Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) E17.5-E18.5 cortices were dissociated and plated on poly-D-lysine. After 10d, cultures were stained for pan neuronal (MAP2), astrocyte (GFAP) and microglia (IBA1) markers, and cell type composition was determined by quantitative analysis of immunofluorescence images. Based on 6 Rad21 +/+ Nex Cre and 8 Rad21 lox/lox Nex Cre different samples analysed in 4 independent experiments. b) Immunofluorescence staining of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre neuronal explant cultures for RAD21 and MAP2 (left) and distribution of RAD21 expression by MAP + neurons (right). Note the discontinuous distribution of RAD21 expression in Rad21 lox/lox Nex Cre neurons. Three independent experiments per genotype. DAPI marks nuclei. Scale bar = 60 μm. c) Immunofluorescence staining for RAD21, MAP2, and the marker of GABAergic inhibitory neurons, GAD67 (left). Distribution of RAD21 expression in GAD67 + and GAD67 - neurons (right). Note that the discontinuous distribution of RAD21 expression in Rad21 lox/lox Nex Cre neuronal explant cultures is due to GAD67 + GABAergic inhibitory neurons. Three independent experiments for Rad21 +/+ Nex Cre and 6 independent experiments for Rad21 lox/lox Nex Cre . DAPI marks nuclei. Scale bar = 20 μm. d) Quantitative RT-PCR analysis of Rad21 mRNA expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures (mean ± SEM, n=18). Hprt and Ubc were used for normalization (left). RAD21 protein expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures was quantified by fluorescent immunoblots (mean ± SEM, n=6) and normalised to LaminB (center). Nex Cre RiboTag RNA-seq of analysis of Rad21 mRNA expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures (right, 3 independent biological replicates). e) 5C heat maps of a 1.72 Mb region on chromosome 2, comparing Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures. CTCF ChIP-seq (Ref. ) and mm9 coordinates are shown for reference. Arrowheads mark the position of CTCF-based loops. Results were consistent across two replicates and 3 chromosomal regions Histograms below show the quantification of representative CTCF-based loops (arrowheads) in two independent biological replicates for control and Rad21 lox/lox Nex Cre neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Staining, Immunofluorescence, Expressing, Marker, Quantitative RT-PCR, Western Blot, RNA Sequencing Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Nex Cre -dependent Rpl22-HA (RiboTag) expression is restricted to RAD21-negative cells in Rad21 lox/lox Nex Cre neurons. Immunofluorescence staining for RAD21, the pan-neuronal marker MAP2 and HA (RiboTag) in explant culture. DAPI marks nuclei. Scale bar = 40 μm. b) Nex Cre RiboTag captures excitatory neuron-specific transcripts such as Slc17a7 and Camk2a and depletes cell type-specific transcripts expressed in inhibitory neurons ( Gad1, Gad2, Slc32a1 ), astrocytes ( Gfap, Aqp4, Mlc1 ), and microglia ( Aif ). Transcript enrichment (or depletion) was calculated using the normalized counts from Nex Cre RiboTag versus standard RNA-seq in Rad21 +/+ Nex Cre neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Immunofluorescence, Staining, Marker, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Volcano plot representing log2 fold-change (FC) versus significance (-log10 of adjusted P values) of downregulated genes (1028) and upregulated genes (572) in RiboTag RNA-seq of Rad21 lox/lox Nex Cre versus Rad21 +/+ Nex Cre neurons. Red marks Rad21 . b) Analysis of gene ontology of biological functions of deregulated genes in Rad21 lox/lox Nex Cre neurons. Enrichment is calculated relative to expressed genes. c) The percentage of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in explant culture at baseline as determined by RiboTag RNA-seq. The P -value (Fisher Exact Test) and Odds ratio indicate that activity-dependent genes are more frequently deregulated than constitutive genes.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: RNA Sequencing Assay, Activity Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Examples of deregulated genes in Rad21 lox/lox Nex Cre neurons. Genes associated with autism spectrum disorders are highlighted in red. b) GSEA for downregulated genes in Nex Cre/+ Rad21 lox/lox neurons using gene sets derived from (i) KEGG pathway database, (ii) GO Biological Process Ontology, (iii) GO Molecular Function Ontology, (iv) GO Cellular Component Ontology in the Molecular Signatures Database (MSigDB). c) Overlap between human genes associated with autism spectrum disorders from the SFARI database and differentially expressed genes (left), downregulated genes (middle) and upregulated genes (right) in Rad21 lox/lox Nex Cre cortical neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expected Mendelian ratios and observed percentages of live Rad21 +/+ Nex Cre , Rad21 lox/+ Nex Cre , Rad21 lox/lox Nex Cre mice at the indicated developmental stages, n = 217. b) Immunofluorescence analysis shows neither the apoptosis marker activated caspase 3 (CC3) nor the DNA damage marker γH2AX in E16.5 (top) and E18.5 Rad21 lox/lox Nex Cre (bottom, white lines demarcate the cortex). Wild type E16.5 thymi are shown as positive controls for CC3 and γH2AX. Two biological replicates. Scale bar = 100 μm. Photomicrographs of coronal brain sections at gestational age E16 modified from the Atlas of the prenatal mouse brain are shown for orientation. c) Quantitative RT-PCR analysis of gene expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre E17.5/18.5 cortical explant cultures 10 d after plating. Hprt and Ubc were used for normalization. Mean ± SEM of 3 cultures per genotype. d) Brain weights of Rad21 +/+ Nex Cre and Rad21 lox/+ Nex Cre , Rad21 lox/lox Nex Cre mice at birth (P0). Mean ± SEM of between 3 and 13 mice per genotype. e) Embryonic cortices from wild-type and Rad21 lox/lox Nex Cre mice were dissected at E17.5 and E18.5 and dissociated. Cortical cell numbers were determined by counting in Neubauer chambers. Each symbol denotes an independent experiment. Mean ± SEM are also shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Immunofluorescence, Marker, Modification, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Schema of cortical layers showing subplate (SP), layer 6 (VI), layer 5 (V), the cortical plate (CP), and the marginal zone (MZ). Immunofluorescence analysis of the neuronal transcription factors CUX1, TBR1, and CTIP2 at E16.5. Representative of 3 biological replicates. Scale bar = 100 μm. b) Morphology of E18.5 neurons after 1 d in explant culture. Immunofluorescence staining for the pan-neuronal marker MAP2, tubulin beta 3 (TUBB3), and DAPI. Scale bar = 20 μm. c) Morphology of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical neurons in explant culture on rat glia . Cultures were sparsely labeled with GFP to visualize individual cells and their processes, and stained for GAD67 to exclude GABAergic neurons. Dendritic traces of GFP + neurons. Scale bar = 50 μm. d) Sholl analysis of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical neurons in explant cultures shown in c). Shown is the number of crossings, dendritic length, terminal points, branch points and spines per 10 μm. Three independent experiments, 32 Rad21 lox/lox Nex Cre and 28 Rad21 +/+ Nex Cre neurons except for the number of spines (two independent experiments, 10 Rad21 lox/lox Nex Cre and 10 Rad21 +/+ Nex Cre neurons). * adj. P <0.05, ** adj. P <0.01, *** adj. P <0.001, **** adj. P <0.0001. Scale bar = 10 μm.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Immunofluorescence, Staining, Marker, Labeling

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) GSEA of the gene set downregulated (DEseq2, adj. P < 0.05) in RAD21-TEV neurons in Rad21 lox/lox Nex Cre neurons (left) and GSEA of genes downregulated in Rad21 lox/lox Nex Cre neurons (DEseq2, adj. P < 0.05) in RAD21-TEV neurons. b) Scatter plots of gene expression within aggregate GO terms, comparing RAD21-TEV with Rad21 lox/lox Nex Cre neurons. Genes that were found deregulated in at least one of the genotypes are shown. P -values and odds ratios refer to the probability of finding the observed patterns of co-regulation by chance. R S : Spearman’s rank coefficient. c) Deregulation of constitutive and activity-dependent genes 24h after acute cohesin depletion by inducible proteolytic cleavage of RAD21-TEV; adj. P <0.05 based on DEseq2 analysis of 3 RNA-seq replicates per experiment. Two independent experiments are shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Activity Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Preferential deregulation in Rad21 lox/lox Nex Cre neurons of genes near constitutive and KCl-inducible neuronal enhancers . Based on 3 RiboTag RNA-seq replicates per genotype. b) Enrichment of inducible activity-dependent genes near constitutive and KCl-inducible neuronal enhancers . c) CTCF binding at neuronal genes and enhancers. All genes: all expressed genes in total RNA-seq; Activity-dependent genes: Previously defined activity-dependent genes (Kim et al., 2010) that are inducible by KCl in our experiments (KCl minus TTX adj. P < 0.05); Constitutive genes: Expressed genes that are not inducible by KCl in our experiments (KCl minus TTX adj. P > 0.05); Enhancers: Previously defined forebrain enhancers ; CTCF binding: Previously defined CTCF binding peaks within 1kb of TSS or enhancer. d) Only a minority of activity-dependent gene promoters are directly bound by CTCF, and most activity-dependent genes that lack CTCF promoter binding are nevertheless deregulated in cohesin-deficient neurons. e) Models of gene regulation by direct (left) versus domain-wide chromatin contacts (right). CD: contact domain.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: RNA Sequencing Assay, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Top: The percentage of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in explant culture at baseline as determined by RNA-seq. Analysis of previously defined activity-dependent genes . Middle: Fraction of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in the presence of TTX and D-AP5 (TTX). Bottom: Fraction of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons after 6h stimulation with KCl. b) Expression of activity-dependent genes in explant cultures of Rad21 +/+ and Rad21 lox/lox Nex Cre neurons under baseline conditions that allow for cell-cell communication versus TTX/D-AP5 (TTX). Comparison by two-sample Kolmogorov-Smirnov test showed that Rad21 +/+ Nex Cre neurons showed stronger expression of activity-dependent genes than Rad21 lox/lox Nex Cre neurons ( P = 5.89e-11). c) Fraction of activity-dependent genes significantly induced by KCl in Rad21 lox/lox Nex Cre neurons at 1 and 6h. In wild-type neurons, 117 and 810 genes were induced ≥2-fold at 1 and 6h of KCl treatment, respectively. d) Fraction of activity-dependent genes that were significantly induced by BDNF at 30 and 120min in RAD21-TEV neurons 24h after RAD21 cleavage. In control RAD21-TEV neurons, 16 and 261 activity-dependent genes were induced ≥2-fold 30 and 120min after BDNF treatment, respectively. Most of these remained inducible 24h after RAD21-TEV cleavage. Dark orange: Strongly induced: log2 FC>1, adj. P <0.05; light orange: moderately induced (log2 FC > 0.5, adj. P <0.05); grey: weakly induced (adj. P <0.05); white: not induced (adj. P >0.05).

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Activity Assay, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expression of the activity-dependent Fos gene at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (left, mean log2-transformed counts from 3 biological replicates, * adj. P < 0.05). b) Enhancer transcripts in control and Rad21 lox/lox Nex Cre neurons were quantified based on normalized RNA-seq reads within 1kb of the eRNA transcription start site. An intergenic region on chr11 was used as a negative control (71.177.622-71.177.792). c) H3K27ac ChIP normalized to H3 in control and Rad21 lox/lox Nex Cre neurons at a control site, Fos enhancer 1 and Fos enhancer 2 after TTX/D-AP5 (TTX) or 1h KCl (KCl). d) Interaction score heatmaps of the 65 kb region immediately surrounding Fos obtained by 5C. Black frames highlight interactions between the Fos gene and upstream enhancers 1 and 2. Previously published CTCF-ChIP-seq is shown for orientation and H3K27ac ChIP-seq in inactive (TTX-treated) and activated neurons is shown to annotate enhancer regions . RNA-seq in TTX-treated and 1h KCl-activated control and Rad21 lox/lox Nex Cre neurons shows KCl-inducible transcription of Fos enhancers in wild-type and cohesin-deficient neurons. Two independent biological replicates are shown in . e) Quantification of 5C contacts between the Fos promoter and Fos enhancer 1 (top), the Fos promoter and Fos enhancer 2 (middle), and CTCF-marked boundaries of the sub-TAD containing Fos (bottom). Two replicates per genotype and condition. f) Model for how cohesin-mediated domain-wide contacts alter the probability of enhancer-promoter contacts, and in this way fine-tune the transcription of activity-dependent genes at baseline and in response to activation. In the absence of cohesin, many activity-dependent genes are expressed at inappropriate levels, but most remain responsive to inducing activation signals. At the Fos and Arc loci, cohesin is not required for chromatin contacts between inducible enhancers and their target promoters. See text for details.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Activity Assay, Transformation Assay, RNA Sequencing Assay, Negative Control, ChIP-sequencing, Activation Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expression of Arc mRNA at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (top, mean log2-transformed counts from 3 biological RNA-seq replicates, * adj. P < 0.05) and at the indicated time after BDNF stimulation (bottom, data points represent biological RT-PCR replicates). P- values refer to induction relative to 0 min. * P < 0.05, *** P <0.001. b) Interaction score heatmaps of the ∼40 kb region immediately surrounding Arc obtained by 5C for resting (TTX) and 1h KCl-activated wild-type (top) and Rad21 lox/lox Nex Cre neurons (bottom). Black frames highlight interaction between the Arc gene (y-axis) and a nearby downstream enhancer (x-axis). Previously published CTCF-ChIP-seq is shown . H3K27ac ChIP-seq in inactive (TTX-treated) and activated neurons is shown to annotate enhancer regions . Two independent biological replicates are shown in . c) Quantification of 5C data. Arc enhancer-promoter loop (top). A CTCF-based loop that braces the Arc locus (arrowhead marked with * in panel b) is quantified for comparison (bottom).

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Transformation Assay, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, ChIP-sequencing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Top: Interaction frequency zoom-in heatmaps of 250 kb region surrounding the Fos gene. Dashed lines and arrow heads mark major CTCF binding sites at the boundaries of the domain that contains Fos . Note the weakening of these contacts in Rad21 lox/lox Nex Cre neurons. Bottom: Interaction score heatmaps of the 65 kb region immediately surrounding the Fos gene. Black frames highlight interactions between the Fos gene and upstream enhancers 1 and 2. H3K27ac ChIP-seq data from Bicuculline-treated (active) and TTX-treated (inactive) neurons annotate enhancer regions. Two independent biological replicates are shown. b) Top: Interaction frequency zoom-in heatmaps of ∼200 kb region surrounding the Arc gene. Dashed lines and arrow heads mark major CTCF binding sites at the boundaries of domains that contain the Arc locus. Note the weakening of these contacts in Rad21 lox/lox Nex Cre neurons. Bottom: Interaction score heatmaps of the ∼40 kb region immediately surrounding the Arc gene. Black frames highlight interaction between the Arc gene (y-axis) and a nearby downstream enhancer (x-axis). H3K27ac ChIP-seq data from Bicuculline-treated (active) and TTX-treated (inactive) neurons annotate enhancer regions. Two independent biological replicates are shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Binding Assay, ChIP-sequencing

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Identification of the cohesin interactome with proximity ligation mass spectrometry. A Schematic of the experimental setup for cohesin proximity ligation followed by mass spectrometry. Transgenic NIH-3T3 mouse fibroblast cell lines were generated containing a 3xHA-TurboID or RAD21-3xHA-TurboID transgene with a tetracycline responsive element (TRE). Cell lines were treated with doxycycline for 36 h to promote expression of the transgenes. Additionally, RAD21-3xHA-TurboID cells that were not treated with doxycycline serve as a No TurboID control. Following doxycycline treatment, cells were treated ± biotin for 2 h. Cells were collected, lysed, and nuclear proteins were extracted. A streptavidin enrichment was performed, and a quality control check was done on 10% of the protein sample by western blotting for biotinylated proteins, while the remaining 90% of the streptavidin enriched sample was used for LC–MS/MS. Proteins identified by mass spectrometry were analyzed by two methods to identify proteins enriched relative to controls. Proteins enriched in the RAD21-3xHA-TurboID biotin treated sample relative to the: (1) 3xHA-TurboID biotin treated sample (Tier 1; p < 0.05, FC > 2); or (2) No TurboID biotin treated sample (Tier 2; p < 0.05, FC > 2). Each condition was performed in triplicate. B Co-purification of RAD21-3xHA-TurboID protein with endogenous cohesin subunits. IgG (negative control), anti-SMC3, and anti-HA antibodies were used to purify cohesin complexes in nuclear extracts from RAD21-3xHA-TurboID expressing cells under high stringency conditions. Cohesin subunits were detected by western blotting (SMC3, SMC1, RAD21, and HA). Arrows indicate endogenous RAD21 (black) and transgenic RAD21-3xHA-TurboID proteins (green). Box contains a depiction of core cohesin complex with RAD21-3xHA-TurboID. C Venn diagram showing the overlap of proteins detected in the Tier 1 cohesin interactome, Tier 2 cohesin interactome, and cohesin IP-MS experiments [ – ]. D , E Volcano plots showing proteins detected in the Tier 1 ( D ) and Tier 2 ( E ) cohesin interactomes. Proteins significantly enriched in Tier 1 and Tier 2 shown in red and blue, respectively. Known cohesin subunits and interactors are indicated with green dots and labels

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: Ligation, Mass Spectrometry, Transgenic Assay, Generated, Expressing, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Copurification, Negative Control, Protein-Protein interactions

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Cohesin co-localizes with SWI/SNF at enhancers, promoters, and CTCF sites. A Genome browser view of the Sox2 gene and super enhancer. ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) is shown. DNA interactions were detected by Micro-C and are shown at the top with a contact frequency heatmap and arcs for DNA loops (5 kb resolution). RAD21 peaks overlapping BRG1 peaks highlighted in purple, RAD21 peaks not overlapping BRG1 highlighted in green, and BRG1 peaks not overlapping RAD21 highlighted in orange. B Heatmaps showing ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) at sites where cohesin peaks do not overlap SWI/SNF peaks (top), sites where cohesin peaks overlap SWI/SNF peaks (middle), and sites where SWI/SNF peaks do not overlap cohesin peaks (bottom). Signal is z-score normalized. C UpSet plot of cohesin peaks overlapping CTCF, H3K27ac, H3K4me3, and/or BRG1

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: ChIP-sequencing

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Altered cohesin occupancy at CTCF sites and enhancers upon SWI/SNF perturbation . A The SWI/SNF cBAF complex and three small molecules that inhibit or lead to degradation of the BRG1 subunit (dark orange). The ACBI1 PROTAC (red) targets the bromodomain of BRG1, recruiting an E3 ubiquitin ligase for poly-ubiquitination and proteasomal degradation. PFI-3 (purple) targets the bromodomain of the BRG1 subunit. BRM014 (blue) targets the ATPase active site of BRG1. The levels of SWI/SNF subunits shown in light orange were assessed following ACBI1 PROTAC treatment. B Genome browser views of RAD21 peaks that increase (up) or decrease (down) following ACBI1 PROTAC treatment. ChIP-seq signal for RAD21 with DMSO or ACBI1 treatment, BRG1, H3K4me3, H3K27ac, and CTCF is shown. Differential RAD21 peaks are indicated with a gray box. C Violin plots showing differential RAD21 signal in ACBI1 PROTAC treated mESCs relative to DMSO treated mESCs. The RAD21 peaks with significantly increased signal following ACBI1 treatment are shown in red, while peaks with significantly decreased signal are shown in gray. D Violin plots showing BRG1 (orange), H3K27ac (pink), and CTCF (dark blue) ChIP-seq signal at all RAD21 peaks, peaks with increased RAD21 signal (up) following ACBI1 treatment, and peaks with decreased RAD21 signal (down) following ACBI1 treatment. Significance determined by non-parametric Mann–Whitney test (****p-value < 0.0001). E Percentage of differential RAD21 peaks (up on left, down on right) upon ACBI1 treatment that overlap with the anchors of DNA loops detected by Micro-C, and percentage of non-differential RAD21 peaks with similar RAD21 signal intensity shown as a control. F Same as ( C ) for PFI-3 treatment. The RAD21 peaks with significantly increased signal following PFI-3 treatment are shown in purple, while peaks with significantly decreased signal are shown in gray. G Same as ( D ) for all RAD21 peaks and peaks with significantly different RAD21 signal following PFI-3 treatment ( n.s. not significant, *p-value < 0.01). H Same as ( E ) for PFI-3 treatment. I Same as ( B ) for BRM014 treatment. DNA loop anchor and long-range DNA interaction is depicted on top. J Same as ( C ) for BRM014 treatment. The RAD21 peaks with significantly increased signal following BRM014 treatment are shown in blue. K Same as ( D ) for all RAD21 peaks and peaks with increased RAD21 signal (up) following BRM014 treatment (****p-value < 0.0001). L Same as ( E ) for BRM014 treatment

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: Ubiquitin Proteomics, ChIP-sequencing, MANN-WHITNEY, Control

Journal: EMBO Reports

Article Title: STAG2 loss in Ewing sarcoma alters enhancer-promoter contacts dependent and independent of EWS::FLI1

doi: 10.1038/s44319-024-00303-6

Figure Lengend Snippet: ( A ) Chromatin-bound levels of cohesin subunits and regulators assessed by Chromoflow flow cytometry. A representative experiment comparing A673 WT and STAG2 KO#1 is shown. Similar results were obtained comparing WT or Parental cells and STAG2 KO#1 or KO#2 in two independent experiments. ( B ) Immunoprecipitation of cohesin with anti-SMC1 from the indicated cell extracts followed by immunoblotting of cohesin and regulators. Colored dots below the panels identify each sample. ( C ) Quantification of the amount of the indicated proteins co-immunoprecipitated with anti-SMC1, relative to the amount of RAD21, in these four samples. Gray dots often overlap with black dots. .

Article Snippet: Mouse monoclonal anti-RAD21 clone 53A303; FC: 2 µg/ml , Merck , cat#05-908.

Techniques: Flow Cytometry, Immunoprecipitation, Western Blot

Journal: EMBO Reports

Article Title: STAG2 loss in Ewing sarcoma alters enhancer-promoter contacts dependent and independent of EWS::FLI1

doi: 10.1038/s44319-024-00303-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse monoclonal anti-RAD21 clone 53A303; FC: 2 µg/ml , Merck , cat#05-908.

Techniques: Recombinant, Sequencing, Reverse Transcription, SYBR Green Assay, Software, Isolation

Journal: bioRxiv

Article Title: Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

doi: 10.1101/2020.06.06.136432

Figure Lengend Snippet: a) PCR analysis of Rad21 alleles from Rad21 Tev/WT , Rad21 Tev/Tev and Rad21 WT/WT mice. b) Schematic of ERt2-TEV-dependent RAD21-TEV degradation c) Western blot of RAD21-TEV protein expression over a time course of 4-OHT treatment. Bar plot of RAD21-TEV protein expression normalised to LAMIN B (n = 7, h = hours). d) Volcano plot of gene expression fold-change versus adjusted P value in RNA-seq of RAD21-TEV neurons transduced with ERt2-TEV and treated with 4-OHT for 24h (n = 3). 463 genes were down- and 287 genes were upregulated (adj P < 0.05 shown in red, DE = differentially expressed). e) Bar graph of individual GO terms and broad categories in RNA-seq of RAD21-TEV neurons treated as in d). Terms represented by downregulated genes are shown in blue. Upregulated genes showed no GO term enrichment at P < 1 × 10 −4 . f) Enrichment of shared deregulated genes (adj P < 0.05) in response to acute cohesin depletion induced by ERt2-TEV and NLS-TEV. Fisher’s exact test was applied for the odds ratio and P value. g) Scatter plot of gene expression, comparing log 2 fold-change of deregulated genes (adj P < 0.05) in response to acute cohesin depletion induced by ERt2-TEV and NLS-TEV (DE = differentially expressed, R = Pearson correlation coefficient). h) 4C analysis of chromatin interactions at the Pcdhb locus. Top: Hi-C representation of domain structure at the Pcdhb locus in mouse cortical neurons , with selected genes and enhancers. Bottom: contact profiles of enhancers HS18-20 in control cells (top panel) and RAD21-TEV cleaved cells (bottom panel). A dashed line indicates the enhancer site and viewpoint. A grey band displays the 20 th to 80 th percentiles and the black line within shows mean values for 40kb windows. The colour panel shows the mean contact intensities for multiple window sizes from 2kb to 5kb, n = 4 independent biological replicates. i) Genes significantly deregulated (adj P < 0.05) by ERt2-TEV mediated RAD21-TEV degradation are enriched for the binding of cohesin (top) CTCF (center) and proximity to neuronal enhancers (bottom, see methods). Fisher’s exact test was applied for the odds ratio and P value. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Primary antibodies were goat polyclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-6216) and mouse monoclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-374015), and mouse monoclonal anti-myc tag for detection of RAD21-TEV (1:500, Santa Cruz biotechnology, sc-40).

Techniques: Western Blot, Expressing, Gene Expression, RNA Sequencing, Transduction, Hi-C, Control, Binding Assay

Journal: bioRxiv

Article Title: Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

doi: 10.1101/2020.06.06.136432

Figure Lengend Snippet: a) Immunofluorescence staining for TUJ1 and DAPI in explant RAD21-TEV neurons showing neuronal maturation over the course of ten days (scale bar = 50μm). b) Immunofluorescence staining for NeuN and DAPI in ten-day old explant RAD21-TEV neuron cultures treated with AraC at day 5. Bar graph plots mean % of NeuN+ and NeuN-nuclei in ten-day old explant RAD21-TEV neuron cultures treated with AraC at day 5. (n =10, error bars = range, scale bar = 50μm). c) Immunofluorescence staining for ERt2-TEV and DAPI, with GFP expressed from ERt2-TEV lentiviral transduction. Left: ERT2-TEV is retained in the cytoplasm with vehicle treatment. Right: ERt2-TEV translocates to the nucleus following 4-OHT exposure. White arrow heads indicate example cells. (Scale bar = 25μm).

Article Snippet: Primary antibodies were goat polyclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-6216) and mouse monoclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-374015), and mouse monoclonal anti-myc tag for detection of RAD21-TEV (1:500, Santa Cruz biotechnology, sc-40).

Techniques: Immunofluorescence, Staining, Transduction

Journal: bioRxiv

Article Title: Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

doi: 10.1101/2020.06.06.136432

Figure Lengend Snippet: a) Schematic of lentiviral construct containing an all-in-one doxycycline inducible NLS-TEV system. Tet-On advanced transactivator (rtTA) and RFP are constitutively expressed under control of the ubiquitin promoter. NLS-TEV expression is in turn controlled by tet response element (TRE) promoter upon addition of doxycycline. b) Schematic of doxycycline dependent RAD21-TEV degradation by NLS-TEV. c) Western blot of RAD21-TEV protein expression 24 hours after Dox pulse (6h, 100ng/ml). Bar plot of RAD21-TEV protein expression normalised to LAMIN B 24 hours after Dox exposure, ~30% RAD21-TEV protein remained (n = 4, h = hours). d) Volcano plot of gene expression log 2 fold-change versus adjusted P value in RAD21-TEV +Dox (24 hours after 6-hour 100ng/ml pulse, n = 3). 570 genes were down- and 150 genes were upregulated (adj P < 0.05, shown in red, DE = differentially expressed). e) Top individual GO terms and broad categories for downregulated genes (blue). There was no significant enrichment for upregulated genes.

Article Snippet: Primary antibodies were goat polyclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-6216) and mouse monoclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-374015), and mouse monoclonal anti-myc tag for detection of RAD21-TEV (1:500, Santa Cruz biotechnology, sc-40).

Techniques: Construct, Control, Ubiquitin Proteomics, Expressing, Western Blot, Gene Expression

Journal: bioRxiv

Article Title: Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

doi: 10.1101/2020.06.06.136432

Figure Lengend Snippet: a) Bar graph of overlap between deregulated genes in RAD21-TEV neurons (NLS-TEV and ERt2-TEV combined) and other animal models of neuronal dysfunction including Nipbl +/− (ref. 19), deletion of PRC2 components Ezh1 and Ezh2 ( Ezh1 - 2 −/− ) , Mecp2 +/− (ref. 34), Fmrp −/− (ref. 35), mutant Htt (m Htt ), a mouse model of Huntington’s disease and mouse mutant Pten m3m4/m3m4 (ref. 37). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, Fisher’s exact test. b) Bubble plot of number of represented gene ontologies in different mouse models of neuronal dysfunction and RAD21-TEV depleted neurons separated into broad categories. GO analysis conducted on both down- and upregulated genes for each condition indicated in the respective columns. For each condition either all significant GO ( P < 1×10 −4 ) or only top 100, where more than 100 terms reach significance, are plotted. Triangle size is proportional to the number of terms in that category, and direction of point indicates direction of deregulation.

Article Snippet: Primary antibodies were goat polyclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-6216) and mouse monoclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-374015), and mouse monoclonal anti-myc tag for detection of RAD21-TEV (1:500, Santa Cruz biotechnology, sc-40).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

doi: 10.1101/2020.06.06.136432

Figure Lengend Snippet: a) GSEA of RAD21-TEV downregulated genes in CdLS NeuN-positive RNAseq (left). GSEA of CdLS NeuN-positive downregulated genes in RAD21-TEV (right). NES = normalised enrichment score, FDR = false discovery rate. b) Heatmap of log 2 fold-change for selected genes from CdLS NeuN-positive and RAD21-TEV samples separated into broad functional categories. Genes shown were significantly deregulated in CdLS and their mouse orthologs deregulated in at least one RAD21-TEV (ERt2-TEV or NLS-TEV) system. Genes highlighted in red were identified in the SFARI database, those in blue were deregulated in ASD, genes highlighted in orange fulfil both criteria. c) Comparison of RAD21 (cohesin) binding (top), CTCF binding (center) and enhancer proximity (bottom, see methods) of genes deregulated (adj P < 0.05) in NeuN-positive CdLS (left) compared to genes deregulated in both NeuN-positive CdLS and RAD21-TEV (right). Up- and downregulation indicates the direction of deregulation in CdLS NeuN-positive RNAseq. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Primary antibodies were goat polyclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-6216) and mouse monoclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-374015), and mouse monoclonal anti-myc tag for detection of RAD21-TEV (1:500, Santa Cruz biotechnology, sc-40).

Techniques: Functional Assay, Comparison, Binding Assay

Journal: bioRxiv

Article Title: Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

doi: 10.1101/2020.06.06.136432

Figure Lengend Snippet: Overlap of deregulated genes between RAD21-TEV neurons (NLS-TEV and ERt2-TEV combined) and neuronal or whole cortical tissue RNAseq of human neurological diseases including NeuN-positive CdLS, Rett Syndrome , Huntington’s disease , Alzheimer’s disease , and Down Syndrome . * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, Fisher’s exact test.

Article Snippet: Primary antibodies were goat polyclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-6216) and mouse monoclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-374015), and mouse monoclonal anti-myc tag for detection of RAD21-TEV (1:500, Santa Cruz biotechnology, sc-40).

Techniques:

Journal: bioRxiv

Article Title: Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

doi: 10.1101/2020.06.06.136432

Figure Lengend Snippet: a) Schematic of NLS-TEV mediated RAD21-TEV degradation and rescue. b) Western blot of RAD21-TEV protein expression over a time course of Dox treatment (6h, 100ng/ml). Bar plot of RAD21-TEV protein expression normalised to LAMIN B (n = 4). c) Volcano plots of gene expression log 2 fold-change versus adjusted P -value in RAD21-TEV +Dox treated neurons before and after rescue of RAD21-TEV expression (n = 3). Left: 570 genes were down- and 150 genes were upregulated (adj P < 0.05, shown in red) following RAD21-TEV depletion. Right: 43 genes were down- and 44 genes were upregulated after rescue of RAD21-TEV expression (adj P < 0.05, shown in red and orange). Genes in red are significantly deregulated in both RAD21-TEV depletion and rescue, genes in orange are significantly deregulated only after rescue (DE = differentially expressed). d) Log 2 fold-change of significantly deregulated genes (adj P < 0.05) following RAD21-TEV depletion (left) and after rescue (right). e) The number of significantly deregulated genes (adj P < 0.05) after RAD21-TEV depletion (in green) and after rescue of RAD21-TEV expression (in orange). DE = differentially expressed. f) Heatmap of de novo deregulated genes following RAD21-TEV rescue (adj P < 0.05) and their maturation trajectory in control (left) and +Dox treated samples (right). g) Barcode plot of de novo deregulated genes following RAD21-TEV rescue (adj P < 0.05) and their enrichment for directionality in maturation. Top: significantly de novo upregulated genes following rescue are enriched for genes upregulated during neuronal maturation. Bottom: de novo downregulated genes after cohesin rescue are enriched for genes that are downregulated during neuronal maturation.

Article Snippet: Primary antibodies were goat polyclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-6216) and mouse monoclonal to LAMIN B (1:5000, Santa Cruz Biotechnology, sc-374015), and mouse monoclonal anti-myc tag for detection of RAD21-TEV (1:500, Santa Cruz biotechnology, sc-40).

Techniques: Western Blot, Expressing, Gene Expression, Control