Fig 2: CBX2 targets Lef1 for repression in XY gonads.(A) ChIP-qPCR for CBX2 on embryonic gonads (2 replicates containing 50 pooled E13.5 and E14.5 XY gonads). Values represent mean ± SEM. (B) Gene expression profile of Lef1 in XX (pink) and XY (blue) supporting cells throughout sex determination from [41]. (C) Genome browser tracks showing ChIP-seq profiles for H3K4me3 (green) and H3K27me3 (red) at the Lef1 locus. Note the spreading of H3K27me3 over the gene body in XY E13.5. Bold black lines represent significant enrichment when compared to flanking regions as determined by HOMER.

Fig 3: CBX2 knockdown enhances the therapeutic efficiency of tazemetostatA549 cells transfected with control or CBX2 siRNAs were treated with DMSO or TAZ (200 nmol/L). (A) MTT assays were applied to examine the viability of the indicated cells (n = 4). (B) Transwell assays were applied to detect the invasion of A549 cells (n = 3). The images represent one field under a microscope. Scale bar, 200 µm. The invaded cells were counted.

Fig 4: SAHA induces CBX2 degradation. a Western blot analysis of CBX2 in K562, U937, and HL-60 cells untreated (-) or treated with 5 µM SAHA at indicated times. ERK1/2 was used as loading control. b Real-time qPCR analysis for CBX2 expression in K562, U937 and HL-60 cells untreated (-) or treated with 5 µM SAHA at indicated times. Error bars indicate standard deviation (STD) of three biological replicates. p-value is not significant. c Western blot of CBX2 in 3 primary blasts derived from AML patients untreated (-) or treated with 5 µM SAHA at 24 h. d Real-time qPCR analysis of CBX2 expression levels (relative to ctr) in three primary blasts derived from AML patients untreated or treated with 5 µM SAHA at 24 h. Results show CBX2 relative fold change in SAHA-treated blasts compared to untreated counterpart. Error bars represent STD of three technical replicates. p-value is not significant

Fig 5: Knockdown of let-7 can reverse Hox gene dysregulation.(A) Morphology (top panels) and alkaline phosphatase activity (bottom panels) of Wt and Lin28a–/– ES-like cells. (B) Western blot analysis of Lin28a in ES-like cells. ß-actin is shown as a loading control. (C) q-PCR analysis of stemness factors. (D) q-PCR analysis of let-7-family members. The level of expression relative to total let-7 amount in Wt is shown. (E) q-PCR analyses of Hoxa11 and Hoxd12 over a time course of 3, 6, 9, 12 days following embryoid body formation. (F) Precursor sequences of let-7-family members and guide RNAs for let-7 targeting Let-7 mature microRNAs are shown in red. The protospacer adjacent motif (PAM) sequence for hCas9 is highlighted in yellow, and targeting sequences are underlined. (G) Let-7 expression in Wt, Lin28a–/– and Lin28a–/–; let-7 KD cells. The level of expression relative to total let-7 amount in Wt is shown. (H) Let-7 knockdown rescues Hox gene dysregulation in Lin28a–/– cells. (I) Cbx2 expression level of Wt, Lin28a–/– and Lin28a–/–; let-7 KD derived EBs. ß-actin is shown as a loading control. (J) Schematic diagram of let-7 target site deletion from Cbx2 3’UTR and genotyping via PCR of mutant clones. (K) q-PCR analyses of Hoxa11 and Hoxd12 following embryoid body formation. (L) Schematic diagram of Lin28a/let-7 mediated Hox gene regulation. All data are expressed as the mean ± SEM (n = 3). n.s., not significant.Figure 5—source data 1.Source data related to panel C-E, G-H, and K.