Journal: Science Advances

Article Title: Genomic context– and H2AK119 ubiquitination–dependent inheritance of human Polycomb silencing

doi: 10.1126/sciadv.adl4529

Figure Lengend Snippet: ( A ) ChIP-qPCR analysis of H3K27me3 enrichment at the reporter locus and surrounding regions at the WT1 locus before establishment (−Dox), after establishment (+Dox), and during the maintenance phase (Dox removal). GAPDH served as a negative control and the native Polycomb-repressed MYT1 gene as a positive control. Results for two biological replicates are presented. Error bars represent SDs. The location of PCR amplicons and CGIs are highlighted in the map at the top. ( B ) Genome browser snapshots of H3K27me3 ChIP-seq reads at the reporter locus inserted near WT1 and the endogenous Polycomb-silenced PCDH10 genes. Expanded scale (bottom) highlights the spreading of H3K27me3 to the promoter of WT1 and its maintenance. ( C ) Same as (A) but H2AK119ub1 ChIP-qPCR. ( D ) ChIP-qPCR analysis of H3K27me3 enrichment at the reporter locus inserted near B2M and surrounding regions before establishment (−Dox), after establishment (+Dox), and during the epigenetic maintenance of silencing (Dox removal). GAPDH served as a negative control and the native Polycomb-repressed PCDH10 gene as a positive control. Results for two biological replicates are presented. Error bars represent SDs. ( E ) Same as (D) but H2AK119ub1 ChIP-qPCR; HOXA3 served as a positive control.

Article Snippet: For H3K27me3 ChIP-Seq, Drosophila S2 chromatin (Active Motif, no. 53083) and histone H2Av antibody (Active Motif, no. 61686) were added as spike-in controls.

Techniques: ChIP-qPCR, Negative Control, Positive Control, ChIP-sequencing

Journal: Science Advances

Article Title: Genomic context– and H2AK119 ubiquitination–dependent inheritance of human Polycomb silencing

doi: 10.1126/sciadv.adl4529

Figure Lengend Snippet: ( A ) Flow cytometry histograms showing CITRINE expression, with the reporter inserted at the Polycomb target locus, WT1 , after establishment of silencing (+Dox) and 8 days after removal of doxycycline (Dox removal) in control ( EED +/+ ) and EED -deleted cell line ( EED −/− ). ( B ) Schematic diagram showing the wild-type EED ( HA-EED ) and the three residues mutated [F97A, Y148A, and Y365A] in the aromatic cage of EED ( HA-EED-3A ) (top). A snapshot of the structure of EED aromatic cage with bound H3K27me3 peptide. The EED is colored in gray, the F97, Y148, and Y365 residues are in blue, and the H3K27me3 peptide is red (bottom) (PBD3IIW) . ( C ) Western blot showing H3K27me3 levels in EED +/+ , EED −/− , and EED −/− overexpressing HA-EED or HA-EED-3A. Histone H3 is used as a loading control. ( D ) Flow cytometry histograms showing CITRINE expression 8 days after removal of doxycycline (Dox removal) in HA-EED and HA-EED-3A cells. ( E ) ChIP-qPCR analysis of H3K27me3 enrichment at the reporter locus and surrounding regions before establishment (−Dox), after establishment (+Dox), and 8 days after removal of doxycycline (Dox removal) in EED +/+ , EED −/− , HA-EED , and HA-EED-3A cells . GAPDH served as a negative control and the native Polycomb-repressed MYT1 gene as a positive control. Error bars represent SDs.

Article Snippet: For H3K27me3 ChIP-Seq, Drosophila S2 chromatin (Active Motif, no. 53083) and histone H2Av antibody (Active Motif, no. 61686) were added as spike-in controls.

Techniques: Flow Cytometry, Expressing, Control, Western Blot, ChIP-qPCR, Negative Control, Positive Control

Journal: Science Advances

Article Title: Genomic context– and H2AK119 ubiquitination–dependent inheritance of human Polycomb silencing

doi: 10.1126/sciadv.adl4529

Figure Lengend Snippet: ( A ) Flow cytometry histograms showing CITRINE expression, inserted at the Polycomb target locus, WT1 , after establishment of silencing (+Dox) and at the indicated days after removal of doxycycline (Dox removal) in wild-type control (WT) and MTF2 -deleted cell line ( MTF2 −/− ) . ( B ) Schematic diagram showing the wild-type MTF2 ( Flag-MTF2 ) and the two residue mutations [K338A and K339A] generated in the extended homology (EH) domain of MTF2 ( Flag-MTF2-EH ) (top). A snapshot of the structure of MTF2 EH domain with bound DNA. MTF2 is colored in green, the K338 and K339 residues are in red, and the DNA is multicolored (PDB: 5XFR) . ( C ) Flow cytometry histograms showing CITRINE expression at day 8 after removal of doxycycline (Dox removal) in MTF2 −/− and MTF2 −/− cells overexpressing Flag-MTF2 or Flag-MTF2-EH . ( D ) ChIP-qPCR analysis of Flag enrichment at the reporter locus and surrounding regions before establishment (−Dox), after establishment (+Dox), and 8 days after removal of doxycycline (Dox removal) in MTF2 −/− and MTF2 −/− overexpressing Flag-MTF2 and Flag-MTF2-EH. GAPDH served as a negative control and the native Polycomb-repressed PTF1A gene as a positive control. Error bars represent SDs. ( E ) Same as (D) but ChIP for H3K27me3 in WT, MTF2 −/− and MTF2 −/− overexpressing Flag-MTF2 or Flag-MTF2-EH. ( F and G ) Same as (D) but ChIP for MTF2 at the WT1 (F) and TFRC (G) loci.

Article Snippet: For H3K27me3 ChIP-Seq, Drosophila S2 chromatin (Active Motif, no. 53083) and histone H2Av antibody (Active Motif, no. 61686) were added as spike-in controls.

Techniques: Flow Cytometry, Expressing, Control, Residue, Generated, ChIP-qPCR, Negative Control, Positive Control

Journal: Science Advances

Article Title: Genomic context– and H2AK119 ubiquitination–dependent inheritance of human Polycomb silencing

doi: 10.1126/sciadv.adl4529

Figure Lengend Snippet: ( A ) Flow cytometry histograms showing CITRINE expression after establishment of silencing (+Dox) and 8 days after removal of doxycycline (Dox removal) in wild-type (WT) and RING1A/B -deleted cell line ( RING1A/B −/− ) with the reporter inserted at the Polycomb target locus, WT1. ( B ) ChIP-qPCR analysis of H3K27me3 enrichment at the reporter locus and surrounding regions before establishment (−Dox), after establishment (+Dox), and 8 days after removal of doxycycline (Dox removal) in control and RING1A/B −/− cells. GAPDH served as a negative control and the native Polycomb-repressed MYT1 gene as a positive control. Error bars represent SDs. ( C ) Schematic diagram showing the wild-type RYBP ( RYBP-Flag ) and the two residues mutated [T31A and F32A] generated in the ubiquitin binding domain of RYBP ( RYBP-2A-Flag ) (top). Flow cytometry histograms showing CITRINE expression 4 days after removal of doxycycline (Dox removal) in EED +/+ and HA-EED-3A cells overexpressing RYBP-Flag or RYBP-2A-Flag. (−) refers to nontransfected cells (bottom).

Article Snippet: For H3K27me3 ChIP-Seq, Drosophila S2 chromatin (Active Motif, no. 53083) and histone H2Av antibody (Active Motif, no. 61686) were added as spike-in controls.

Techniques: Flow Cytometry, Expressing, ChIP-qPCR, Control, Negative Control, Positive Control, Generated, Ubiquitin Proteomics, Binding Assay

Journal: Science Advances

Article Title: Genomic context– and H2AK119 ubiquitination–dependent inheritance of human Polycomb silencing

doi: 10.1126/sciadv.adl4529

Figure Lengend Snippet: ( A ) Diagram showing the expected rTetR-CBX7-initiated recruitment of PRCs (cPRC1, vPRC1, and PRC2) leading to H2AK119 mono-ubiquitination (H2AK119ub1) and H3K27 trimethylation (H3K27me3). ( B ) Summary of interactions in wild-type cells at a developmental gene targeted by Polycomb. ( C ) In the absence of H3K27me3 in EED-3A cells, intact PRC2 and PRC1 complexes mediate heritable silencing. ( D ) At housekeeping genes, Polycomb silencing is not heritable due to the absence of extended CpG islands (eCGI) and/or other differences between housekeeping genes and developmental genes.

Article Snippet: For H3K27me3 ChIP-Seq, Drosophila S2 chromatin (Active Motif, no. 53083) and histone H2Av antibody (Active Motif, no. 61686) were added as spike-in controls.

Techniques: Ubiquitin Proteomics