# Presentation: 514539

## Diet and Epigenetics 

**Sharon Ross**

**Nutritional Science Research Group**
**Division of Cancer Prevention**
**National Cancer Institute**
**Bethesda, MD 20892**

## Epigenetic Regulation of Cancer

**Epigenetic Regulation of Cancer**

**Site Specific**
**Hypermethylation**

**Global**
**Hypomethylation**

**Histone  Modifications**

**Regulating Factors**
- Dietary
- Hormonal
- Genetic

**Verma M, Srivastava S. Lancet Oncol. (2002) 3:755-63****.**

**DNA Methyltransferases**
**Histone Methyltransferases**
**Histone Acetylases/Deacetylases**

**Epigenetics Regulates****:**
**Cell Cycle Control**
**DNA Damage**
**Apoptosis**
**Invasion**
**X-Chromosome Inactivation**
**Imprinting**
**Aging**

## -

**SAH**

**SAM**

**methionine**

**MS**

- methylated-DNA

- DNA

**MTHFR**

-  cystathionine

-  cysteine

** ****GSH**

**CBS**

- (B6)

- (B12)

- PE

**  ****PC**

**PEMT**

**MAT**

**betaine**

**choline**

**BHMT**

- THF

- 5-MTHF

- 5,10-MTHF

- DHF

- dUMP

- dTMP

- DNA

-  gly

-  ser

- (B6)

**dietary**
**folates**

**dietary**
**folates**

- SAM

-  DMG

- SAM

**+**

- Ach

**choline-P**

- CDP-choline

## Slide 4

## Bioactive Food Components in Epigenetic Processes

**Bioactive Food Components in Epigenetic Processes**

## Yellow Mouse

**Yellow Mouse**

**Agouti Mouse**

- Cooney  et al. (2002)  J Nutr. 132:2393S

**Maternal Supplements **
**with**
**zinc**
**methionine**
**choline**
**folate**
**B12**

**High risk cancer, diabetes, obesity **
**Reduced lifespan**

**Lower risk of cancer, diabetes,  obesity**
** ****Prolonged life**

**LTR Hypomethylated**

**LTR Hypermethylated**

## Prevalence of promoter methylation for high and low methyl intake (classified by alcohol and folate intake) 

|  | High Methyl Intake* | Low Methyl Intake* |
| --- | --- | --- |
| Promoter Methylation |  |  |
| APC | 33 % | 44 % |
| p14ARF | 28 | 38 |
| p16INK4 | 31 | 32 |
| hMLH1 | 28 | 30 |
| O6-MGMT | 36 | 47 |
| RASSF1A | 15 | 25 |
| Number of Genes CH3 |  |  |
| 0 | 30 % | 16 % |
| 1 | 20 | 21 |
| 2 | 21 | 26 |
| 3 | 13 | 16 |
| 4 | 15 | 12 |
| 5 | 2 | 5 |
| 6 | 0 | 3 |
| At least 1 CH3 | 70 % | 84 % |

- *n=61             van Engeland et al. Cancer Research: 63: 3133-3137, 2003

**Prevalence of promoter methylation for high and low methyl intake (classified by alcohol and folate intake) **

## Tea Polyphenol Epigallocatechin-3-Gallate Inhibits DNA Methyltransferase Activity

- Fang et al. Cancer Res. 63(22): 2003

## Demethylation: reversal of hypermethylation of p16INK4a, RARβ, (MGMT), and hMLH1 genes

**Demethylation: reversal of hypermethylation of p16INK4a, RARβ, (MGMT), and hMLH1 genes**
**Reactivation: increased expression of mRNA for p16INK4a, RARβ, (MGMT), and hMLH1 genes**

**Consequences of Epigallocatechin-3-Gallate Inhibition of DNA Methyltransferase Activity**

- Fang et al. Cancer Res. 63(22): 2003

## Sensitization by 5-Aza-deoxycytidine of Leukemia Cells to Differentiation by Retinoic Acid 

**Sensitization by 5-Aza-deoxycytidine of Leukemia Cells to Differentiation by Retinoic Acid **

- 20 -

- 40 -

- 10 -

- 30 -

**Untreated**
**Retinoic Acid**
**5-AZA**
**5-AZA + Retinoic Acid****-**

**SN-1**

**KOCL33**

**KOCL51**

**Differentiation Ratio**

*Nitsu, et al. British Journal of Hematology 112: 315, 2001.*

## Dietary Factors Influencing Histone Modifications

## Histone Modifications

**Acetylation of lysines**
**Methylation of lysines and arginines**
**Phosphorylations of serines and threonines **
**ADP-ribosylation of glutamic acids **
**Ubiquitination of lysine residues**
**Sumolyation of lysine residues**
**Biotinylation of lysines**

**Histone Modifications**

## 							Johnstone 2002Epigenetic Mechanisms

**Notes:**
Depending on what groups are attached to the DNA and the histones, the overall chromatin will be compacted or open....
       closed chromatin ------------ transcriptional repression
       open chromatin --------------transcriptional activation

## Chromatin-Level Regulation of Gene Expression

- Garfinkel and Ruden Nutrition:20:56-62, 2004

**Inactive Chromatin**

**SFN, DADS**

## Effects of Fatty Acids on Mammalian Cells in Culture

| No. of carbons in fatty acid | Effect on fibroblast growth % control | Induction of alkaline phosphatase (HeLa) % control | Inhibition of estradiol-induced synthesis of transferring mRNA | Inhibition of histone deacetylase (calf thymus) |
| --- | --- | --- | --- | --- |
|  |  |  |  |  |
| C2, acetate | 82 | 170 | 18 | 10 |
| C3, propionate | 45 | 160 | 77 | 60 |
| C4, butyrate | 0 | 630 | 95 | 80 |
| C5, valeroic | 71 | 420 | — | 65 |
| C6, caproate | — | 120 | — | 30 |

**Davie JR. J. Nutr. 133: 2485S-2493S, 2003**

## A model for butyrate induction of p21Waf1/Cip1 gene expression and inhibition of cell cycle 

***A model for butyrate induction of p21Waf1/Cip1 gene expression and inhibition of cell cycle***

**Davie JR. J. Nutr. 133: 2485S-2493S, 2003**

## Sulforaphane inhibits HDAC activity in HCT116 cells

**The *****wedge symbol*****, 0, 3, 9, 15 μM SFN**

- Myzak et al. Cancer Research: 64: 5767-5774, 2004

## Sulforaphane increases histone acetylation and p21Cip1/Waf1 protein 

**Myzak et al. Cancer Research: 64: 5767-5774, 2004**

**The *****wedge symbol*****, 0, 9, 15 μM SFN**

**Sulforaphane increases histone acetylation and p21Cip1/Waf1 protein **

## DADS Influences p21waf1/cip1 mRNA and Protein Levels in HT-29 Cells

**Druesne N et al. Carcinogenesis 25: 1227-1236, 2004**

## Effects of DADS on the acetylation of histones in HT-29 cells

- Druesne N et al. Carcinogenesis 25: 1227-1236, 2004

**Effects of DADS on the acetylation of histones in HT-29 cells**

## Chromatin-Level Regulation of Gene Expression

- Garfinkel and Ruden Nutrition:20:56-62, 2004

**Inactive Chromatin**

**SFN, DADS**

**resveratrol**

## Sirtuin 1 (SIRT1): a protein deacetylase

**Expression associated with longevity, cell survival**
**NAD+-dependent histone deacetylase **
**SIRT1 deacetylates histone polypeptides with a preference for histone H4 lysine 16 and H3 lysine 9 in vitro. **
**RNAi-mediated decreased expression of SIRT1 in human cells resulted in hyperacetylation of H4-K16 and H3-K9 in vivo. **
**SIRT1 protein deacetylates non-histone substrates (p53, MyoD), as well as histone substrates**

**Sirtuin 1 (SIRT1): a protein deacetylase**

**Vaquero A, et al. Mol Cell. 16:93-105, 2004. **

## Caloric restriction increases SIRT1 expression in a variety of rat tissues

**Cohen HY, et al. Science 305:390-392, 2004.**

## Resveratrol increases NAD-dependent HDAC activity

**Resveratrol increases NAD-dependent HDAC activity**

**Yeung F, et al. Embo J. 23:2369-2380, 2004**

## SIRT1-mediated deaceytlation

**Yeung F, et al. Embo J. 23:2369-2380, 2004**

**SIRT1-mediated deaceytlation**

## Chromatin-Level Regulation of Gene Expression

- Garfinkel and Ruden Nutrition:20:56-62, 2004

**SFN, DADS**

**resveratrol**

## Histone Methyltransferases

**Transfer methyl groups from the methyl donor S-adenosylmethionine to the arginine or lysine residues in histones or other proteins **
**Three classes based on the sequence and structure of their catalytic domain:  protein arginine methyltransferases, SET domain containing lysine methyltransferases and Dot1 class lysine methyltransferases. **
**The SET domain family has been linked with mitotic inheritance of gene activity and carcinogenesis. **
**Methylation is among the most stable histone modifications. **
**The initiation and maintenance of heterochromatin is carried out by histone H3 lysine 9 methylation, whereas euchromatin is characterized by H3 lysine 4 methylation.**
**Six HMTs methylate H3-K9 (SET domain family)**

## Inhibition of histone methylation activity by increasing SAH levels

**Inhibition of histone methylation activity by increasing SAH levels**

**Kim K, et al. Cancer Research 63:7619-7623, 2003**

## Chromatin-Level Regulation of Gene Expression

- Garfinkel and Ruden Nutrition:20:56-62, 2004

**SFN, DADS**

**resveratrol**

## 1,25-Dihydroxyvitamin D(3) Stimulates Cyclic Vitamin D Receptor/Retinoid X Receptor DNA-Binding, Co-activator Recruitment, and Histone Acetylation in Intact Osteoblasts. 

**Kim S, Shevde NK, Pike JW.** **J Bone Miner Res. 2005 Feb;20(2):305-17. Epub 2004 Nov 16.**

**1,25(OH)(2)D(3) induces rapid association of the VDR and RXR with both the Cyp24 and the Opn gene promoters in both MC3T3-E1 osteoblasts and MOBs**
**1,25(OH)(2)D(3) treatment also induces rapid recruitment of co-regulators such as SRC-1, -2, and -3, CBP, and p300 to both promoters**
**Recruitment leads to acetylation of histone 4 on Cyp24 but not the Opn. **

## Slide 31

## Histone Acetylation                              P.Marks 2001

## 							P.Marks 2001                 Histone Modification:

**Notes:**
Histones = basic proteins
5 key histones: 2 dimers: H2B-H2A and H4-H3 are present in duplicate in the nucleosome
		    H1 (H1o) is in linker region
DNA loops around nucleosome 2 times
H3 tail has a lysine (K) 9 ---gets acetylated and methylated 

## Overview of Chromatin-Level Regulation of Gene Expression

- Garfinkel and Ruden Nutrition:20:56-62, 2004