Journal: Journal of proteome research

Article Title: Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

doi: 10.1021/pr300760p

Figure Lengend Snippet: Analysis of cohesin binding at the human c-MYC locus. (A) Schematic of human c-MYC gene. Black solid boxes indicate translated regions. Cohesin binding to MINE and first exon is denoted by an asterisk. Arrows indicate transcriptional start sites of the P1 and P2 promoter. Location of primer pairs A-C are indicated, ChIP assays with antibody against RAD21 were analysed by real-time PCR with primers pairs B and C specific for the promoter region of the c-MYC locus. Binding at each site was determined relative to primer A, where no RAD21 binding was predicted. (B) Results showed that RAD21 co-localized to the promoter region of c-MYC in all SMC1A- and SMC3-mutated CdLS cell lines, with the exception of CdL057, whereas bound cohesin was dramatically reduced in exon 1 in CdLVH, CdLSS, CdL057, CdL060, CdL107 CdLS cell lines. CdL203, which shares the same mutation with CdL060, showed a decrease close to significant (p = 0.07). Since no difference was found in control cell lines, data was pooled. Results shown are the averages of three independent experiments. The graphs show the average and the standard error of the normalized values and *p < 0.05.

Article Snippet: Antibody against RAD21 (Bethyl Laboratories) was used.

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Mutagenesis, Control

Journal: Journal of proteome research

Article Title: Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

doi: 10.1021/pr300760p

Figure Lengend Snippet: Mutated SMC1A and SMC3 co-immunoprecipitate with RAD21 in the CdLS cell lines. (A) SMC1A (and SMC3) was found to be co-precipitated with RAD21, (B) whereas no RAD21 signal was detected in the IPs using IgG-coated beads. (C) In addition, RAD21co-precipitated with SMC1A (and SMC3) and (D) no SMC specific signal was detectable in the IPs using IgG-coated beads.

Article Snippet: Antibody against RAD21 (Bethyl Laboratories) was used.

Techniques:

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Log2 fold change (log2fc) of spatial distance between adjacent TADs versus -log10 false discovery rate (FDR) for each perturbation. Each dot represents a perturbation in the screen library. In all volcano plots, the top hits (nuclear proteins with the largest log2fc and FDRs<0.1) in both directions are indicated with blue (knockout leads to upregulation) and red dots (knockout leads to downregulation), respectively. The top candidate genes which when knocked out led to increased adjacent TAD distances are: RB1, MRVI1 and PIP5K1B; the top candidate genes which when knocked out caused decreased adjacent TAD distances are: GLDC, NR4A1 and ZNF114. Positive controls (NIPBL and CTCF) are marked in black. ( B ) Log2 fold change of adjacent TAD distance across chr22 for selected hits. ( C ) Spatial distances between adjacent TADs for non-targeting control and selected hits. ( D ) Log2 fold change of long-range A-A contact frequency versus -log10 FDR for each perturbation. Top three hits in both directions including NR4A1, PDE1A, HOXB9, RB1, PCBP1 and LRRC10B are labeled. ( E ) Long-range A-A contact frequencies for non-targeting control and selected hits. ( F ) Log2 fold change of long-range A-B contact frequency versus -log10 FDR for each perturbation. Top three hits in both directions, including RFESD, HOXB9, FAM69B, C2CD2, CHD7 and FAM13C, are labeled. ( G ) Long-range A-B contact frequencies for non-targeting control and selected hits. ( H ) Log2 fold change of long-range B-B contact frequency versus -log10 FDR for each perturbation. Top hits in both directions, including FOS, NR4A1, DDX24 and MYBPH, are labeled. ( I ) Long-range B-B contact frequencies for non-targeting control and selected hits. ( J ) Log2 fold change of overall inter-TAD distances versus -log10 FDR for each perturbation. Top three hits in both directions, including PCBP1, RB1, CHD7, GLDC, HOXB9 and CUL1, are labeled. ( K ) Overall inter-TAD distances for non-targeting control and selected hits. ( L ) Log2 fold change of individual overall inter-TAD distances in chr22 for selected hits. P values in (C) and (K) were calculated by two-sided Wilcoxon signed rank test. P values in (E), (G) and (I) were calculated by two-sided Wilcoxon rank sum test. In all box plots throughout the manuscript, the boxes cover the 25 th to 75 th percentiles, the whiskers cover the 10 th to 90 th percentiles, and the line in the middle of the boxes represents the median value. For all relevant panels, significance is represented as *p<0.1. **p<0.05. ***p<0.01.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Knock-Out, Labeling

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Western blot of siCtrl- and siCHD7-treated A549-Cas9 nuclear extracts. Top: anti-CHD7 antibody; bottom: anti-Actin B antibody. ( B ) A-B compartment profile of chr22 in siCtrl cells. ( C ) A-B compartment profile of chr22 in siCHD7 cells. ( D ) Polarization indices of chr22 A-B compartments of siCtrl (white) and siCHD7 (orange). Shadowed boxes show the polarization indices from randomized controls, where the compartment identities of TADs are scrambled. ( E ) Long-range contact frequency of siCtrl and siCHD7 (shadowed) among A compartments (red), between A and B compartments (purple), and among B compartments (blue). ( F ) Overall inter-TAD distance of siCtrl and siCHD7. ( G ) Radii of gyration of siCtrl and siCHD7. P values in (D), (E) and (G) were calculated by two-sided Wilcoxon rank sum test. P value in (F) was calculated by two-sided Wilcoxon signed rank test.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Western Blot

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Log2 fold change of inter-TAD distance of siCHD7 compared to siCtrl. Number of traces analyzed: 3,558 (siCtrl), 4,134 (siCHD7). ( B ) Log2 fold change of short-range (defined as spatial distances between genomic regions that are less than 3Mb apart) and long-range (defined as spatial distances between genomic regions that are more than 3Mb apart) inter-TAD distances between siCHD7 and siCtrl. ( C ) Log2 fold change of inter-TAD distance of CHD7 overexpression compared to GFP overexpression. Number of traces analyzed: 3,157 (GFP OE), 1,174 (CHD7 OE). ( D ) Log2 fold change of short-range and long-range inter-TAD distances between CHD7 and GFP overexpression. ( E ) Log2 fold change of inter-TAD distance of TSA-treated cells compared to DMSO-treated cells. Number of traces analyzed: 1,214 (DMSO), 2,223 (TSA). ( F ) Log2 fold change of short-range and long-range inter-TAD distances between cells with TSA and DMSO treatment. ( G ) Log2 fold change of inter-TAD distance of siCTCF compared to siCtrl. Number of traces analyzed: 5,768 (siCtrl), 4,226 (siCTCF). ( H ) Log2 fold change of short-range and long-range inter-TAD distances between siCTCF and siCtrl. ( I ) Log2 fold change of inter-TAD distance of double knockdown of CTCF and CHD7 (doubleKD) compared to siCTCF. Number of traces analyzed: 3,560 (doubleKD). ( J ) Log2 fold change of short-range and long-range inter-TAD distances between doubleKD and siCTCF. ( K ) Working model of CHD7 compacting chromatin over long range (cartoon created with Biorender.). All chromatin tracing experiments in this figure were done in the A549 cell background, targeting chr22. P values were calculated by two-sided Wilcoxon signed rank test. For all violin plots, the median log2 fold change values are indicated by colored dashed line, the quartiles are indicated by colored dotted line, and value 0 is marked by black dashed line.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Over Expression

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) A-B compartment profile of chr22 in A549-Cas9 cells with GFP overexpression. ( B ) A-B compartment profile of chr22 in A549-Cas9 cells with CHD7 overexpression. ( C ) Polarization indices of cells with GFP (white) and CHD7 (orange) overexpression and the according randomized controls (shadowed). ( D ) Long-range contact frequency of cells with GFP of CHD7 (shadowed) overexpression in A compartments (red), across A and B compartments (purple) and in B compartments (blue). ( E ) Overall inter-TAD distance of chr22 in cells with GFP and CHD7 overexpression. ( F ) Radii of gyration of chr22 in cells with GFP and CHD7 overexpression. P values in (C), (D) and (F) were calculated by two-sided Wilcoxon rank sum test. P value in (E) were calculated by two-sided Wilcoxon signed rank test.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Over Expression

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Top 10 gene ontology terms up and down in siCHD7 cells versus siControl cells. Gene ontology was performed using Enrichr. ( B ) Volcano plot of RNA-seq of siCHD7 and siControl. Top differentially expressed genes are displayed on the graph as labels. CHD7 is highlighted and is a top differentially downregulated gene in the siCHD7 cells, validating the knockdown. ( C ) Heat map of other proteins/epigenetic mark localized to CHD7 peaks by CUT&RUN. ( D ) Example tracks of CUT&RUN peak profiles of CHD7 and other proteins/epigenetic mark over a gene. ( E ) Peak annotation for all CHD7 peaks. ( F ) Overlap of CUT&RUN peaks of CTCF, RAD21, and H3K4me3 with CHD7 peaks on promoter regions.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: RNA Sequencing Assay

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Western blot of siCtrl, siCHD7, siCTCF and doubleKD (siCHD7+siCTCF) in A549-Cas9 cells. Top: anti-CHD7 antibody; middle: anti-CTCF antibody; bottom: anti-Actin B antibody. ( B ) Short-range inter-TAD distance of chr22 in siCtrl, siCTCF and doubleKD cells. ( C ) Long-range inter-TAD distance of chr22 in siCtrl, siCTCF and doubleKD cells. ( D ) Overall inter-TAD distance of chr22 in siCtrl, siCTCF and doubleKD cells. ( E ) Radii of gyration of chr22 in siCtrl, siCTCF and doubleKD cells. P values in (B) to (D) were calculated by two-sided Wilcoxon signed rank test. P values in (E) was calculated by two-sided Wilcoxon rank sum test.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Western Blot

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: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Gene targeting efficiencies in mESC and HAP1 clones in the example of the AtAFB2 system.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Haploid and diploid HAP1 clones could be reliably separated by cell size using FACS or visual clues. (A) FACS plots for haploid and diploid HAP1 clones. (B) Mosaic HAP1 clone consisting of two subpopulations in equal proportions. Blue/red highlight indicates haploid/diploid subpopulations, respectively. (C) Two eGFP-positive RAD21-miniIAA7-eGFP clones that show significant cell size differences, making it easy to pick the correct clone (haploid clone is on the right).

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Degradation efficiency and dynamics of POI depletion in mESC and HAP1 clones with randomly integrated OsTIR1 and AtAFB2 constructs. POI degradation efficiency in mESC clones (A) and HAP1 clones (B) . Each dot represents one independent cell clone derived after AFB protein gene integration. For each clone, the measurement was carried out once. The black horizontal line shows the median of the group. (C) POI degradation dynamics for selected mESC clones (Rad21-mAID-Clover, Smc2-mAID-Clover, Rad21-miniIAA7-eGFP, Smc2-miniIAA7-eGFP) with OsTIR1 and AtAFB2. For each clone, the experiment was carried out once. (D) POI degradation dynamics for HAP1 clones (RAD21-miniIAA7-eGFP, RAD21-mAID-Clover) with AtAFB2 and OsTIR1. For each clone, the experiment was carried out once. (E) POI degradation dynamics for HAP1 clones (SMC2-miniIAA7-eGFP, SMC2-mAID-Clover) with AtAFB2 and OsTIR1. Numbers in brackets are individual clones’ designations. For each clone, the experiment was carried out once.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay, Construct, Derivative Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Comparison of degradation efficiency with AFB protein driven by the same EF1a promoter. (A) Scheme of genetic constructs used. (B) Dynamics of POI depletion in mESC clones (Rad21 colored in red; Smc2 colored in blue). Vectors expressing AFB proteins: pSH-EFIRES-P-OsTIR1-mCherry-weak NLS and pSH-EFIRES-P-AtAFB2-mCherry-weak NLS, were randomly integrated. For this analysis, all clones were grown in puromycin, and the proportion of Cherry-positive cells in each of them was more than 95%. For each clone, the measurement was carried out twice.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Comparison, Construct, Clone Assay, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

doi: 10.3389/fmolb.2021.757394

Figure Lengend Snippet: Effects of pH on AtAFB2 and OsTIR1 AID degradation efficiency. (A) High pH levels inhibit auxin-induced eGFP degradation in HAP1 clones with RAD21 and SMC2 modifications. Experiments were performed in duplicate. (B) IAA and NAA auxin analogs show similar sensitivity to pH changes (HAP1 clones). Experiments were performed in duplicate. (C) eGFP levels in the mosaic HAP1 clone SMC2-miniIAA7-eGFP. Blue color indicates eGFP levels in Cherry pos subpopulation, and purple color indicates Cherry neg subpopulation. The shift between two peaks illustrates minor basal degradation induced by AtAFB2 in Cherry pos cells. (D) Shift of mES cell population (OsTIR1 system) toward left on the FITC-A axis in acidic media.

Article Snippet: After blocking in 5% milk/TBST for 2 h, the membrane was incubated with primary antibodies against RAD21 and SMC2 (#12673/#8720, Cell Signaling Technology) at 4°C overnight.

Techniques: Clone Assay