Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 1. A genome-wide CRISPR-Cas9 genetic screen identifies an essential requirement for CRAMP1 and histone H1.4 in PRC2-mediated reporter repression (A) Schematic representation of GFP reporter repression by the PRC2 complex. (B) The GFP reporter is derepressed upon CRISPR-Cas9-mediated gene disruption of any of the three core PRC2 subunits, as assayed by flow cytometry. (C) A genome-wide CRISPR-Cas9 screen to identify factors required for PRC2 function. Following Cas9 expression in KBM-7 cells harboring the PRC2-sensitive GFP reporter, genome-wide mutagenesis was carried out with the Sabatini/Lander single guide RNA (sgRNA) library, 36 and GFP + cells isolated through two sequential rounds of FACS. ‘‘Significance’’ on the y axis represents the negative log of the ‘‘pos|score’’ metric reported by Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK). 37

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: Genome Wide, CRISPR, Disruption, Flow Cytometry, Expressing, Mutagenesis, Isolation, Knock-Out

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 5. Linker histones are not enriched at regions marked by H3K9me3 (A–D) Lack of linker histone enrichment at H3K9me3-marked genomic regions. (A) Tornado plots depicting linker histone CUT&Tag signal across H3K9me3 peaks from the ENCODE project; average signal intensity is shown in (B). (C) Heatmap depicting the lack of correlation between linker histone occupancy and H3K9me3. Cells are annotated with pairwise Spearman correlation coefficients. An example locus is shown in (D). (E) CUT&Tag faithfully profiles H3K9me3. Example loci comparing CUT&Tag versus H3K9me3 ChIP-seq data (ENCODE) are shown. (F and G) Linker histone insufficiency does not impair H3K9me3-dependent LINE-1 silencing by the HUSH complex. (F) Schematic representation of the dual- color reporter cell line designed to monitor both H3K9me3-dependent repression by the HUSH complex and linker histone-mediated PRC2-reporter repression. (G) HUSH-mediated LINE-1 silencing is unaffected upon CRAMP1 depletion. The indicated CRISPR sgRNAs were expressed in the dual-color reporter cell line, and GFP and iRFP fluorescence assayed by flow cytometry. See also Figure S5 and Table S2.

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: ChIP-sequencing, CRISPR, Fluorescence, Flow Cytometry

Journal: Molecular cell

Article Title: The Integrator complex attenuates promoter-proximal transcription at protein-coding genes

doi: 10.1016/j.molcel.2019.10.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies IntS1 Ezzeddine et al., 2011 IntS9 Ezzeddine et al., 2011 IntS11 Ezzeddine et al., 2011 alpha-tubulin abcam ab15246 AB_301787 H3K4me3 EpiCypher 13-0028 Normal Rabbit serum Invitrogen 01-610-1 Chemicals, Peptides, and Recombinant Proteins AzNTPs TriLink Technologies Cat# K-1005 Biotin-11-NTPs Perkin Elmer Cat# NEL54(2/3/4/5)001 Critical Commercial Assays NEBNext Ultra II DNA library kit NEB Cat# E7103S Deposited Data Raw and analyzed data This paper GEO: {"type":"entrez-geo","attrs":{"text":"GSE114467","term_id":"114467"}} GSE114467 Start-seq from Triptolide-treated S2 cells Krebs et al., 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE77369","term_id":"77369"}} GSE77369 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from S2 cells Henriques et al., 2018 GEO: {"type":"entrez-geo","attrs":{"text":"GSE85191","term_id":"85191"}} GSE85191 Start-seq from Control and Rrp40-depleted S2 cells Henriques et al., 2013 GEO: {"type":"entrez-geo","attrs":{"text":"GSE49078","term_id":"49078"}} GSE49078 Chromatin RNA-seq from IntS11-depleted HeLa cells Lai et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE68401","term_id":"68401"}} GSE68401 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from HeLa cells Gerstein et al., 2012 GEO: {"type":"entrez-geo","attrs":{"text":"GSE29611","term_id":"29611"}} GSE29611 PRO-seq from DMSO-treated HeLa cells Nilson et al. 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE100742","term_id":"100742"}} GSE100742 Chromatin RNA-seq from Xrn2 or CPS73-depleted HeLa cells Nojima et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE60358","term_id":"60358"}} GSE60358 Experimental Models: Cell Lines DL1 cells Dr. Sara Cherry, UPenn S2-DGRC clone 6 DGRC Stock number 6 DL1 FLAG-eGFP This paper DL1 FLAG-IntS11WT This paper DL1 FLAG-IntS11E203Q This paper Oligonucleotides Table S3 This paper Recombinant DNA pUB-3xFLAG vector Chen et al., 2012 pMT-puro David Sabatini lab Addgene #17923 pMT-FLAG-dIntS11-WT-puro This paper pMT-FLAG-dIntS11-E203Q-puro This paper pMT-FLAG-eGFP-puro This paper Software and Algorithms bowtie 1.2.2 Langmead et al., 2009 R v3.3.1 www.r-project.org Rstudio v1.0.136 www.rstudio.com featureCounts Liao et al., 2014 DESeq2 Love et al. , 2014 MISO Katz et al., 2010 Prism v8.1.2 GraphPad Partek Genomics Suite v6.15.0127 www.partek.com Open in a separate window KEY RESOURCES TABLE Integrator inhibits transcription elongation at ~15% of mRNA genes and enhancers Integrator targets promoter-proximally paused Pol II for termination The RNA endonuclease of Integrator subunit 11 is critical for gene repression Integrator-depletion increases productive elongation and Histone H3 K4 methylation

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Circulation research

Article Title: Efficient Gene Disruption in Cultured Primary Human Endothelial Cells by CRISPR/Cas9

doi: 10.1161/CIRCRESAHA.117.306290

Figure Lengend Snippet: (A) Unmodified EC stimulated with IFN-γ increase expression of MHC class I (HLA-A,B,C) and class II (HLA-DR), whereas a high proportion of EC transduced with TetOn-Cas9/sgCIITA vectors upregulate MHC class I but show either reduced (HLA-DRmid) or complete loss of MHC class II (HLA-DRneg) expression, (B) qRT-PCR analysis of FACS isolated WT and HLA-DRneg EC for CIITA, HLA-DRA, and CXCL10 transcripts, (C) HLA-DRneg EC clonally sorted by single-cell FACS, expanded, and sequenced across sgRNA target site demonstrates bi-allelic indel mutations in CIITA but not in the likeliest off-target site (SLC6A9). Representative plots shown from multiple donors from 3 independent experiments.

Article Snippet: CRISPR/Cas9 mutagenesis The tetracycline-inducible Cas9 lentiviral vector (pCW-Cas9, also produced by Eric Lander & David Sabatini, and available through Addgene as plasmid: #50661) was used to transduce ECFC-derived EC to create stable inducible Cas9 expressing EC.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Isolation