 1. "World Cancer Report". International Agency for Research on Cancer. 2008. 2. “Breast Cancer Statistics”. Susan G. Komen for the cure. 2013.
- 3. Hereditary Breast Cancer Approximately 5-10% of all Breast Cancer cases are believed to have a genetic basis3 Mutations in the BRCA gene complex have been identified as a leading cause of hereditary breast cancer Inherited BRCA gene mutations are responsible for about 5 percent of total breast cancers4 The BRCA gene complex includes tumor suppressor genes BRCA1 and BRCA2 3 Statistics from: 3. “Hereditary Cancer Syndromes”. The University of Texas MD Anderson Cancer Center. 4. “BRCA Gene test for breast cancer”. The Mayo Clinic.
- 4. BRCA2 BRCA2 is a gene in the BRCA complex that acts as a tumor suppressor by performing an essential role in DNA repair Located on the q arm of the Chromosome 13 in humans The human BRCA2 gene contains 27 exons that code for a protein of 3418 amino acids Experiments have established mice as a suitable model system for the study of BRCA2, despite significant amino acid differences between murine and human genes BRCA2 has been experimentally verified as essential to the viability of mice ES cells 4
- 5. The BRCA Paradox A loss of both copies of BRCA2 leads to cell death A loss of both copies of BRCA2 leads to uncontrollable cell division 5 ES cells Mature Tissue Cells BRCA2 BRCA2 BRCA2 BRCA2 BRCA2 BRCA2
- 6. MSCV Retroviral Screening for BRCA2 interactors MSCV (Murine Stem Cell Virus) was used as a retroviral vector to induce the expression of possible BRCA2 interactors The vector had two essential qualities: 1. The viral genome was modified to prevent the standard replication of the viral DNA and subsequent lysing of the cell 2. The MSCV LTR acts as an enhancer- increasing or inducing transcription of the gene adjacent to it 6
- 7. Conditional allele Knocked-out HP RT1 HATR; Not viable Cre Recombinase Brca2 conditional ES cell (F7) Brca2 deficient ES cell MSCV retrovirus HP RT1 Gene X OR OR Brca2 deficient mutant ES cell Brca2 Brca2 Cre Recombinase HPRT1 Brca2 HPRT1 Brca2 Brca2 MSCV MSCV Gene X MSCV Brca2 MSCV Gene X Gene X Screening for genetic interactors in Brca2 Deficient tumorgenesis 7
- 8. Table 1. Insertion sites and nearby genes of the MSCV-rescued clones Clonec Chromosome Candidate Gene Comments 1 11qE2 SocS3 Suppressor of cytokine signaling family 3 5qB1 BRE Antiapoptotic, BRCA1/2 interacting, TNFa 4 10qC1 GIPC3 Growth factor signaling, cell adhesion 5 XqA1.3 Araf Activates MEK1 7 7qF3 NFAT Cip 8 5qE5 PTPN13 Protein tyrosine phosphatase, 9 17qb1 DAXX Enhances fas mediated apoptosis 10 2QE5 ZFP106 12 6qF3 FKbp4 22a 8qA1.1 Fbox-25 22b 12qA1.1 NCoA1 Steroid receptor coactivator-3 25 7qD2 ZFP710 27 2qH3 NCoA3 30 7qD2 ZFP710 8 Figure provided by Dr. Kajal Biswas
- 9. BRE(Brain and Reproductive Organ Expressed) I focused my project on one identified interactor- BRE (brain and reproductive organ expressed) Also known as BRCC4, BRCC45 BRE was found to rescue the lethality of BRCA2 deficient cells BRE is located on chromosome 2 in humans and chromosome 5 in mice There are 11 BRE splice variants the largest of which is 1.852 kb in length5 9 5. “Gene: BRE ENSG00000158019”. Ensemble.org. 2013.
- 10. 0 0.5 1 1.5 2 2.5 LevelofBREmRNApresent F7 Clone 3d 2.5 2.0 1.5 1.0 0.5 Increased expression of BRE in the rescued clone 3d 10 Figure provided by Dr. Kajal Biswas
- 11. Brca cko/++Cre HAT Brca cko/ko +BRE+Cre M15 BRE overexpression partially rescues Brca2 deficient ES cells 11 Figure provided by Dr. Kajal Biswas
- 12. Generation of BRE overexpressed Transgenic line A transgenic line of BRE overexpressed mice is being generated An inducible transgenic construct was prepared for generation of said line Construct DNA was injected into mice embryos Theoretically, BRE overexpressed mice should be able to rescue the lethality of BRCA2 deficient mice 12
- 13. BRE transgenic Construct 13 Actin Promoter Lac Z Neo HA-BREloxP loxP Neo Fwd Bre Rev Actin Promoter HA-BREloxP Cre
- 14. Generation of transgenic mice 14 Superovulated Female Gene of interest Injection of Gene into Fertilized Eggs Insertion of egg into Psuedopregnant female Birth of Transgenic Mice Genotyping
- 15. Genotyping The inducible BRE transgenic construct was microinjected into pronuclei of mice of mixed genetic background. Three weeks after the pups were born, a small portion of tail tips (~0.5 inches) was clipped. DNA was isolated from the tails and analyzed through polymerase chain reaction (PCR) using the primers Neo-Fwd and Bre-Rev to identify transgenic pups. The PCR products were then separated on 1% agarose gel. 15
- 17. Future Crosses for Transgenic Mice Line 17 Tg X BRCA2 KO/+ • Generate KO/+;Tg mice KO/+;Tg X KO/+;Tg • Cross heterozygotes KO/KO;Tg • Rescue lethality of BRCA2 null mice with trans gene
- 18. BRE rescued ES cells show higher Cdc25A level after IR C 0.5 1 2 C 0.5 1 2 Time After IR (Hrs) Cdc25A Actin Brca2cko/- (F7) Brca2-/-;Bre (3d) 18 • Cdc25A is an oncogene that induces mitosis in cells, and is normally degraded following DNA damage • However, BRE overexpressed cells show increased levels of Cdc25a despite exposure to Ionizing radiation
- 19. 19 Cdc25a • Cdc25a (Cell division cycle 25 homologue a) is a protein that regulates the cell at the G1 checkpoint, preceding the S stage • In healthy cells Cdc25a levels rise and activate CDK2, leading to the continuation of the cell cycle • However when DNA damage is present Cdc25a is degraded via ubiquitination leading to cell cycle arrest
- 20. BRE up-regulates Cdc25A transcriptionally after IR 0 0.5 1 1.5 2 2.5 3 3.5 4 F7 F7 IR F7-BRE F7-BRE IR RelativemRNAlevel Cells Cdc25A RNA level 20 Figure provided by Dr. Kajal Biswas
- 21. BRE interacts with transcription factor ATF3 to induce transcription of Cdc25a Bre was found to interact with Cyclic AMP-dependent transcription factor ATF-3 via Co-Immunoprecipitation ATF3 has multiple known binding sites on the Cdc25a promoter An experiment was set up to identify the effect of these ATF3 sites on Cdc25a transcription 21
- 22. Cdc25a promoter Cdc25a promoter Cdc25a promoter Cdc25a promoter Cdc25a promoter Cdc25a promoter Luciferase Luciferase Luciferase Luciferase Luciferase Luciferase 22 Reporter assay using different ATF3 binding site deletion of Cdc25a promoter
- 23. 23 Initial PCR Plan Cdc25a promoter fragment Fwd x Cdc Rev 1. Amplify desired Cdc25a promoter fragment via PCR 2.Ligate into luciferase containing plasmid, amplify in bacteria Amp 3. Cut out Cdc promoter/luciferase, insert into mammalian cell line
- 24. 24 However… • Initial PCRs were unsuccessful or did not yield sufficient product for ligation • Electroporation of competent cells failed • The experiment was revised to amplify the Cdc25a promoter fragment separately, prior to ligation with luciferase
- 25. 25 New PCR Plan Cdc25a promoter fragment Fwd x Cdc Rev 1. Amplify desired Cdc25a promoter fragment via PCR Amp 2. Ligate into Topo-Vector 3. Transduce Plasmid into bacteria, plate bacteria and amplify plasmid 4. Pick colonies and perform colony PCR to identify positives 5. Isolate plasmid, cut out Cdc promoter fragment Amp Amp Luciferase 6. Ligate Cdc promoter fragment into luciferase plasmid, finish original plan A A T T
- 26. Results of Colony PCR for FWD6 and FWD8 26
- 27. Eventual Goals Place Cdc25a promoter/luciferase in human cell lines and analyze the relationship between number of ATF3 binging sites on promoter to Luciferase expression 27
- 28. Acknowledgements 1."World Cancer Report". International Agency for Research on Cancer. 2008. 2.“Breast Cancer Statistics”. Susan G. Komen for the cure. 2013 3.“Hereditary Cancer Syndromes”. The University of Texas MD Anderson Cancer Center. 4.“BRCA Gene test for breast cancer”. The Mayo Clinic. 5. “Gene: BRE ENSG00000158019”. Ensemble.org. 2013 Several figures provided by Dr. Kajal Biswas Research done in the Genetics of Cancer Susceptibility Section, headed by Dr. Shyam Sharan, Mouse Cancer Genetics Program, Frederick National Laboratory Thank you all for a great summer! 28
Editor's Notes
- #3: More deadly in men
- #6: Something must be over or underexpressed
- #8: Mention overexpression to generate
- #9: Q and p arms, a b c d sections
- #11: Mention quantitative arty PCR to determine level of mRNA present
- #12: M15 is normal, bottom rightis control
- #14: Lac Z: allows us to see integration Neo: selectable marker Ha : hemoaglutinase antibody binding Inducible to prevent cell from silencing it
- #17: amplified with PCR, red means positives Positive control (+ve) was plasmid DNA used for injection and negative control (- ve) was tail DNA from a C57BL/6 mice 17 positives here
- #21: No degradation, more transcription
- #23: About 2.5 kb
- #24: Digestion after pcr, make sticky ends
- #26: Why couldn’t we electroportate? Ligation needs salt
- #27: Possible artifact in pcr