![Identification of LRR1 as a regulator of S-phase progression based on RNA-Seq analysis and a targeted CRISPR screen. (A) Cell-cycle entry pathway in mammalian cells. Rb, retinoblastoma protein. (B) Schematic of experimental setup for RNA-Seq. (C) Examples of gene expression time course measured by RNA-Seq. Each time point contains three independent experiments. RPKM, reads per kilobase of transcript per million mapped reads. (D) Volcano plot from differential expression analysis of three independent experiments. Differentially expressed genes (|log2[fold-change]| > 1, adjusted P value < 0.1) are highlighted. Labeled genes are examples of reported E2F targets (Bracken et al., 2004). FDR, false discovery rate. (E) Venn diagram of CDK4/6–dependent genes that are targets of E2F1, FOXM1, or MYBL2 (ENCODE ChIP-Seq datasets; see Materials and methods for details). (F) Manually annotated function of CDK4/6–dependent genes. (G) Top: Experimental setup. Bottom: Schematic of gating for S-phase cells (n = 3,000 random cells displayed). A.U., arbitrary unit; R.F.U., relative fluorescence unit. (H) Rate of EdU incorporation was measured in S-phase cells as the median nuclear intensity and was normalized to control cells (sgCNTL). Error bars are population medians with 95% confidence intervals (n ≥ 8,659 cells per condition). (I) LRR1 gene expression time course measured by RNA-Seq. Each time point contains three independent experiments (two experiments for 24-h time point). (J) Left: Serum-starved cells were released in the presence of DMSO or palbociclib, harvested, and blotted for LRR1 and known E2F targets TOP2A and GINS2. Arrowhead indicates LRR1-specific band; the other band is a nonspecific band. Right: Quantification of Western blots (relative to actin loading control), normalized to DMSO conditions. Paired Student’s t test; P = 8.0 × 10−3, 3.7 × 10−3 (n = 3 independent experiments). (K) E2F1 ChIP-Seq signal (blue plot, ENCODE ENCFF009LGS; data are representative of two independent experiments) and detected peaks (green line, ENCODE ENCFF998YJY, irreproducible discovery rate cutoff = 0.05). In all panels, **, P < 0.01; ***, P < 0.001.](https://cdn.rupress.org/rup/content_public/journal/jcb/220/8/10.1083_jcb.202009147/3/jcb_202009147_fig1.png?Expires=1767703514&Signature=CYn9II58OKOSX6DoK2PQzjg-d~DJrC5Ntr23P4Xmi9WvPSihn~6d3DYM1UEZf6D2R8j0flDDQarTpouRNxV2M1H40bLvSqa2elvKyFxo4lhiSuqaE~K5fVAvflxA6uijk2K1eXggGXMOOrMHkPqvstQfZQcKsfre1bKgjalzvhXpb6Ob50QQkWrFVVVkmo~elqFnxm8dKtV3rHniwxYIgozoxcTkyyf-TleuyfjPGJrDGB-TDCHyHzw71RQMzO9RjwuoTvYXjrzUJUdTazgppsGDWBDivLNNKCbnuYDqYJbxTbgYRmp5~bIkxOpGnniNA8L0J~wGAiIbpF1pLbwu6A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Identification of LRR1 as a regulator of S-phase progression based on RNA-Seq analysis and a targeted CRISPR screen. (A) Cell-cycle entry pathway in mammalian cells. Rb, retinoblastoma protein. (B) Schematic of experimental setup for RNA-Seq. (C) Examples of gene expression time course measured by RNA-Seq. Each time point contains three independent experiments. RPKM, reads per kilobase of transcript per million mapped reads. (D) Volcano plot from differential expression analysis of three independent experiments. Differentially expressed genes (|log2[fold-change]| > 1, adjusted P value < 0.1) are highlighted. Labeled genes are examples of reported E2F targets (Bracken et al., 2004). FDR, false discovery rate. (E) Venn diagram of CDK4/6–dependent genes that are targets of E2F1, FOXM1, or MYBL2 (ENCODE ChIP-Seq datasets; see Materials and methods for details). (F) Manually annotated function of CDK4/6–dependent genes. (G) Top: Experimental setup. Bottom: Schematic of gating for S-phase cells (n = 3,000 random cells displayed). A.U., arbitrary unit; R.F.U., relative fluorescence unit. (H) Rate of EdU incorporation was measured in S-phase cells as the median nuclear intensity and was normalized to control cells (sgCNTL). Error bars are population medians with 95% confidence intervals (n ≥ 8,659 cells per condition). (I) LRR1 gene expression time course measured by RNA-Seq. Each time point contains three independent experiments (two experiments for 24-h time point). (J) Left: Serum-starved cells were released in the presence of DMSO or palbociclib, harvested, and blotted for LRR1 and known E2F targets TOP2A and GINS2. Arrowhead indicates LRR1-specific band; the other band is a nonspecific band. Right: Quantification of Western blots (relative to actin loading control), normalized to DMSO conditions. Paired Student’s t test; P = 8.0 × 10−3, 3.7 × 10−3 (n = 3 independent experiments). (K) E2F1 ChIP-Seq signal (blue plot, ENCODE ENCFF009LGS; data are representative of two independent experiments) and detected peaks (green line, ENCODE ENCFF998YJY, irreproducible discovery rate cutoff = 0.05). In all panels, **, P < 0.01; ***, P < 0.001.