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Myc regulation of histone Myc regulation of histone acetylationacetylation
TCA cycle
[U-13C] Glucose
[U-13C]-Pyruvate 13C-FA, 13C-acetate and 13C-acetoacetate
13C-Ac13C-Ac
GCN5
[U-13C]-Acetyl-CoA
[U-13C]-Citrate
Nuclear histones
Analysis of Analysis of 1313C acetyl group C acetyl group transfer to Histone H4transfer to Histone H4
Incubate myc-/- and myc+/+ cells in [U-13C] glucose
Acid extract and RP-HPLC purify histones
Identify fractions enriched with H4 by LC-MS
Detect 12C, 13C, and deuterium labeled K16-H4 by ESI-MS/MS
In vivo label with deuterium acetic anhydride
Histone H4 acetylation Histone H4 acetylation analysisanalysis
Acetylated peptides
Myc increases Myc increases 1313C labeling of C labeling of acetyl groups on Histone H4 acetyl groups on Histone H4
20.6%
12.7%
Morrish et al. J. Biol Chem 2010
Myc regulates the supply of Myc regulates the supply of metabolic substrates for histone metabolic substrates for histone
acetylationacetylation
Rapid modification of chromatinRapid modification of chromatin
Increased gene transcriptionIncreased gene transcription
Induce rapid cellular re-programmingInduce rapid cellular re-programming
Regulation of metabolism Regulation of metabolism during tumor progression during tumor progression
and regressionand regression
Induction/regression of islet tumors
21 day +Tamoxifen
pInsMyc/Bcl-XL
Bcl-XL
8 day no tamoxifen
Islet tumor
Glutamate Glucose
KetonesLactatePyruvate Plasma
Acetyl CoA HMG CoA
Cholesterol
SphingolipidsKetones.Isoleucine, leucine and valine
CellSlc1a1 Slc2a5 Slc16a1
Slc25a1Palmitoyl CoA
TCA cycle
Glud1
Eno1Fbp1G6p
IdhCS
Fasn
HmgcrMvdMvkFdpsDhcr7Sqle
Ppap2aDegs2Sgpl1
Oxct1
Oxct1AcadmBcat2BckdhaAox1Aldh2Aldh7a1
Genes for substrate transport and pathways Genes for substrate transport and pathways for conversion to lipids induced by Myc in for conversion to lipids induced by Myc in
cellscells
1
211
21
TCA
Data compiled from results in Lawlor et al 2006
Time-course analysis for Time-course analysis for neoplasia induction and neoplasia induction and
regressionregression
48 hrs 16 days 21 days
6 days
Regression
Induction of neoplasia
•Each time point performed 5 times with 5 MycER/Bcl-XL mice and 5 Bcl-Xl mice
Daily tamoxifen injections
1313C labeling of islets C 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
Incubate in 20 mM 13C glucose
1 hr
2 hr
Quench in -700 methanol
Extract and derivatize
Stearate
Summary of changes in Summary of changes in de novode novo metabolismmetabolism
Summary of events Summary of events Glucose
Pyruvate
Succinate
Lactate
Alanine
Glucose
Pyruvate
Malate
Lactate
Alanine
2 days 16 days
Glutamate
Glutamine
Citrate PalmitateStearate
Pyruvate
Succinate
Lactate
Alanine
Glutamate
Glutamine
Citrate PalmitateStearate
Glucose
Pyruvate
Succinate
Lactate
Alanine
Glutamate
Glutamine
Citrate PalmitateStearate
Glucose
Glutamate
Citrate PalmitateStearate
Glutamine
21 days 8 day regression
Lipid metabolism on Lipid metabolism on regressionregression
18:2n-6
18:3 n6
20:3 n-6
20:4 n-6
C20:2 n-6
Elongation
Delta6-desaturationFADS2
Phospholipids
*
* *
*
**
• Suppression of miR34a• Esr1
Lipotoxicity protection mechanisms
*
**
Switching off Myc alters Switching off Myc alters cellular FAO capacity and ATP cellular FAO capacity and ATP
productionproductionATP generation with palmitateOxygen consumption with palmitate
ConclusionsConclusions Myc is a dual regulator of cell cycle Myc is a dual regulator of cell cycle
genes and metabolism genes and metabolism Myc regulates anabolic and catabolic Myc regulates anabolic and catabolic
metabolism via the TCA cyclemetabolism via the TCA cycle Myc links metabolome and proteome Myc links metabolome and proteome
via protein post-translational via protein post-translational modificationmodification
Myc-induces metabolic switching Myc-induces metabolic switching providing metabolic flexibility for providing metabolic flexibility for tumor progression tumor progression
Myc-induced lipid production Myc-induced lipid production provides an “Achilles Heel” for provides an “Achilles Heel” for targeted drug therapytargeted drug therapy
AcknowledgementsAcknowledgementsFHCRCDavid HockenberyJhoanna Noonan
Marc VanGilst Carissa Perez
Phillip Gafken
University of Washington DERCIan Sweet Ben ReedChemistry Mass SpectrometryMartin Sadelik
PNNLNancy Isern
UT South WesternMark Jeffrey
MITJoanne Kelleher
Funding sources: NIH/NCI
UCSFGerard Evan
Myc temporally regulates genes Myc temporally regulates genes related to acetyl-CoA metabolismrelated to acetyl-CoA metabolism
Sirt3 Acat1
Myc regulation of the histone Myc regulation of the histone acetyltransferase GCN5acetyltransferase GCN5
GCN5
Myc increases Myc increases 1313C labeling of C labeling of acetyl groups on Histone H4acetyl groups on Histone H4
FM012908_021308_TGR #4508-4758 RT: 21.24-22.15 AV: 84 NL: 6.00E5F: ITMS + c NSI d Full ms2 [email protected] [185.00-1460.00]
524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
533.39
530.36 532.40
534.40
531.39535.41
536.42 539.46537.40529.41 538.49526.30524.37 528.40527.41525.35
FM030308_031908_KO #4149-4398 RT: 19.75-20.66 AV: 84 NL: 1.15E6F: ITMS + c NSI d Full ms2 [email protected] [185.00-1460.00]
524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540
m/z
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
533.45
530.42534.45
532.52531.46
535.48 536.48 539.43537.46 538.48525.33 528.42526.35524.43 527.42 529.28
MS2 Analysis y5 fragment (K16)(provides % acetylation occupancy at specific lysines )
TGR
KO
0 Ac
1 Ac 1 Ac (2 x 13C)
0 Ac
1 Ac
1 Ac (2 x 13C)
20.6%
12.7%
Myc+/+
Myc-/-
Morrish et al J. Biol Chem 2010
Function of metabolic and Function of metabolic and bioenergetic cell cycle bioenergetic cell cycle
checkpointscheckpoints
Quiescent cell
Stimulus
Nutritional status?
Good
Inadequate
No response to stimulus
Cell division