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1.
Fig 7

Fig 7. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

(A) Morphology of the mouse embryonic stem cell colonies at day 5 after the transfection with siLSD1. R1 ES cells were transfected with siControl or siLSD1 and stained with LSD1 antibody (green). (B) LSD1 knockdown affects growth rate of mouse embryonic stem cells. Cell growth was determined with the CellTiter-Blue assay after 5 days of transfection. Data are the means ± SEM of two independent experiments.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
2.
Fig 2

Fig 2. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

LSD1 and H3K4me2 are enriched at a group of active genes in mouse ES cells. (A and B) LSD1 (A) and H3K4me2 (B) peaks are enriched at active genes and correlate with gene expression in ES cells. The LSD1 and H3K4me2 levels were calculated for the gene sets and are represented as high, medium, or low levels. (C) The overrepresented set of LSD1 target genes is associated with transcriptional regulation. Gene ontology analysis was performed using tools provided by DAVID Bioinformatics Resources, and highly significant groups are shown. P values are shown in parentheses.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
3.
Fig 1

Fig 1. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

ChIP-seq analysis demonstrates significant overlap between LSD1 and H3K4me2 genomic regions. (A) Tag density plot shows strong enrichment of LSD1 and H3K4me2 near the TSS. Distribution of LSD1 and H3K4me2 at the gene was defined as the intervals observed from 2 kb upstream from the TSS to the TES. (B) Summary of genome-wide distribution of LSD1 and H3K4me2 ChIP-seq peaks. The percentages of sites mapped to promoter, intragenic, and distal genomic regions are shown in parentheses. (C) LSD1 matrices predicted by the de novo motif discovery algorithm Weeder. (D) The top predicted LSD1 motif is localized within the center of the LSD1 binding peaks. The locations of the motifs were deduced from the Weeder output, and these values were used to compute distance to the center of the input sequences. (E) LSD1 binds to the genome in close proximity to H3K4me2. Approximately 91% of the LSD1 intervals overlap H3K4me2 intervals (marked by red line).

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
4.
Fig 4

Fig 4. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

(A and B) Stability of LSD1 protein was not affected by nocodazole treatment. R1 ES cells were treated with either vehicle or nocodazole (100 ng/ml for 16 h), and whole-cell extracts were prepared and subjected to immunoblotting for LSD1/GAPDH (A) and Jarid1b (B). Lanes 1, molecular mass marker; lanes 2, control; lanes 3, nocodazole-treated cells; lanes 4, vehicle-treated cells. (C) Synchronization of cells at G2/M phase by nocodazole (100 ng/ml for 16 h) had no effect on the levels of H3K4 and H3K9 methylations. Immunoblots of histones prepared from control (lane 1), nocodazole-treated (lane 2), and vehicle-treated (lane 3) cells were probed with indicated antibodies. (D) LSD1 interaction with CoREST is impaired in nocodazole-treated cells. Cell extracts from vehicle-treated (lane 1) and nocodazole-treated (lane 2) cells were immunoprecipitated (IP) with anti-LSD1 antibody and immunoblotted (IB) for CoREST. The same blot was stripped and reprobed for LSD1. (E) Western immunoblot showing the stability of CoREST in nocodazole-treated cells. Lane 1, vehicle-treated cells; lane 2, nocodazole-treated cells. (F) Immunoblots of chromatin prepared from vehicle-treated (lane 1) and nocodazole-treated (lane 2) cells were probed with LSD1 and H3K4me2. Note the reduced amount of LSD1 in chromatin prepared from nocodazole-treated cells. All of the blots shown are representative of two independent experiments.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
5.
Fig 3

Fig 3. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

LSD1 is regulated in a cell cycle-dependent manner. (A) Immunofluorescence analysis showing the association of LSD1 (green) with nuclei (blue) in G1, S, and G2 phases and its displacement from chromatin to cytoplasm of M-phase ES cells (arrows). DMSO, dimethyl sulfoxide. (B) LSD1 is excluded from the chromatin of cells at all phases of mitosis. R1 ES cells growing under self-renewal conditions were stained with antibodies to H3K4me2 (red; top) or LSD1 (green; bottom). Nuclei were stained with DAPI (blue). Arrows show cells at different phases of mitosis as indicated. Note the cytoplasmic localization of LSD1 in M-phase cells. (C) Synchronization of cells at G2/M phase by nocodazole treatment (100 ng/ml for 16 h) significantly increased the amount of cells with LSD1 (green) excluded from the chromatin (blue) (arrows). (D) Quantitation of cells in G1/S/G2 and M phase in R1 ES cultures. Cells grown in vehicle or nocodazole (100 ng/ml for 16 h) were stained with antibodies to LSD1, and nuclei were stained with DAPI. The nuclear morphology and the subcellular localization of LSD1 were analyzed by fluorescence microscopy. The numbers of cells analyzed for subcellular localization of LSD1 and nuclear morphology characteristics in G1/S/G2 and M phase in each group are shown in parentheses. (E) Flow cytometry analysis of R1 ES cells stained with Hoechst 33342. (F) Representative Amnis ImageStream flow cytometry images showing different stages of the cell cycle as indicated.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
6.
Fig 8

Fig 8. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

(A to C) UCSC Genome Browser maps show LSD1 peaks at the promoter regions of Oct4 and Sox2 in mouse ES cells. The locations of LSD1 peaks within the genes were defined as the intervals observed from 3 kb upstream to 2 kb downstream from the TSS. Amplicons are numbered relative to their TSS along the gene. The schematics at the bottom show the locations of the LSD1 peaks and at the top (numbered) show the locations of the primer sets used to detect the ChIP-enriched DNA fragments within the context of the genomic structures of mouse Oct4, Sox2, and Nanog. (D to F) High-resolution mapping of LSD1 binding sites across the Oct4, Sox2, and Nanog promoters in mouse ES cells by ChIP analysis. Fold enrichment is the relative abundance of DNA fragments at the indicated regions compared to the abundance at a control region (intergenic) as quantified by real-time PCR. Rabbit IgG was used as a control. *, P < 0.001. (G to L) ChIP analysis of H3K4me2 (G to I) and Oct4 (J to L) occupancy on the Oct4, Sox2, and Nanog promoters in LSD1 knockdown ES cells using the respective antibodies. LSD1 knockdown significantly increased H3K4me2 levels without affecting the levels of Oct4 at Oct4 and Sox2 promoters. Data are the means ± SD from three independent experiments. *, P < 0.01.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
7.
Fig 9

Fig 9. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

Cell cycle-dependent expression of selected LSD1 target genes. (A) Nocodazole and vincristine treatment significantly increased the number of cells in M phase. Cells were treated with 100 ng/ml of nocodazole or vincristine for the indicated period of time, and cells at different phases of the cell cycle were analyzed by imaging flow cytometry. (B to G) ChIP analysis of LSD1 (B to D) and H3K4me2 (E to G) on Oct4, Sox2, and Nanog promoter regions in R1 ES cells treated with nocodazole or vehicle for the indicated period of time. Data are the means ± SD from three independent experiments. Treatment of ES cells with nocodazole for 16 h significantly reduced the occupancy of LSD1 and increased H3K4me2 at Oct4 and Sox2 promoters. *, P < 0.01. (H to V) Expression of selected LSD1 target genes (H to Q) and nontargets (R to V) in nocodazole- and vincristine-treated ES cells. R1 ES cells were treated with 100 ng/ml of nocodazole or vincristine for the indicated period of time, and mRNA levels were analyzed by qRT-PCR with fold differences measured against the results for control ES cells (0 h) and normalized using β-actin, α-tubulin, and rps11 levels in these cells (mean ± SEM, n = 6; *, P < 0.01). Treatment with nocodazole and vincristine increased the expression of selected LSD1 target genes without affecting the expression of nontarget genes.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
8.
Fig 5

Fig 5. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

The corepressor activity of LSD1 is regulated in a cell cycle-dependent manner. (A) Schematic of the plasmid constructs pBIND-LSD1, c-terminal-deletion mutant pBIND-LSD1Δ, pBIND-PGC1α, and c-terminal-deletion mutant pBIND-PGC1αΔ. G4 DBD, GAL4 DNA binding domain; AO, amine oxidase; RBM, RNA binding motif. (B) pBIND-LSD1 represses transcription. Various amounts of pBIND-LSD1 or pBIND-LSD1Δ plasmid were transfected into R1 ES cells together with pG5luc reporter gene, and the cell extracts were prepared for luciferase activity measurements. In each sample, firefly luciferase activity was normalized to the Renilla luciferase value, and the results were expressed as the means ± standard errors of the means (SEM). The reporter activity in the presence of the pBIND vector was designated 100. The data are the means of three independent experiments. (C) pBIND-LSD1Δ mutant is defective in repression. The repression activity was expressed as the fold repression (mean ± SEM, n = 8) relative to the results for the pBIND vector (100%). pBIND-PGC1α and pBIND-PGC1αΔ constructs were used as controls. (D) Effect of nocodazole on LSD1 corepressor activity was tested by cotransfecting pBIND-LSD1 or pBIND-LSD1Δ plasmids along with the pG5luc reporter gene into R1 ES cells. In all, cells were treated with 100 ng/ml nocodazole 24 h after transfection. Luciferase activity was analyzed 16 h after the addition of nocodazole and compared with that in vehicle-treated cells (mean ± SEM, n = 8). For the experiments whose results are shown in panels C and D, 0.5 μg of plasmid constructs were used in all transfections. (E) Nocodazole treatment causes displacement of pBIND-LSD1 from the chromatin. R1 ES cells were transfected with pBIND-LSD1 construct and treated with vehicle or 100 ng/ml of nocodazole for 16 h. The cells were stained with LSD1 antibody (green) and nuclei with DAPI (blue). Note the dissociation of pBIND-LSD1 (green) in the cell with a condensed nucleus in the bottom panels. *, P > 0.001.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.
9.
Fig 6

Fig 6. From: Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells.

LSD1 represses the expression of LSD1 target genes in ES cells. (A) Effect of LSD1 siRNA on endogenous levels of LSD1 protein in ES cells. Cells were transfected with siLSD1 and harvested at the indicated times. The LSD1 protein level was confirmed by Western blotting. GAPDH was used as a loading control. (B) Expression of selected LSD1 target genes and nontargets (Jarid1b, Clcn1, c-Myc, Nanog, and Tbx3) in ES cells at 48 h after siLSD1 transfection. A nonspecific control siRNA was included. cDNAs were prepared from the LSD1 knockdown cells and analyzed by real-time PCR, with fold differences measured against the results for control ES cells and normalized using β-actin, α-tubulin, and rps11 levels in these cells. Data are the means ± SEM from triplicate measurements (n = 6 to 8). (C) Increased expression of Oct4 in LSD1 knockdown cells was further confirmed by droplet digital PCR (ddPCR). Data are the means ± standard deviations (SD) (n = 4). (D) LSD1 enzymatic activity is required for the suppression of Oct4. Western blot analysis of LSD1 protein in ES cells transiently transfected with LSD1-FLAG or the catalytically inactive (K661A) form of LSD1 for 48 h is shown. The blots were probed with anti-LSD1 and anti-FLAG antibodies. Oct4 mRNA levels were quantified by ddPCR and normalized to β-actin levels in these cells. Data are the means ± SD (n = 4). *, P < 0.01 by one-way ANOVA followed by Tukey's post hoc comparisons. (E to G) Oct4 protein levels are shown to increase in LSD1 knockdown ES cells. R1 ES cells were transfected with LSD1 siRNA or control siRNA. After 48 h, total cell lysates were prepared, and Oct4 (E), GAPDH (F), and Nanog (G) levels were determined by Western immunoblotting. Lanes 1, molecular mass markers; lanes 2, control (nontransfected); lanes 3, siLSD1-treated cells; lanes 4, siControl-treated cells. Representative immunoblots from two independent experiments are shown.

Venugopalan D. Nair, et al. Mol Cell Biol. 2012 December;32(23):4861-4876.

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