Fig. 3: Methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation sequencing (ChIP-seq) reveal the alternative splicing of DNMT1 regulated by the CCCTC-binding factor (CTCF) as the downstream target of PARP1/TET1. | Nature Communications

Fig. 3: Methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation sequencing (ChIP-seq) reveal the alternative splicing of DNMT1 regulated by the CCCTC-binding factor (CTCF) as the downstream target of PARP1/TET1.

From: TRIM37–PARP1–TET1 axis maintains stemness and prevents osteoporosis by inhibiting DNMT1 alternative splicing via 5hmC regulation

Fig. 3: Methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation sequencing (ChIP-seq) reveal the alternative splicing of DNMT1 regulated by the CCCTC-binding factor (CTCF) as the downstream target of PARP1/TET1.The alternative text for this image may have been generated using AI.

a Venn diagram showing overlap of genes enriched in MeDIP analysis (Late > Early), PARP1 ChIP-seq analysis (Early > Late) and TET1 ChIP-seq analysis (Early > Late). Numbers indicate the number of overlapping genes or regions. b ChIP-polymerase chain reaction (PCR) showing the binding or PARP1 and TET1 at the transcription-start site (TSS) of DNMT1 in early MSCs. c Early MSCs were lentivirally transduced with scrambled (CTR), PARP1 (PARP1-KD) or TET1 short hairpin RNAs (shRNAs) (TET1-KD), followed by western blotting showed that the knockdown of PARP1 or TET1 decreased the levels of the DNMT1 full-length protein. d Representative MeDIP peaks shown in early and late MSCs for genes encoding DNMT1. The high methylation region of late MSCs is located in exons 30 to 37, which is the catalytic domain of DNMT1. e 5’-rapid amplification of cDNA ends (RACE)-PCR revealed the increase of alternative splicing forms of DNMT1 in late MSCs. Arrow indicates the PCR product of the alternative splicing isoform. f CTCF ChIP real-time PCR showing the binding of CTCF in exon30 and exon37 in early and late MSCs. H19 serving as the positive control. g Early MSCs were lentivirally transduced with scrambled (CTR) and CTCF shRNAs (#1” and “#2” refer to two different shRNA constructs), followed by western blotting, which showed that the knockdown of CTCF decreased the DNMT1 full-length protein levels. h Realtime PCR showed that the knockdown of CTCF increased the levels of the alternative splicing isoform of DNMT1 in early MSCs. Late MSCs served as the control. i Plasmids carrying full-length (Full) or alternative splicing isoform (AS) of DNMT1 were transfected into 293 T cells, followed by immunoprecipitation of exogenous DNMT1 to determine the in vitro DNMT1 activity. Compared to Full, AS reduced the DNMT1 activity. Moreover, the addition of AS reduced the DNMT1 activity of Full in a dose-dependent manner. j Dot-blot assay shows that the 5mC level in plasmids carrying Full or AS of DNMT1 transfected 293 T cells. Addition of AS reduced the 5mC level in 293 T cells. Quantitation of dot-blot assay (j lower panel). k–m Early MSCs were inserted in lentiviral overexpression control vectors (CV) or vectors carrying AS DNMT1, followed by (k) western blotting analysis of senescence marker levels (AS M.W. size: around 130 kDa), (l) growth curve analysis, and (m) differentiation potential analysis. Cells were induced for osteogenic and adipogenic differentiation for 14 d, followed by ARS and Oil Red staining, respectively. Extracts of dyes were subjected to optic measurement. mright Quantification of optical measurements. Overexpression of AS-DNMT1 in MSCs inhibited their proliferation and differentiation potential, and increased the senescence marker levels. Results are expressed as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, unpaired two-tailed t-test, or ordinary one-way ANOVA). Exact p-values are shown in the Source Data file. Scale bar: 500 μm.

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