Valproic Acid and Trichostatin a Induce Transcriptional Silencing of a Subset of Genes

Deacetylase inhibition, estrogen signalling and promoter repression. Supplemental Material

Valproic acid and trichostatin A induce transcriptional silencing of a subset of genes that include estrogen receptor a.

George Reid1,*, Raphaël Métivier1,2, Chin-Yo Lin3, Stefanie Denger1, David Ibberson1, Heike Brand1, Vladimir Benes1, Edison T. Liu3 and Frank Gannon1.

*: Corresponding author. E-mail:

1 European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Phone: +49 6221 889 1102. Fax: +49 6221 889 1200.

2 Present Address: Equipe EMR, UMR CNRS, 6026 (ICM), Universite de Rennes I, Batiment 13, 35042 Cedex, Rennes, France.

3Genome Institute of Singapore, Genome Building, #02-01, 60 Biopolis Street, Singapore 138672.

Supplemental Figure S1: VPA and TSA are cytotoxic and cytostatic and induce apoptosis and differentiation. VPA has a cytotoxic effect on both ER-a positive and ER-a negative cells. There is little difference in the cytotoxicity of VPA (IC50 values between 1 and 3 mM) and TSA (IC50 values between 1 and 3 nM) between the ER-a positive breast carcinoma cell line MCF-7, and the ER-a negative cell lines MDA MB231 and HeLa. Moreover, at 40 hours after the addition of VPA, around 25% of cells were found to have a DNA content less than in Go/G1 (figure S1(b)), indicating that a proportion of cells are apoptotic. VPA also induces differentiation of MCF-7 cells, as indicated by the extensive number of lipid droplets in cells treated for 48 hours with 6 mM VPA (figure S1(c)). TSA also induces apoptosis and differentiation in MCF-7 cells (data not shown). The general growth inhibitory effect of VPA and TSA suggests that multiple changes in gene expression occur on inhibition of deacetylase activity.

Supplemental Figure S2: VPA and TSA induce cell cycle arrest. Flow cytometry indicates that treatment with 6 mM VPA or with 300 nM TSA arrests MCF-7 cells in the Go/G1 and G2/M phases of the cell cycle, in contrast to the Go/G1 arrest induced by the pure antiestrogen ICI 182,780. The analysis with TSA was restricted to 30 hours of treatment due to excessive cell death occurring beyond this time. Arrest in both Go/G1 and G2/M indicates that multiple key regulatory components, in addition to ERa, are affected by VPA and TSA treatment.

Supplemental Figure S3: Comparison of the response MCF-7 cells make to VPA and to TSA.

Supplemental Figure S4: Cycloheximide blocks the antiestrogenic effect of VPA or TSA on MCF-7 cells. The response of the estrogen responsive gene set, as described for figure 3, was analysed with regard to the effect that concomitant addition of cycloheximide (CHX) has on the response to VPA and to TSA. The addition of CHX prevents VPA or TSA induced down-regulation or ERa, and in keeping with this effect, VPA and TSA no longer have a predominantly anti-estrogenic effect when de novo protein synthesis is blocked by CHX.