Cohesin-dependence of neuronal gene expression relates to chromatin loop length
- MRC London Institute of Medical Sciences, Imperial College London, United Kingdom
- Institute of Clinical Sciences, Faculty of Medicine, Imperial College, United Kingdom
- Department of Bioengineering, University of Pennsylvania, United States
- Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, United States
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, United States
Figures
Figure 1 with 2 supplements
Conditional cohesin deletion in post-mitotic neurons.
(a) E17.5–E18.5 cortices were dissociated and plated on poly-D-lysine. After 10 days, cultures were stained for pan neuronal (MAP2), astrocyte (GFAP), and microglia (IBA1) markers, and cell type …
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Figure 1—source data 1
Figure 1: Conditional cohesin deletion in post-mitotic neurons.
- https://cdn.elifesciences.org/articles/76539/elife-76539-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Rad21 NexCre RiboTag validation.
(a) RAD21 and LAMIN B protein expression in Rad21+/+ NexCre and Rad21lox/lox NexCre cortical explant cultures was analyzed by fluorescent immunoblots. One representative blot of six is shown here, …
Figure 1—figure supplement 2
Restoration of cohesin rescues chromatin loops.
(a) Transient RAD21 depletion by Dox-inducible TEV expression and recovery after Dox washout (Weiss et al., 2021). RAD21-TEV western blots (left) and heat maps of chromatin contacts at the …
Figure 2 with 1 supplement
Loss of cohesin from immature post-mitotic neurons perturbs neuronal gene expression.
(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 Rad21lox/lox NexC…
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Figure 2—source data 1
Figure 2: Loss of cohesin from immature post-mitotic neurons perturbs neuronal gene expression.
- https://cdn.elifesciences.org/articles/76539/elife-76539-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Gene expression in Rad21lox/lox NexCre neurons.
(a) Examples of deregulated genes in Rad21lox/lox NexCre neurons. Genes associated with autism spectrum disorders are highlighted in red. (c) Overlap between human genes associated with autism …
Figure 3 with 1 supplement
Cohesin contributes to the maturation of post-mitotic neurons.
(a) Top: Schema of cortical layers (Greig et al., 2013) showing subplate (SP), layer 6 (VI), layer 5 (V), the cortical plate (CP), and the marginal zone (MZ). Middle: Immunofluorescence analysis of …
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Figure 3—source data 1
Figure 3: Cohesin contributes to the maturation of post-mitotic neurons.
- https://cdn.elifesciences.org/articles/76539/elife-76539-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Impact of cohesin loss in immature post-mitotic neurons in vivo.
(a) Expected Mendelian ratios and observed percentages of live Rad21+/+ NexCre, Rad21lox/+ NexCre, Rad21lox/lox NexCre mice at the indicated developmental stages, n=217. (b) Immunofluorescence …
Figure 4
Acute cohesin depletion deregulates ARG expression.
(a) Western blot documenting acute RAD21 depletion by 4-OHT-inducible RAD-TEV cleavage (left). GSEA of the gene set downregulated (DEseq2, adj. p<0.05) in RAD21-TEV neurons in Rad21lox/lox NexCre …
Figure 5 with 1 supplement
Activity-regulated neuronal gene (ARG) classes differ in their reliance on cohesin.
(a) Pie charts show the expression of constitutive genes (top), immediate early genes (IEGs) (center), and late response genes (LRGs) (bottom) in Rad21 NexCre neurons under four different …
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Figure 5—source data 1
Figure 5: Activity-regulated neuronal gene (ARG) classes differ in their reliance on cohesin.
- https://cdn.elifesciences.org/articles/76539/elife-76539-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Gene expression and genotype interaction analysis.
(a) The expression of constitutive and previously defined activity-regulated genes (Kim et al., 2010) was determined by RNA-seq in explant cultures of Rad21lox/lox NexCre neurons at baseline, in the …
Figure 6 with 1 supplement
The genomic distance traversed by chromatin contacts formed by neuronal genes predicts whether or not cohesin is required for their full expression.
(a) The span of Hi-C loops (left), Hi-C loops with CTCF bound to at least one of the loop anchors (middle) and Hi-C loops between promoters and inducible enhancers (right) for Immediate early genes …
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Figure 6—source data 1
Figure 6: The genomic distance traversed by chromatin contacts formed by neuronal genes predicts whether or not cohesin is required for their full expression.
- https://cdn.elifesciences.org/articles/76539/elife-76539-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Promoter binding of CTCF or cohesin does not distinguish cohesin-dependent from cohesin-independent genes.
(a) Presence of CTCF (Bonev et al., 2017), RAD21 (Fujita et al., 2017), or cohesin-non-CTCF binding at gene promoters (TSS ± 2 kb) at immediate early genes (IEGs), late response genes (LRGs) that …
Figure 7 with 3 supplements
Fos enhancer-promoter contacts are robustly induced in cohesin-deficient neurons.
(a) Expression of the immediate early genes (IEG) Fos at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (left, mean log2-transformed counts from 3 biological replicates, * adj. p<0.05). (b) …
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Figure 7—source data 1
Figure 7: Fos enhancer-promoter contacts are robustly induced in cohesin-deficient neurons.
- https://cdn.elifesciences.org/articles/76539/elife-76539-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Inducible gene expression in cohesin-deficient neurons.
(a) Examples of Activity-regulated neuronal gene (ARG) expression at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation. Mean log2-transformed counts from three biological replicates (* adj. p<0.05…
Figure 7—figure supplement 2
Contacts between the Arc promoter and an inducible enhancer in wild -type and cohesin-deficient neurons.
(a) Expression of Arc mRNA at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (top, mean log2-transformed counts from three biological RNA-seq replicates, * adj. p<0.05) and at the indicated …
Figure 7—figure supplement 3
Replicate 5 C experiments.
(a) Top: Interaction frequency zoom-in heatmaps of 250 kb region surrounding the Fos gene obtained by 5C analysis of chr12 86201802–87697802. Dashed lines and arrow heads mark major CTCF binding …
Figure 8 with 1 supplement
Bdnf enhancer-promoter contacts are weakened in the absence of cohesin.
(a) Total Bdnf transcripts at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (left, mean log2-transformed counts from three biological replicates, * adj. p<0.05). (b) Bdnf promoter IV …
Figure 8—figure supplement 1
Quantification of observed and distance-corrected loop strength.
(a) Observed values loop strength, as presented throughout the paper. (b) Observed values loop strength corrected for the distance-dependent background signal and shown on the same y-axis scale for …
Author response image 1
No scaling between gene length and cohesin-dependence in Rad21 NexCre neurons.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Rad21lox Rad21tm1.1Mmk | DOI: 10.1038/nature10312 | MGI:5293824 | |
| Genetic reagent (Mus musculus) | NexCre Neurod6tm1(cre)Kan | DOI: 10.1002/dvg.20256 | MGI:2668659 | |
| Genetic reagent (Mus musculus) | Rpl22(HA)lox (RiboTag) Rpl22tm1.1Psam | DOI: 10.1073/pnas.0907143106 | MGI:4355967 | |
| Genetic reagent (Mus musculus) | Rad21tev Rad21tm1.1Kktk | DOI: 10.1101/gad.605910 | MGI:4840469 | |
| antibody | anti-RAD21 (rabbit polyclonal) | Abcam | Cat #. ab154769 | WB: (dilution 1:1000) IF: (dilution 1:500) |
| antibody | anti-LAMIN B (goat polyclonal) | Santa Cruz Biotechnology | Cat #. sc-6216 | WB: (dilution 1:10,000) |
| antibody | anti-rabbit IgG (H + L) Alexa Fluor 680 (goat polyclonal) | ThermoFisher Scientific | Cat #. A-21109 | WB: (dilution 1:10,000) |
| antibody | anti-goat IgG (H + L) Alexa Fluor 680 (donkey polyclonal) | ThermoFisher Scientific | Cat #. A-21084 | WB: (dilution 1:10,000) |
| antibody | anti-HA (rabbit polyclonal) | Sigma | Cat #. H6908 | polysome immunoprecipitation |
| antibody | anti-GFAP (rabbit polyclonal) | Wako | Cat #. Z0334 | IF: (dilution 1:500) |
| antibody | anti-MAP2 (chicken polyclonal) | Abcam | Cat #. ab611203 | IF: (dilution 1:5000) |
| antibody | anti-GAD67 (mouse monoclonal) | Millipore | Cat #. MAB5406 | IF: (dilution 1:500) |
| antibody | anti-HA (mouse monoclonal) | Covance | Cat #. MMS-101R | IF: (dilution 1:1000) |
| antibody | IBA1 (rabbit polyclonal) | Wako | Cat #. 019–19741 | IF: (dilution 1:250) |
| antibody | anti-TUBB3 (Tuj1, mouse monoclonal) | Biolegend | Cat #. 801,202 | IF: (dilution 1:500) |
| antibody | anti-gamma-H2AX (rabbit polyclonal) | Bethyl Laboratories | Cat #. A300-081A | IF: (dilution 1:3000) |
| antibody | anti-Cleaved Caspase-3 (Asp175) (rabbit polyclonal) | Cell signalling | Cat #. 9,661 | IF: (dilution 1:400) |
| antibody | anti-TBR1 (rabbit polyclonal) | Abcam | Cat #. ab31940 | IF: (dilution 1:1000) |
| antibody | anti-CTIP2 (25B6, rat monoclonal) | Abcam | Cat #. ab18465 | IF: (dilution 1:500) |
| antibody | anti-CUX-1 (rabbit polyclonal) | Santa Cruz Biotechnology | Cat #. sc-13024 | IF: (dilution 1:400) |
| antibody | anti–Phospho-Histone H3 S10 Alexa Fluor 647 conjugate (rabbit polyclonal) | Cell signalling | Cat #. 9,716 | IF: (dilution 1:50) |
| antibody | anti-rabbit IgG (H + L) Alexa Fluor 647 (goat polyclonal) | ThermoFisher Scientific | Cat #. A-21244 | IF: (dilution 1:500) |
| antibody | anti-Rabbit IgG (H + L) Alexa Fluor 568 (goat polyclonal) | ThermoFisher Scientific | Cat #. A-11011 | IF: (dilution 1:500) |
| antibody | goat anti-mouse IgG (H + L) Alexa Fluor 488 (goat polyclonal) | ThermoFisher Scientific | A-11001 | IF: (dilution 1:500) |
| antibody | anti-chicken IgY (H + L) Alexa Fluor 568 (goat polyclonal) | Abcam | ab175711 | IF: (dilution 1:500) |
| Software, algorithm | ImageJ software | (http://imagej.nih.gov/ij/) | ||
| Software, algorithm | GraphPad Prism software | (https://graphpad.com) | ||
| Software, algorithm | FilamentTracer, Imaris software, Bitplane AG | https://imaris.oxinst.com | ||
| Software, algorithm | GSEA Desktop v3.0 | https://www.gsea-msigdb.org/gsea/index.jsp | ||
| Software, algorithm | Leica Application Suite X (LAS X, v2.7) software | https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/ | ||
| Software, algorithm | CellProfiler v2.2 | https://cellprofiler.org | ||
| Software, algorithm | Image Studio Software (v5.2) | Li-cor Image Studio https://www.licor.com/bio/image-studio/ |
Additional files
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Supplementary file 1
Gene expression analysis NexCre Ribotag RNA-seq Rad21 lox/lox minus wild-type.
- https://cdn.elifesciences.org/articles/76539/elife-76539-supp1-v2.xlsx
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Supplementary file 2
Gene expression analysis total RNA-seq NexCre Rad21 lox/lox minus wild-type baseline ('at rest').
- https://cdn.elifesciences.org/articles/76539/elife-76539-supp2-v2.xlsx
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Supplementary file 3
Gene ontology analysis of NexCre Ribotag RNA-seq Rad21 lox/lox minus wild-type.
- https://cdn.elifesciences.org/articles/76539/elife-76539-supp3-v2.xlsx
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Supplementary file 4
Gene expression analysis RAD21-TEV 24 h cleaved vs control at baseline.
- https://cdn.elifesciences.org/articles/76539/elife-76539-supp4-v2.xlsx
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Supplementary file 5
Gene expression analysis total RNA-seq NexCre Rad21 lox/lox minus wild-type TTX, KCl 1 h, KCl 6 h.
- https://cdn.elifesciences.org/articles/76539/elife-76539-supp5-v2.xlsx
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Supplementary file 6
Gene expression analysis RAD21-TEV 24 h cleaved vs control BDNF 30 min and BDNF 120 min.
- https://cdn.elifesciences.org/articles/76539/elife-76539-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/76539/elife-76539-transrepform1-v2.docx
