FIG. 6.

brachyury, a direct target gene of LSD1, is derepressed in Lsd1^{β-geo/β-geo} gene trap embryos. (A) Quantitative RT-PCR shows upregulation of brachyury mRNA in Lsd1^Δ3/Δ3 ES cells, with (day 0) and without (day 3) LIF. Values are expressed relative to the level of transcript in Lsd1^Lox/Δ3 cells with LIF. (B) The Western blot shows an increase in brachyury protein levels in Lsd1^Δ3/Δ3 ES cells with (day 0) and without (day 3) LIF. The fold induction of brachyury protein was calculated relative to Sin3A. (C) A schematic of the brachyury gene shows relative position of primers used for quantitative PCR following chromatin immunoprecipitation (ChIP). A region −600 bp from the transcriptional start site is enriched in anti-LSD1 ChIPs from Lsd1^Lox/Δ3 but not Lsd1^Δ3/Δ3 cells, showing specific enrichment of LSD1 protein (left panel). Loss of LSD1 protein results in increased histone H3 K4 dimethylation at the −600-bp region (right panel). (D) Quantitative RT-PCR analysis of e6.5 embryos. Homozygous (Lsd1^{β-geo/β-geo}) embryos have a 20-fold increase in brachyury mRNA compared with wild-type and heterozygous (Lsd1^+/β-geo) embryos. RasGRP3 transcription (5-fold) and CDA transcription (10-fold) are similarly increased in Lsd1^{β-geo/β-geo} embryos. Assays were performed in triplicate with individual embryos of each genotype. Values are expressed relative to the level of transcript in wild-type embryos; mean values (n = 3) ± SEM are plotted.