| Literature DB >> 36043052 |
Yaping Sun1, Gabrielle A Dotson2, Lindsey A Muir2, Scott Ronquist2, Katherine Oravecz-Wilson1, Daniel Peltier1, Keisuke Seike1, Lu Li1, Walter Meixner2, Indika Rajapakse2,3, Pavan Reddy1.
Abstract
WAPL, cohesin's DNA release factor, regulates three-dimensional (3D) chromatin architecture. The 3D chromatin structure and its relevance to mature T cell functions is not well understood. We show that in vivo lymphopenic expansion, and alloantigen-driven proliferation, alters the 3D structure and function of the genome in mature T cells. Conditional deletion of WAPL, cohesin's DNA release factor, in T cells reduced long-range genomic interactions and altered chromatin A/B compartments and interactions within topologically associating domains (TADs) of the chromatin in T cells at baseline. WAPL deficiency in T cells reduced loop extensions, changed expression of cell cycling genes and reduced proliferation following in vitro and in vivo stimulation, and reduced severity of graft-versus-host disease (GVHD) following experimental allogeneic hematopoietic stem cell transplantation. These data collectively characterize 3D genomic architecture of T cells in vivo and demonstrate biological and clinical implications for its disruption by cohesin release factor WAPL.Entities:
Keywords: Genetics; Genomics; Molecular biology
Year: 2022 PMID: 36043052 PMCID: PMC9420521 DOI: 10.1016/j.isci.2022.104846
Source DB: PubMed Journal: iScience ISSN: 2589-0042
Figure 1Generating conditional Wapl knockout T cells utilizing CRISPR-CAS9 system
(A) Map of two sgRNAs targeting exon 2 of the Wapl gene on Chromosome 14.
(B) Genotyping and confirmation of Wapl knockout. Mice tail DNA was processed for PCR genotyping to confirm expression of CRE and sgRNA-Wapl insert and Cas9-stop signal expression or cleavage. GFP expression was determined by flow cytometry, and WAPL protein deletion was determined by Western Blotting.
(C) Confirmation of Wapl KO in T cells on multiple pups according to the screen procedures in (B) by western blotting.
(D) RNA-seq confirmation across biological triplicates of sgRNA-mediated deletion of exon 2 in Wapl.
Figure 2Genome-wide effects of WAPL knockout in unstimulated näıve T cells and after syngeneic/allogeneic transplantation
(A) Illustration of hierarchical Hi-C analysis workflow.
(B) Chromosome-level features and contact maps for chromosomes 1 and 2 from WT and Wapl knockout T cells. Gene expression (top track) is shown as a vector of log2(TPM) values binned at 100 kb resolution, and chromatin accessibility (bottom track) is shown as signed values from the Fiedler vector of the Hi-C contact map where positive values (red) denote A compartments and negative values (blue) denote B compartments. ICE and O/E normalized Hi-C contact maps are shown at 100-kb resolution and log-scale. Matrix dissimilarity between WT and KO conditions was detected by the Larntz-Perlman procedure (see STAR Methods) and genomic regions with dissimilarity in the 99th percentile are shaded in green. These areas indicate the largest perturbations to the chromatin architecture upon Wapl knockout.
Figure 3Comparison of internal TAD organization between WT and KO T cells
(A) Schematic of TADs illustrating how “corner peaks”—interactions between opposite TAD boundaries—were characterized in terms of their local neighborhoods. The nonzero mean of absolute read counts in each local corner peak neighborhood (CPN) was used to define the strength of TAD boundary interactions.
(B) Change in corner peak signal from WT (blue axis) to KO (orange axis) for each TAD (individual gray lines) on Chromosome 11. The average change in corner peak signal between the two conditions is plotted as a solid black line in each panel to capture the overall trend across settings. TADs and their corner peak signals are represented at 50 kb resolution.
(C) (Top center) Chromosome 11 observed contact map pooled across samples at 50 kb resolution. (Bottom left) TAD region of Chromosome 11 extending from 105.5 Mb to 107.2 Mb capturing corner peak increase from WT to KO most notably in the syngeneic and allogeneic settings; differential expression of gene Wipi1 occurs in the centermost TAD in this region. (Bottom right) TAD region of Chromosome 11 extending from 109.4 Mb to 111.1 Mb capturing corner peak increase from WT to KO; differential expression of gene Cdc42ep4 occurs in the centermost TAD in this region.
(D) Heatmap of differentially expressed cell-cycle genes residing on Chromosome 11.
Figure 4Cell-cycle gene network across T cells
(A) Hi-C-derived 5C contact maps representing the cell-cycle gene network. Rows and columns correspond to genomic bins across all chromosomes that contain cell-cycle genes. Maps are shown at 1-Mb resolution as a Pearson correlation of normalized (observed/expected) contacts. The purple and green boxes highlight subgroups of interest that are assessed in C. The purple subgroup is comprised of 10 loci (1Mb length) ranging noncontiguously from 18 Mb to 197 Mb and containing 10 cell-cycle genes. The cell-cycle gene loci in WT and KO T cells in each subgroup is comprised of 12 loci (1Mb length) ranging noncontiguously from 1,489 Mb to 1,606 Mb and containing 16 cell-cycle genes.
(B) Structural and functional differences between all cell-cycle gene loci in WT and KO T cells in each setting. Difference is measured as the Frobenius norm of the difference between WT and KO data (see STAR Methods). The left y axis (blue) reflects the Frobenius norm of the difference between WT and KO Hi-C matrices (measure of structural change) in each setting and the right y axis (orange) reflects the Frobenius norm of the difference between WT and KO gene expression vectors (measure of functional change) in each setting.
(C) Degree centrality (i.e. the row sum of a matrix) of subgroups of interest.
Figure 5WAPL deficiency impairs T cell development, proliferation, and cell cycling
(A) Total numbers of thymocytes isolated from WT and Wapl KO thymii were counted. Data were combined from WT (9) and Wapl KO (10) mice (mean ± SEM).
(B) Total T cell numbers of spleens from WT and Wapl KO mice were counted. Data were combined from WT (23) and Wapl KO (26) mice (mean ± SEM).
(C) WAPL protein expression was upregulated in T cells when co cultured with allogeneic DCs in vitro. A representative western blot was shown.
(D) mRNA expression of cell-cycle genes was analyzed through GSEA. Gene sets of WT and Wapl KO T cells from näıve, syngeneic, and allogeneic conditions were sorted by enrichment scores (ESs). All groups were in biological triplicates.
(E–H) According to ESs in (D), the mRNA expressions of the top or bottom two scored genes were analyzed by quantitative real-time PCR. Data were combined from three independent experiments (mean ± SEM).
(I) H3-TdR incorporation in WT and Wapl KO T cells were analyzed by mixed lymphocyte reaction when stimulated by allogeneic DCs for 4 days. Data were combined from three independent experiments (mean ± SEM).
(J) Cell proliferation was analyzed by Far-Red dilution for either WT or Wapl KO T cells when stimulated with allogeneic DCs for up to 7 days. Data were combined from two independent experiments (mean ± SEM).
(K) Cell apoptosis was analyzed by FACS after An- nexin V staining for WT and Wapl KO T cells when stimulated with allogeneic DCs for up to 7 days. Data were combined from two independent experiments (mean ± SEM).
(L) After WT and Wapl KO T cells were stimulated with CD3/CD28Ab for 2–4 days in vitro, cells were stained with FxCycle™Far-Red, DNA contents were separated as 2C and >2C by flow cytometry. Combined data were obtained from three independent experiments (mean ± SEM).
(M) WT and Wapl KO T cells were isolated from recipient spleens on day 7 after allogeneic BMT in vivo and were stained with FxCycle™Far-Red; DNA contents were separated as 2C and >2C by flow cytometry. Combined data were obtained from two independent experiments (mean ± SEM).
Figure 6WAPL-deficient T cells mitigate GVHD severity in allogeneic BMT
(A) Wapl expression was upregulated in T cells on day 7 after allogeneic BMT. A representative western blot was shown.
(B) Allogeneic MHC mismatched B6 (H2b) into BALB/c(H2d) GVHD model. Survival percentages were recorded daily. p-values were obtained using the log rank test based on comparisons between animals that received either TCD BM plus WT T cells (blue circles) or TCD BM plus Wapl KO T cells (red squares). Combined data were from two independent experiments.
(C) GVHD score was assessed by a standard scoring system as before (Sun et al., 2015). The Mann Whitney U test was used for the statistical analysis of clinical scores between animals that received either TCD BM plus WT T cells (blue circles) or TCD BM plus Wapl KO T cells (red squares). Combined data were from two independent experiments.
(D) Histopathological analysis of the B6 into BALB/c BMT model. Bowel (small and large intestine), liver, and skin were obtained after BMT on day 21. GVHD scores were from two independent experiments (mean ± SEM).
(E and F) Sera were collected from recipient mice on day 21 after allogeneic BMT as in (B); the concentrations of IFN-γ and TNF-α were measured by ELISA. Significant lower concentrations of INF-γ and TNF-α were detected in the sera collected from mice transferred with Wapl KO T cells. Combined data were from two independent experiments (mean ± SEM).
(G) In vivo T cell expansion was determined by isolating transferred WT and Wapl KO in BALB/c recipients on day 7 after allogeneic BMT. Data were combined from three independent experiments (mean ± SEM).
(H) Transferred donor T cells (WT and Wapl KO) were isolated from recipient’s spleens on day 7 after allogeneic BMT and stained for CD3+ and CD69+. The absolute numbers of activated Wapl KO T cells (CD3+CD69+) were significantly lower than WT T cells. Data were combined from three independent experiments (mean ± SEM).
(I and J) Donor T cells were isolated as in (H) on day 7 after allogeneic BMT and stained for CD4+ 25+ FoxP3+ for identifying T regulatory cells. The absolute numbers of Wapl KO Treg cells were significantly lower than WT Treg cells (I), although the percentages were the same between WT and Wapl KO T cells (J). Data were combined from three independent experiments (mean ± SEM). p-values were obtained using unpaired t test.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| anti-Wapl rabbit polyclonal Ab | Proteintech group Rosemont, IL | #16370-1-AP |
| anti–β-actin mouse mAb | Abcam Waltham, MA | ab8226 |
| rat Anti-Mouse CD16/CD32 | BD Pharmingen™ San Diego, CA | #553142 |
| anti-mouse CD4 APC | BioLegends San Diego, CA | #100412 |
| anti-mouse CD8 APC | BioLegends San Diego, CA | #100712 |
| anti-mouse CD3 APC | BioLegends San Diego, CA | #100312 |
| anti-mouse CD45.2 APC | BioLegends San Diego, CA | #109814 |
| anti-mouse CD45.1 APC | BioLegends San Diego, CA | #110714 |
| anti-mouse CD25 APC | BioLegends San Diego, CA | #102012 |
| anti-mouse CD69 APC | BioLegends San Diego, CA | #104514 |
| anti-mouse CD3 PE | BioLegends San Diego, CA | #100206 |
| anti-mouse CD4 PE | BioLegends San Diego, CA | #100512 |
| anti-mouse CD8a PE | BioLegends San Diego, CA | #100708 |
| anti-mouse CD25 PE | BioLegends San Diego, CA | #102008 |
| anti-mouse TCR β PE | BioLegends San Diego, CA | #109208 |
| anti-mouse CD3 PerCP/Cyanine5.5 | BioLegends San Diego, CA | #100218 |
| anti-mouse CD4 PerCP/Cyanine5.5 | BioLegends San Diego, CA | #100434 |
| anti-mouse CD8a PerCP/Cyanine5.5 | BioLegends San Diego, CA | #100734 |
| Mouse anti-rabbit IgG-HRP | Santa Cruz Biotechnology Santa Cruz, CA | SC-2357 |
| Mouse-IgGk BP-HRP | Santa Cruz Biotechnology Santa Cruz, CA | SC-516102 |
| Mouse anti- FoxP3 antibody PE | eBioscience, SanDiego, CA | #126403 |
| PE Annexin V | BioLegends San Diego, CA | #640934 |
| H3-Thymidine | Perkin Elmer | NET027005MC |
| CellTraceTM Far Red | Invitrogen | C34564 |
| Dynabeads Mouse T-Activator CD3/CD28 | Gibco | 11452D |
| FxCycleTM Far Red Stain | Invitrogen | F10348 |
| RNase A | Roche | #70294823 |
| anti-CD90.2 microbeads | Miltenyi Biotec Inc., Auburn, CA | #130-121-278 |
| CD11 c MicroBeads | Miltenyi Biotec Inc., Auburn, CA | #130-108-338 |
| Miltenyi LS Columns | Miltenyi Biotec Inc., Auburn, CA | #130-042-401 |
| PVDF membrane | Thermo Scientific Rockfood IL | #88518 |
| IGEPAL® CA-630 | MilliporeSigma Burlington, MA | #9002-93-1 |
| Protease Inhibitor Cocktail powder | MilliporeSigma Burlington, MA | P2714 |
| Sodium dodecyl sulfate | Sigma-Aldrich St. Louis, MO | #436143 |
| Triton™ X-100 | Sigma-Aldrich St. Louis, MO | #9036-19-5 |
| Restriction enzyme MboI | NEB Ipswich, MA | R0147S |
| Biotin-14-dATP | ThermoFisher Scientific, Ann Arbor, MI | #19524016 |
| DNA Polymerase I, Large (Klenow) Fragment | NEB Ipswich, MA | M0210S |
| T4 DNA Ligase | NEB Ipswich, MA | M0202S |
| Proteinase K | NEB Ipswich, MA | P8107S |
| Dynabeads™ MyOne™ Streptavidin C1 | ThermoFisher Scientific, Ann Arbor, MI | #65002 |
| T4 Polynucleotide Kinase | NEB Ipswich, MA | M0201L |
| T4 DNA Polymerase | NEB Ipswich, MA | M0203L |
| Klenow Fragment (3′→5′ exo-) | NEB Ipswich, MA | M0212L |
| PfuUltra II Fusion High-fidelity DNA Polymerase | Agilent Technologies, Inc Santa Clara, CA | #600387 |
| IC Fixation Buffer | Biolegend San Diego, CA | #420801 |
| Permeabilization Buffer | Biolegend San Diego, CA | #421002 |
| Pan T Cell Isolation Kit II | Miltenyi Biotec Inc., Auburn, CA | #130-095-130 |
| IFNg DuoSet ELISA Kit | BD Systems | DY485 |
| TNFalpha ELISA Set | BD OptEIATM | 555268 |
| PierceTM ECL Western | Thermo Scientific Rockfood IL | #32106 |
| AMPure XP beads | Beckman Coulter Indianapolis IN | C63510 |
| LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit | ThermoFisher Scientific, Ann Arbor, MI | L10120 |
| AllPrep DNA/RNA Mini Kit | Qiagen Germantown, MD | #80204 |
| RNA-seq mouse donor T-cells 7 days post HSCT | This paper | GEO: |
| T cell chromosome architecture (Hi-C) | This paper | BioBroject |
| C57BL/6, H2b genotype | Charles River Labs; | Strain Code: 027 |
| Rosa26-floxed STOP-Cas9 knockin | The Jackson Laboratory | Stock No:026175 |
| CD4-Cre | The Jackson Laboratory | Stock No: 017336 |
| BALB/c, H2d genotype | Charles River Labs; | Strain Code: 028 |
| Forward primer to determine sgRNA insertion (Wapl-F): 5′-CCGTAAGATGAACCCTTACCC | This paper | N/A |
| Reverse primer to determine sgRNA insertion (Wapl-R): 5′-TCTGAGTAAGATCGGTGTTTCG | This paper | N/A |
| Forward primer to determine if CD4-Cre exists (CD4CRE-F): 5′-GACATGTTCAGGGATCGCCA | This paper | N/A |
| Reverse primer to determine if CD4-Cre exists (CD4CRE-R): 5′-AACCAGCGTTTTCGTTCTGC | This paper | N/A |
| Forward primer to determine stop sign cleavage (LSL-F): 5′-GCAACGTGCTGGTTATTGTG | This paper | N/A |
| Reverse primer to determine stop sign cleavage (LSL-R): 5′-TAGTCTCCGTCGTGGTCCTT | This paper | N/A |
| Forward primer to check Gas2l1 expression (Gas2I1-F): 5′-CGAGCTACCCCTCAG | This paper | N/A |
| Reverse primer to check Gas2l1 expression (Gas2I1-R): 5′-CTGGAGTCTCCCACT | This paper | N/A |
| Forward primer to check Mcm3 expression (Mcm3-F): 5′-AAGAAGGGCTGCTAC | This paper | N/A |
| Reverse primer to check Mcm3 expression (Mcm3-R): 5′-CCGTTTTCAAGTTCCCGCTC-3′ | This paper | N/A |
| Forward primer to check Sphk1 expression (Sphk1-F): 5′-ACTCACCGAACGGA | This paper | N/A |
| Reverse primer to check Sphk1 expression (Sphk1-R): 5′-AGCAGGTTCATGGGT | This paper | N/A |
| Forward primer to check Myh10 expression (Myh10 -F): 5′-GCTTGAACGAAGCCTCTGTCT-3′ | This paper | N/A |
| Reverse primer to check Myh10 expression (Myh10 -R): 5′-CGTGGGCAAGGTACTGAATGA-3′ | This paper | N/A |
| pX330-U6-Chimeric_BB-CBh-hSpCas9 | Addgene Watertown, MA | #42230 |
| FastQC | ||
| RNA-Seq Alignment v1.0 | Illumina, Inc. | |
| Cufflinks v.2.2.1 | ||
| Integrative Genomics Viewer (IGV) | ||
| Gene Set Enrichment Analysis (GSEA) | ||
| 4DNvestigator | ||