Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes

Abstract

Epigenetic chromatin modification is a major regulator of eukaryotic gene expression, and aberrant epigenetic silencing of gene expression contributes to tumorigenesis. Histone modifications include acetylation, phosphorylation, and methylation, resulting in a combination of histone marks known collectively as the histone code. The chromatin marks at a given promoter determine, in part, whether specific promoters are in an open/active conformation or closed/repressed conformation. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by the lysine-specific demethylase 1 (LSD1), a homologue of polyamine oxidases, may broadly repress gene expression. We now report that novel biguanide and bisguanidine polyamine analogues are potent inhibitors of LSD1. These analogues inhibit LSD1 in human colon carcinoma cells and affect a reexpression of multiple, aberrantly silenced genes important in the development of colon cancer, including members of the secreted frizzle-related proteins (SFRPs) and the GATA family of transcription factors. Furthermore, we demonstrate by chromatin immunoprecipitation analysis that the reexpression is concurrent with increased H3K4me2 and acetyl-H3K9 marks, decreased H3K9me1 and H3K9me2 repressive marks. We thus define important new agents for reversing aberrant repression of gene transcription.

Fig. 1.

Inhibition of LSD1 by polyamine analogues. (A) Bisguanidines (1a–1g). (B) Biguanides (2a–2f). (C) Three micrograms of purified LSD1 protein were incubated with 5 μM H3K4me2 (1–21 aa) as substrate in the presence of 1 μM of the indicated analogue. The results represent the mean of three determinations ± SD. (D and E) The effects of increasing concentrations of 1c (D) or 2d (E) on LSD1 activity in the presence of increasing substrate concentrations. Double reciprocal plots indicate inhibition of LSD1 by 1c and 2d to be noncompetitive.

Fig. 2.

Inhibition of LSD1 by polyamine analogues increases global H3K4me1 and H3K4me2, resulting in reexpression of aberrantly silenced genes in treated human colon cancer cells. (A) HCT116 cells were exposed to increasing concentrations of the indicated compound for 48 h, and 30 μg of nuclear protein per lane were analyzed for expression of H3K4me1, H3K4me2, H3K9me2, and PCNA as a loading control. (B) Histograms represent the mean protein expression levels of three determinations relative to PCNA ± SD, as determined by quantitative immunoblotting using infrared detection and analysis. (C) HCT116 cells were treated for 48 h with the indicated compounds, and total RNA was extracted for RT-PCR analysis of SFRP1, SFRP4, SFRP5, and GATA5 expression. GAPDH is included as an internal control. The results shown are from a single experiment repeated at least three times with similar results. (D) RKO cells were exposed to increasing concentrations of the indicated compound for 48 h, and 30 μg of nuclear protein per lane were analyzed for expression of H3K4me2 and PCNA. The histogram represents the mean protein amount of three determinations relative to PCNA ± SD. (E) RKO cells were treated for 48 h with the indicated compounds and total RNA was extracted for RT-PCR analysis of SFRP4 and SFRP5 expression. GAPDH is included as an internal control. The results shown are from a single experiment repeated at least three times with similar results.

Fig. 3.

Relative reexpression of SFRP4 and SFRP5 induced by polyamine analogue inhibitors of LSD1. HCT116 cells were treated with 5 μM 1c, 2d, 1d, or 2b; 1 μM DAC; or 300 nM TSA for 48 h. (A) qPCR analysis of SFRP4 and SFRP5 expression. Results are presented relative to expression induced by DAC and represent the mean of three independent experiments, each performed in triplicate ± SD. (B) Representative qPCR products were analyzed on GelStar-stained 2% agarose gel.

Fig. 4.

Inhibition of LSD1 by polyamine analogues increases activating H3K4me2 and acetyl H3K9 marks and decreases repressive H3K9me1 and H3K9me2 marks at the promoters of reexpressed genes. HCT116 cells were treated with 5 μM of the indicated compound for 48 h. (A–D) ChIP analysis was used to determine the occupancy of the indicated promoters by multiple activating and repressive marks. (E–H) The quantified results are the means of three independent experiments with an SD as indicated.

Fig. 5.

Promoter occupancy by LSD1. (A) HCT116 cells were treated with 5 μM of the indicated compounds for 48 h. ChIP analysis was performed to determine the occupancy of the indicated promoters by LSD1. (B) The quantified results are the mean of three independent experiments ± SD, relative to LSD1 detected at the SFRP1 promoter of untreated cells. (C) After HCT116 cells were treated with 5 μM of the indicated compounds for 48 h, ChIP was used to determine the levels of H3K4me2 and H3K4me1 at the promoters of SFRP2 and GATA4.

Fig. 6.

Knockdown of LSD1 by siRNA leads to specific gene reexpression. (A) HCT116 cells were transfected with scrambled or LSD1-targeted siRNA oligonucleotides for 48 h. Proteins isolated from transfected cells were subjected to quantitative immunoblotting with an antibody to LSD1. (B) The histogram represents the relative LSD1 protein quantity in scrambled and LSD1 siRNA transfectants. (C) ChIP analysis was used to determine the levels of H3K4me2 in the promoters of indicated genes. (D) HCT116 cells were treated for 48 h with 5 μM 1c or 2d or transfected with scrambled or LSD1 siRNA oligonucleotides for 48 h. RNA was extracted for RT-PCR analysis of expression of the indicated genes. GAPDH is included as an internal control.