Fig 2: Co-occupancy of Rinf with Pluripotency Factors and Tet Enzymes at Gene Regulatory Regions(A) Overlapping analysis of Rinf peaks (from this study) with those of pluripotency factors, Tet enzymes, and activating/repressive histone marks (from previous studies, see STAR Methods). Data presented as % of Rinf peaks overlapping with each of the factors.(B) Enrichment of Rinf peaks at ChIP-seq signals of Nanog, Tet1, and Tet2 at promoters (±2 kb of TSS and H3K4me3 positive, left) and enhancers (H3K27ac positive and H3K4me3 low, right).(C) Enrichment of ChIP-seq signals showing co-occupancy of Rinf, pluripotency factors, and Tet enzymes at selected pluripotency and Tet genes. H3K27ac and H3K4me3 tracks are used as reference to depict enhancers and promoters (±2 kb of TSS).(D) Co-immunoprecipitation of exogenously expressed V5-tagged Rinf with Nanog, Oct4, Tet1 catalytic domain (Tet1-CD), and Tet2 in HEK293T cells using anti-V5 antibody.(E) Co-immunoprecipitation of endogenous Rinf with Nanog, Oct4, and Tet2 in mouse ESCs using anti-V5 antibody. Rinf−/− ESCs are used as negative control.(F) Co-immunoprecipitation of in vitro transcribed and translated Rinf, Nanog, and Tet2 catalytic domain (Tet2-CD) from rabbit reticulocyte lysate.(G) Enrichment of Nanog and Tet2 at regulatory elements of indicated genes quantified by ChIP-qPCR in wild-type and Rinf−/− ESCs (data normalized to IgG). Actin and Cyr61 are used as negative controls.(H) Schematic of dual luciferase reporter assay applied to test enhancers targeted by Rinf (top). Transcriptional activity is presented as relative luminescence unit (RLU), which is normalized to the luminescence from cells that express the empty vector only (bottom).Data presented as mean ± SEM. *Statistically significant (p < 0.05). E, enhancer; p, promoter (see also Figure S2).

Fig 3: CXXC5 over-expression suppresses HSC activation. (A–C) LX-2 cells were infected with adenovirus carrying CXXC5 expression vector (Ad-CXXC5) or GFP (Ad-GFP). Gene expression levels were examined by qPCR and Western. Cell proliferation was measured by EdU staining. (D–F) Primary murine hepatic stellate cells were infected with adenovirus carrying CXXC5 expression vector (Ad-CXXC5) or GFP (Ad-GFP). Cell proliferation was measured by EdU staining. All experiments were repeated three times and one representative experiment is shown. *p < 0.05.

Fig 4: Expression and Chromatin Enrichment of Rinf in Mouse ESCs(A) Schematic of protein domains of Rinf and Idax.(B) Schematic of exon and intron structures of Rinf and Idax.(C) Rinf and Idax mRNA levels quantified by qRT-PCR in ESCs. Data normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh).(D) Expression of Rinf in mouse ESCs detected by immunofluorescence.(E) Rinf protein level in soluble and chromatin-bound fractions of ESC lysate.(F) Schematic of gene targeting strategy for generating Rinf knockout ESCs..(G) Rinf mRNA level quantified by qRT-PCR in targeted ESC clones. Data normalized to Gapdh.(H) Quantification of Rinf protein by western blot in targeted ESC clones.(I) Schematic of ChIP-seq strategy (left), and distribution of Rinf peaks in the genome (right).(J) ChIP-seq read densities at Rinf peaks presented as heatmaps (top) and line graphs (bottom). All rows are peaks and centers are the summits of Rinf peaks. The H3K4me3 and H3K27ac data were obtained from a previous study (see STAR Methods).(K) Enrichment of Rinf ChIP-seq signals at regulatory regions of selected pluripotency and Tet genes. H3K27ac and H3K4me3 tracks are used to depict enhancers and promoters (±2 kb of TSS). Selected regions of these peaks (red line) are validated by ChIP-qPCR in (L).(L) Quantification of enrichment of Rinf at regulatory elements of indicated genes by ChIP-qPCR in ESCs (data normalized to immunoglobulin G [IgG]). Rinf KO ESCs are used as control for antibody specificity. Actin is used as a negative control.For all panels, data are presented as mean ± SD. *Statistically significant (p < 0.05). E, enhancer; p, promoter. Scale bars, 50 mm (see also Figure S1).

Fig 5: Loss of Rinf Reduces the Expression of Pluripotency Factors and Tet Enzymes in ESCs and Perturbs Gene Expression Programs(A) Heatmap of differentially expressed genes identified. Colors indicating relative expression.(B) MA plot of the average gene expression level and change, with DEGs in red.(C) GO enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes.(D) Expression patterns of deregulated pluripotency genes, Tets, and Dnmts.(E) Expression patterns of differentially expressed genes that are bound by Rinf.(F) mRNA levels of pluripotency and Tet genes quantified by qRT-PCR. Three ESC lines of each genotype are used. Fgfr1 is used as a negative control (unchanged gene in RNA-seq). Expression of Tet enzymes in the left plot is normalized to the wild-type levels of Tet1.(G) Protein levels of indicated genes quantified by western blot. Quantification of signal intensity of bands by ImageJ is plotted on the right.(H) Restoration of the expression of pluripotency and Tet genes in Rinf−/− ESCs upon expression of exogenous Rinf. Data represent mRNA levels quantified by qRT-PCR.(I) mRNA levels of indicated genes quantified by qRT-PCR. Three independent ESC lines of each genotype are used. Fgfr1 is used as a negative control (unchanged gene in RNA-seq).(J) Protein levels of Dnmt3a and Dnmt3b quantified by western blot in ESCs. Quantification of signal intensity of bands by ImageJ is plotted on the right.(K) Global 5hmC levels in ESC DNA quantified by dot blot. Tet1/2/3 triple knockout (TKO) ESC DNA is used as negative control. Average 5hmC signal intensity was plotted.(L) Global 5mC levels in ESC DNA quantified by dot blot. Average 5mC signal intensity was plotted.Data presented as mean ± SD. *Statistically significant (p < 0.05). n.c., no change (see also Figure S3).