RecQ Helicases and GI Cancers. Mark Derleth, R3 Supervisor: Bill Grady

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1 RecQ Helicases and GI Cancers Mark Derleth, R3 Supervisor: Bill Grady

2 Learn to perform and interpret several lab assays including PCR, MSP, and Bisulfite sequencing Personal Goal

3 Research Goal To examine the significance of DNA methylation as a potential mechanism for gene silencing in the RecQ helicase family of genes

4 RecQ Helicases Essential Caretakers of the Genome and, thus, potential tumor suppressor genes Helicase and Exonuclease Activity

5 RecQ Helicases WRN BLM Werner Syndrome Bloom's Syndrome RecQL RecQL4 Rothmund-Thompson Syndrome (RTS) RecQL5 * These syndromes are characterized by genomic instability and early development of cancers

6 Werner Syndrome

7 Epigenetics Mechanisms for epigenetic control DNA methylation-at CpG dinucleotides Post-translational modifications of histones

8 In mammalian cells: DNA methylation +SAM SAM SAH The CpG dinucleotide is the favored substrate: 5 3 * NNNNNNCGNNNNNNN NNNNNNGCNNNNNNN * 3 5

9 Target Genes of CpG Island Hypermethylation Approximately 20,000 genes Approximately 14,000 of these genes have CpG islands Approximately 20-30% of these will be hypermethylated in cancer Approximately 30-40% of methylated genes will be repressed.

10 Project #1 - MSP Design and test methylation specific PCR (MSP) primers for each of five genes in the RecQ helicase family Use MSP to test methylation status of each gene in a number of colon cancer cell lines

11 Methylation Specific PCR (MSP) Sodium Bisulfite treatment of DNA converts Cytosine to Uracil

12 Methylation Specific PCR (MSP) Methylated Cytosine is not converted in this reaction

13 Methylation Specific PCR Assay (MSP) CGAGCG UGAGUG M U NaHSO 3 PCR M U M U m CGAG m CG m CGAG m CG

14 Methylation Specific PCR (MSP) Design TWO sets of primers: Methyl primers Unmethyl primers Treat DNA (colon cancer cell lines) with sodium bisulfite Run PCR reaction using each set of primers on each DNA sample

15 MSP with CRC Cell Lines RKO SW48 Vaco411 Vaco5 Vaco5 AAC1 AAC1/SB10 H2O sssi-sw48 PBL LS174T PET Lovo SW480 HCT116 Vaco400 + control - control 5/5/08 by Chan M U M U M U M U M U M U M U M U M U M U M U M U M U M U M U M U

16 Summary of MSP Results RecQL RecQL4 BLM WRN RecQL5 Colon cancer cell lines 8% (1/13) 8% (1/13) 22% (2/9) 54% (7/13) 31% (4/13) Colon cancer tissue 0% (0/16) 16% (3/19) 31% (4/13) 33% (7/21) 14% (3/21) Normal colonic mucosa 0% (0/7) 0% (0/10) 38% (3/8) 0% (0/12) 0% (0/8) *Chan s Results

17 Project #2 Bisulfite Sequencing Design primers and perform bisulfite sequencing of RecQ helicase genes in selected colon cancer cell lines

18 Bisulfite Sequencing Design primers to amplify both methylated and unmethylated gene products Treat DNA (colon cancer cell lines) with sodium bisulfite Run PCR reaction Prepare amplicon for sequencing

19 MethPrimer Result for RecQL5 Bisulfite sequencing primer

20 PCR using RecQL5 primer for Bisulfite Sequencing

21 Bisulfite Sequencing of WRN Unmethyl Methyl Methyl Methyl Unmethyl By Slavomir

22 Bisulfite Sequencing of BLM

23 Bisulfite Sequencing of RecQL5

24 Summary of Results Based upon these bisulfite sequencing experiments, hypermethylation of promoter regions does not appear to be a significant mechanism for inactivation of WRN, BLM, or RecQL5

25 Thanks to: Dr. Bill Grady Slavomir Dzieciatkowski Dr. Scott Bornstein Dr. Andrew Kaz Dr. Chan Gyoo Kim

26 The End

27 The following are supplemental slides

28 Werner Syndrome: Clinical Characteristics Adult Onset Segmental Progeroid syndrome Develop normally until the third decade Lack pubertal growth spurt (shortest in graduating highschool class) Early graying and thinning of hair Skin Atrophy Truncal subcutaneous fat deposition (stocky appearance) Limb Osteoporosis High-Pitched voice Flat feet Median age of death 54 (Malignancy and MI most common)

29 Werner Syndrome at Time of Diagnosis Cataracts 100% Hypogonadism 80% Osteoporosis 91% Diabetes 71% Neoplasms 43% Atherosclerosis 40%

30 Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer Agrelo, et al. PNAS 2006 Hypermethylation of promoter CpG islands in WRN leads to gene silencing in human cancer cells Hypermethylation of promoter CpG islands in WRN leads to loss of protein expression as well as hypersensitivity to topoisomerase inhibitors

31 MethPrimer Result for BLM Bisulfite sequencing primer

32 PCR using WRN primer for Bisulfite Sequencing

33 PCR using BLM primer for Bisulfite Sequencing

34 List of Sources Agrelo, et al., PNAS 2006 Muftuoglu, et al., Human Genet 2008 MethPrimer: Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics Nov;18(11):