Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Graphical representation of the Dnmt1 tet/tet cell line. Donated by Chaillet lab. Modified from Borowczyk et al., 2009 – lacking neomycin and puromycin resistance cassettes. (B) Western blot for Dnmt1 and H4 (loading control) proteins over 7 day dox treatment in for Dnmt1 tet/tet cells. M – Molecular ladder. Arrow points to expected band for Dnmt1 protein. (C) Southern blot using methylation sensitive restriction digest (HpyCH4IV enzyme –cut site shown below) followed by hybridisation using probe for minor satellite repeats in Dnmt1 tet/tet cells with (over 7 days) or without dox and in J1 and TKO cells. Methylated DNA cannot be digested and remains as high MW bands. Unmethylated DNA is digested and separates into polymers of satellite repeats. (D) HPLC Mass spectrometry analysis of global 5mC levels as percentage of total Guanines over 7 day dox treatment in for Dnmt1 tet/tet cells and TKO cells as control. Significance was determined by Kruskal-Wallis (one-way Anova) test with FDR correction – Benjamini, Krieger and Yekutieli. (E) Western blot for H3K27me3, H2AK119ub, H3K9me3 and H4 (loading control) over 7 day dox treatment in for Dnmt1 tet/tet cells. M – Molecular ladder. (F) Fixed immunofluorescence (IF) in Dnmt1 tet/tet cells over 7 days dox treatment and TKO cells stained for H2AK119ub (bottom row) and counterstained for DAPI (top row). Representative nuclei from single slice of 3D stack are shown. Z-stacks obtained using 40x air objective on an epifluorescent microscope. Scale bar corresponds to 5µm. Arrowheads point to a representative chromocenter at which the DAPI and antibody signals overlap. (G) Violin plot showing mean signal intensity of H2AK119ub antibody signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells over 7 days dox treatment and TKO cells. Grey dashed line corresponds the threshold above which the signal is considered increased at the chromocenter compared to the nucleoplasm. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001.

Article Snippet: 100ng of gDNA of each sample was digested with methylation-sensitive restriction enzyme HpyCH4IV (also known as MaeII, NEB, R0619S) overnight at 37°C.

Techniques: Modification, Western Blot, Control, Southern Blot, Methylation, Hybridization, Mass Spectrometry, Immunofluorescence, Staining, Microscopy

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Schematic representation of the CRISPR strategy and sgRNA targeting to generate Ezh2 KO. Adapted from (Lavarone, Barbieri and Pasini, 2019). Primer pairs used for genotyping are shown. CRISPR guide PAM sites are shown to be within the exons. (B) Homozygosity PCR screen using two primer pairs in which one of the primers binds inside the deleted region. Successful Ezh2 KO clones are expected to produce no band while wild type alleles would yield 440bp band Exon 2 primer pair (top two panels) and 290bp band for Exon 20 primer pair (bottom two panels). Selected clone (C3) used in this study is marked with red asterisk * (C) Western blot for Dnmt1, Ezh2, H3K27me3 and H4 (loading control) in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox at different time points. (D) Western blot for H2AK119ub and H4 (loading control) in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox at different time points. (E) Mean satellite read counts per million (CPM) from RNA sequencing in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox. Significance was determined by Ordinary one-way Anova with Tukey’s multiple comparisons test, ** p<0.01, *** p<0.001, **** p<0.0001. (F) Western blot for H2AK119ub, H3K27me3, Dnmt1, H3K9me3 and H4 (loading control) in Ring1b-AID cells treated with DMSO, auxin, Dnmt1i or both auxin and Dnmt1i. (G) Southern blot using methylation sensitive restriction digest (HpyCH4IV enzyme –cut site shown below) followed by hybridisation using probe for minor satellite repeats in Ring1b-AID cells treated with DMSO, auxin, Dnmt1i or both auxin and Dnmt1i.

Article Snippet: 100ng of gDNA of each sample was digested with methylation-sensitive restriction enzyme HpyCH4IV (also known as MaeII, NEB, R0619S) overnight at 37°C.

Techniques: CRISPR, Clone Assay, Western Blot, Control, RNA Sequencing, Southern Blot, Methylation, Hybridization

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Graphical representation of the Kdm2b protein containing N-terminal JmjC domain, a ZF-CxxC domain, PHD domain, an F-box domain and 7 Leucine-rich repeats (LRR) donated by Klose lab. Graph shows location of LoxP sites (scissors) flanking the ZF-CxxC domain. Upon OHT treatment cre-recombination leads to excision of the domain. (B) PCR with primers flanking the CxxC domain performed on genomic DNA from Kdm2b cells treated with DMSO, OHT, Dnmt1i or both OHT and Dnmt1i. H20 serves as no DNA control. Primers amplify a 1kb fragment in DMSO and Dnmt1i conditions and a smaller (400bp) fragment following recombination induced by OHT. (C) Southern blot using methylation sensitive restriction digest (HpyCH4IV enzyme –cut site shown below) followed by hybridisation using probe for minor satellite repeats in Kdm2b cells containing the Cbx7-GFP reporter treated with DMSO, OHT, Dnmt1i or both OHT and Dnmt1i. Western blot for H2AK119ub, H3K27me3, H3K9me3 and H4 (loading control) in Ring1b-AID cells treated with DMSO, OHT, Dnmt1i or both OHT and Dnmt1i.

Article Snippet: 100ng of gDNA of each sample was digested with methylation-sensitive restriction enzyme HpyCH4IV (also known as MaeII, NEB, R0619S) overnight at 37°C.

Techniques: Control, Southern Blot, Methylation, Hybridization, Western Blot

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Southern blot using methylation sensitive restriction digest (HpyCH4IV enzyme –cut site shown below) followed by hybridisation using probe for minor satellite repeats in TET WT and TKO cells treated with DMSO or Dnmt1i. (B) Western blot for H2AK119ub, H3K27me3 and H4 (loading control) in TET WT and TKO cells treated with DMSO or Dnmt1i.

Article Snippet: 100ng of gDNA of each sample was digested with methylation-sensitive restriction enzyme HpyCH4IV (also known as MaeII, NEB, R0619S) overnight at 37°C.

Techniques: Southern Blot, Methylation, Hybridization, Western Blot, Control