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Myc regulation of histone acetylation

Myc regulation of histone acetylation. [U- 13 C ] Glucose. [U- 13 C ] - Pyruvate. 13 C - FA, 13 C- acetate and 13 C - acetoacetate. TCA cycle. [U- 13 C ]-Acetyl-CoA. GCN5. [U- 13 C ]-Citrate. 13 C-Ac. 13 C-Ac. Nuclear histones. Analysis of 13 C acetyl group transfer to Histone H4.

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Myc regulation of histone acetylation

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  1. Myc regulation of histone acetylation [U-13C] Glucose [U-13C]-Pyruvate 13C-FA, 13C-acetate and 13C-acetoacetate TCA cycle [U-13C]-Acetyl-CoA GCN5 [U-13C]-Citrate 13C-Ac 13C-Ac Nuclear histones

  2. Analysis of 13C acetyl group transfer to Histone H4 Incubate myc-/- and myc+/+ cells in [U-13C] glucose Acid extract and RP-HPLC purify histones In vivo label with deuterium acetic anhydride Identify fractions enriched with H4 by LC-MS Detect 12C, 13C, and deuterium labeled K16-H4 by ESI-MS/MS

  3. Histone H4 acetylation analysis Acetylated peptides

  4. Myc increases 13C labeling of acetyl groups on Histone H4 12.7% 20.6% Morrish et al. J. Biol Chem 2010

  5. Myc regulates the supply of metabolic substrates for histone acetylation • Rapid modification of chromatin • Increased gene transcription • Induce rapid cellular re-programming

  6. Regulation of metabolism during tumor progression and regression

  7. Induction/regression of islet tumors 21 day +Tamoxifen 8 day no tamoxifen Islet tumor pInsMyc/ Bcl-XL Bcl-XL

  8. Genes for substrate transport and pathways for conversion to lipids induced by Myc in b cells Ketones Lactate Pyruvate Glucose Glutamate Plasma 1 Slc1a1 Slc2a5 Slc16a1 Cell Eno1 Fbp1 G6p 1 21 Oxct1 Ketones. Isoleucine, leucine and valine Glud1 Sphingolipids 21 TCA TCA cycle Ppap2a Degs2 Sgpl1 Cholesterol Idh CS Hmgcr Mvd Mvk Fdps Dhcr7 Sqle Palmitoyl CoA Oxct1 Acadm Bcat2 Bckdha Aox1 Aldh2 Aldh7a1 Slc25a1 Fasn HMG CoA Acetyl CoA Data compiled from results in Lawlor et al 2006

  9. Time-course analysis for neoplasia induction and regression Induction of neoplasia Daily tamoxifen injections 48 hrs 16 days 21 days Regression 6 days • Each time point performed 5 times with 5 MycER/Bcl-XL mice and 5 Bcl-Xl mice

  10. 13C labeling of islets 1,000 islets from MycER/Bcl-XL and Bcl-XL mice Incubate RPMI media O/N Pre-incubate in Krebs Ringer buffer 1 hr Incubate in 20 mM 13C glucose 2 hr Quench in -700 methanol Extract and derivatize

  11. Summary of changes in de novo metabolism Stearate

  12. Summary of events 2 days 16 days Glucose Glucose Lactate Lactate Pyruvate Pyruvate Palmitate Stearate Palmitate Stearate Citrate Citrate Alanine Alanine Malate Succinate Glutamate Glutamate Glutamine Glutamine Glucose Glucose Lactate Lactate Pyruvate Pyruvate Palmitate Stearate Palmitate Stearate Citrate Citrate Alanine Alanine Succinate Succinate Glutamate Glutamate Glutamine Glutamine 21 days 8 day regression

  13. Lipid metabolism on regression * * ** * * * * * 18:2n-6 Elongation Lipotoxicity protection mechanisms 18:3 n6 • Suppression of miR34a • Esr1 C20:2 n-6 20:3 n-6 Delta6-desaturation FADS2 20:4 n-6 Phospholipids

  14. Switching off Myc alters cellular FAO capacity and ATP production Oxygen consumption with palmitate ATP generation with palmitate

  15. Conclusions • Myc is a dual regulator of cell cycle genes and metabolism • Myc regulates anabolic and catabolic metabolism via the TCA cycle • Myc links metabolome and proteome via protein post-translational modification • Myc-induces metabolic switching providing metabolic flexibility for tumor progression • Myc-induced lipid production provides an “Achilles Heel” for targeted drug therapy

  16. Acknowledgements FHCRC David Hockenbery Jhoanna Noonan Marc VanGilst Carissa Perez Phillip Gafken University of Washington DERC Ian Sweet Ben Reed Chemistry Mass Spectrometry Martin Sadelik PNNL Nancy Isern UT South Western Mark Jeffrey MIT Joanne Kelleher UCSF Gerard Evan Funding sources: NIH/NCI

  17. Myc temporally regulates genes related to acetyl-CoA metabolism Sirt3 Acat1

  18. Myc regulation of the histone acetyltransferase GCN5 GCN5

  19. Myc increases 13C labeling of acetyl groups on Histone H4 MS2 Analysis y5 fragment (K16) (provides % acetylation occupancy at specific lysines ) 0 Ac Myc+/+ TGR 20.6% 1 Ac 1 Ac (2 x 13C) 0 Ac Myc-/- KO 12.7% 1 Ac 1 Ac (2 x 13C) Morrish et al J. Biol Chem 2010

  20. Function of metabolic and bioenergetic cell cycle checkpoints Stimulus Cell division Nutritional status? Good Quiescent cell Inadequate No response to stimulus

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