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p53 downregulates Down syndrome-associated DYRK1A through miR-1246 Yu Zhang, Jun-Ming Liao, Shelya X Zeng and Hua

p53 downregulates Down syndrome-associated DYRK1A through miR-1246 Yu Zhang, Jun-Ming Liao, Shelya X Zeng and Hua Lu Indiana University School of Medicine Central South University, Hunan, China EMBO Reports 12:811-817 June 2011. p63. There are three genes in the p53 family

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p53 downregulates Down syndrome-associated DYRK1A through miR-1246 Yu Zhang, Jun-Ming Liao, Shelya X Zeng and Hua

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  1. p53 downregulates Down syndrome-associated DYRK1A through miR-1246 Yu Zhang, Jun-Ming Liao, Shelya X Zeng and Hua Lu Indiana University School of Medicine Central South University, Hunan, China EMBO Reports 12:811-817 June 2011

  2. p63 There are three genes in the p53 family All bind DNA and affect transcription p63 and p73 are essential Nature Rev. Cancer 2:605

  3. p63 Each gene has two alternative promoters and several alternative splicing options The p63 gene encodes six different proteins Notice that DN isoforms lack TA domain Cell Death Diff. 13:962

  4. p63 Many genes have been reported to be activated by one or more family members But what about micro-RNAs (miRNAs)? Naturally occurring form of siRNA Humans encode at least 1,872 miRNAs p53 is known to alter the expression of many miRNAs. What about p63? Cell Cycle 11:2624

  5. p63 Activation of miRNAs Overexpression p63 or controls instead of transfection, cells were infected with recombinant adenoviruses p40 = DN-p63g no transactivation domain! Collect total mRNA from these cells Microarray experiment Targets on gene chips are complementary to miRNAs, not mRNAs Fig. 1A

  6. p63 Activation of miRNAs Collect total RNA from these cells Microarray experiment Targets on gene chips are complementary to miRNAs, not mRNAs Which two RNA samples do you want to compare? or multiple sets? Sent mRNAs to LC Sciences for microarray analysis ~2,500 miRNA probes on the microarray Fig. 1A

  7. p63 Activation of miRNAs TAp63g vs. GFP Table S1

  8. p63 Activation of miRNAs TAp63g vs. GFP 21-fold induction Table S1

  9. p63 Activation of miRNAs TAp63g vs. DN-p63g 28-fold induction Table S1

  10. p63 Activation of miRNAs Confirming that p63 activates miR-1246 in H1299 cells Overexpression or knockdown of p63 But how do we quantify miR-1246? Too small for a Northern blot RT-PCR (with some modifications for the small target size) Fig. 1CD

  11. p63 Activation of miRNAs Unfortunately, PCR isn’t very quantitative. tends to hit a maximal plateau of amplified DNA DNA stains are relatively insensitive and nonlinear need to assay DNA amount before that plateau is hit image from M. Hunt, Univ. South Carolina

  12. p63 Activation of miRNAs Solution is Real-Time PCR [also called quantitative PCR (qPCR)] qPCR monitors the amount of product made each cycle Option 1: SYBR green fluorescent dye binds dsDNA but not ssDNA fluoresces when bound to DNA

  13. p63 Activation of miRNAs Solution is Real-Time PCR [also called quantitative PCR (qPCR)] qPCR monitors the amount of product made each cycle Option 1: SYBR green fluorescent dye binds dsDNA but not ssDNA fluoresces when bound to DNA Option 2: Forward and Reverse PCR primers Also a primer in the middle One end has a Fluor; other end has a Quencher Middle primer is degraded during PCR, releasing the Fluor

  14. p63 Activation of miRNAs 10-fold dilution series of input cDNA. Monitor amplification over cycles Example qPCR with 10-fold serial dilutions of input DNA Notice that after 33 cycles the purple, red and blue dilutions all have the same amount of DNA (Total PCR Product) But if we look at which cycle they cross this threshold, we see the differences. image from M. Hunt, Univ. South Carolina

  15. p63 Activation of miRNAs log scale Ct = threshold cycle

  16. p63 Activation of miRNAs miR-1246 measured by qRT-PCR Does this match the microarray data? Fig. 1CD

  17. Transcription of miR-1246 miR-1246 gene is located on chromosome 2 at 2q31.1 pri-microRNA: mature microRNA: 5’-UGUAUCCUUGAAUGGAUUUUUGGAGCAGGAGUGGACACCUGACCCAAAGGAAAUCAAUCCAUAGGCUAGCAAU-3’ 5’-AAUGGAUUUUUGGAGCAGG-3’ Nature Cell Biol. 11:228

  18. Transcription of miR-1246 194 bp upstream of the miR-1246 gene is a possible p53 enhancer Consensus half-site is RRRCWWGYYY R= purine (A or G) W= A or T Y= pyrimidine (C or T) Good match but does p63 actually bind this site? Fig. 2A

  19. Transcription of miR-1246 Does p63 bind the miR-1246 promoter? Chromatin IP (ChIP) Similar to coIP but detect co-precipitating DNA by PCR or qPCR anti-p63 Image adapted from Lodish et al. “Molecular Cell Biology”

  20. Transcription of miR-1246 p63 was activated by treating cells with etoposide causes DNA damage by inhibiting topoisomerase ChIP with anti-p63 antibody or control immunoglobulin G (IgG) PCR for miR-1246 promoter or catalase promoter (negative control) Fig. 1E

  21. Transcription of miR-1246 ChIP with transfected Flag-tagged p63 Quantify ChIP by qPCR Fig. S3B

  22. Transcription of miR-1246 Also used a reporter gene transfect with firefly luciferase gene under the miR-1246 promoter or mutant miR-1246 promoter with nonfunctional p53 binding sites Luciferase Gene

  23. Transcription of miR-1246 Cotransfect H1299 cells with reporter gene and p63-expression plasmid Lyse, add substrates, measure light production in a luminometer Fig. 2E, S3A

  24. Transcription of miR-1246 Do p53 and p73 also activate miR-1246 transcription? We’ll test several different ways Transfect to overexpress and measure miR-1246 by qRT-PCR Fig. 2B

  25. Transcription of miR-1246 Three cell lines with null p53, wt p53 or inactive p53 Stabilize p53 with etoposide. Measure miR-1246 by qRT-PCR Fig. 2C

  26. Transcription of miR-1246 luciferase assay Fig. 2E

  27. Transcription of miR-1246 p53 knockdown miR-1246 measured by qRT-PCR Fig. S4A

  28. Transcription of miR-1246 Does p53 bind to the miR-1246 promoter? Induce with etoposide and ChIP Fig. 2E, S4C

  29. miR-1246 Targets DYRK1A 5’-AAUGGAUUUUUGGAGCAGG-3’ What gene(s) does miR-1246 target? Difficult question because miRNAs don’t have perfect basepairing! We’re beginning to understand the “rules” Bioinformatic programs can predict which mRNA will be repressed by specific miRNAs

  30. miR-1246 Targets DYRK1A For example, MicroT-CDS predicts 808 potential human mRNAs that may be miR-1246 targets DYRK1A is the 30th best target!

  31. miR-1246 Targets DYRK1A Overexpress miR-1246 on a transfected plasmid qRT-PCR to confirm overexpression Western blot for DYRK1A Fig. 3B

  32. miR-1246 Targets DYRK1A Transfect a miR-1246 mimic (chemically modified, single-stranded RNA of the same sequence) Fig. 4B

  33. miR-1246 Targets DYRK1A Built a reporter construct. Strong promoter driving Luciferase expression. DYRK1A 3’ UTR sequence inserted in 3’UTR of Luciferase Fig. 3C

  34. miR-1246 Targets DYRK1A H1299 cells co-transfected with miR-1246 overexpression plasmid and reporter plasmid (wt or mutant) Lyse and determine luciferase expression p<0.01 Fig. 3D

  35. miR-1246 Targets DYRK1A U2OS cells (wt p53) stabilize p53 with etoposide reporter plasmid (wt or mutant) Lyse and determine luciferase expression Fig. 3D

  36. miR-1246 Targets DYRK1A U2OS cells with wt p53 Knockdown p53 expression What happens to DYRK1A? Fig. 3E

  37. miR-1246 Targets DYRK1A U2OS cells with wt p53 Stabilize p53 with etoposide treatment What happens to DYRK1A? Fig. 3F

  38. miR-1246 Targets DYRK1A Survey of six cell lines Is there a correlation between DYRK1A levels and miR-1246 levels? Fig. S6A

  39. miR-1246 Targets DYRK1A miR-1246 inhibition by chemically modified, single-stranded, complementary RNA p21 (?) Fig. 4A

  40. miR-1246 Targets DYRK1A DYRK1A has several known substrates These should also be affected by miR-1246 NFATc is a transcription factor Nuclear accumulation caused by Ca2+/Calcineurin DYRK1A phosphorylation causes NFATc to leave nucleus

  41. miR-1246 Targets DYRK1A Established a cell line with GFP-NFATc stably expressed Nuclear import caused by thapsigarin (TG; inhibits export of Ca2+ from cytosol thus activating calcineurin) Nuclear export caused by cyclosporin A (CsA; inhibits the phosphatase calcineurin) TG will miR-1246 affect NFATc localization? Fig. 4C

  42. miR-1246 Targets DYRK1A What effect will the miR-1246 mimic have on NFATc localization? Fig. 4C

  43. miR-1246 Targets DYRK1A Reporter plasmid of NFAT-responsive promoter driving luciferase Interleukin-2 promoter is a natural NFATc target Fig. 4D, S5

  44. miR-1246 Targets DYRK1A DYRK1A has been shown to suppress apoptosis phosphorylates Procaspase-9 and Akt Transfect with miR-1246 overexpression plasmid or control Monitor DNA content per cell by FACS Fig. 4E

  45. Fig. 4E

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