1. Patcharawalai Whongsiri1 Wolfgang A. Schulz2 Chanchai Boonla1
Oxidative stress alters histone methylation patterns on LINE-1 elements in bladder cancer cell
Patcharawalai Whongsiri1
Wolfgang A. Schulz2
Chanchai Boonla1
1Department of Biochemistry, Faculty of Medicine, Chulalongkorn University
2Department of Urology, Faculty of Medicine, Heinrich Heine University, Germany

2. Histone modification & Oxidative stress
1. Histone modifications, such as methylation, regulate gene expression.
Active gene markers: H3K4me3, H3K18Ac
Heterochromatin markers: H3K9me3, H3K27me3
Shmoop Editorial Team, "Biology DNA Packaging - Shmoop Biology," Shmoop University, Inc.,
Last modified November 11, 2008,
2. Oxidative stress is implicated in alterations of LINE-1 expression
during bladder cancer progression.

3. Objective
To explore whether oxidative stress alters histone methylation patterns on LINE-1 elements in bladder cancer cells

4. Materials & Methods VMCUB1 bladder cancer cell line
Control (untreated)
30 µM H2O2
30 µM H2O2 +
300 µM Tocopherol acetate
(Antioxidant)
72 hours
Protein and chromatin extraction for analyses

5. Materials & Methods Analyses
Chromatin immunoprecipitation (ChIP): to investigate histone methylations on LINE-1 elements including H3K9me3 (heterochromatin marker) and H3K4me3 (active gene marker)
After ChIP, LINE-1 enrichment was measured by qPCR.
Western blot: to investigate ORF1p expression (LINE-1 encoded protein)
Protein carbonyl assay (Oxidative stress marker)

6. Results: Oxidative stress status
Protein carbonyl level relative to control
control
30 µM H2O2
30 µM H2O2
+ TA
Protein carbonyl level was higher following H2O2 treatment compared
to untreated cells indicating the oxidative stress induced by H2O2.

7. Results: ORF1p expression under oxidative stress
control
30 µM H2O2
30 µM H2O2+ TA
ORF1p
Tubulin
control
30 µM H2O2
+ TA
ORF1p expression was increased
in H2O2-treated cells
and slightly decreased
in antioxidant co-treatment.
ORF1p expression
relative to control

8. Results: Histone methylations
30 µM H2O2
+ TA
control
LINE-1 enrichment relative to input
H3K4me3 IP
(Active gene marker)
H3K9me3 IP
(Heterochromatin marker)
ChIP revealed altered histone methylation patterns on LINE-1 elements under H2O2 exposure. H3K4me3 increased following H2O2 treatment but decreased after co-treatment with antioxidant. Conversely, H3K9me3 decreased during H2O2 treatment but increased after antioxidant co-treatment

9. Conclusion
Oxidative stress
LINE-1 expression is associated with specific histone methylation on lysine residues. Oxidative stress increased H3K4me3 and decreased H3K9me3 on LINE-1 elements correlating with the higher expression of ORF1p in bladder cancer cell. H3 K4 K9 M
Increased H3K4me3
Decreased H3K9me3
ORF1p ↑

10. Acknowledgements DPST scholarship
Thailand Research Fund and Chulalongkorn University
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University
Department of Urology, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany