Epigenetic regulation of Wnt7b expression by the cis-acting long noncoding RNA Lnc-Rewind in muscle stem cells
- Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Italy
- Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR) at Sapienza University of Rome, Italy
- Center for Life Nano Science at Sapienza, Istituto Italiano di Tecnologia, Italy
Figures
Figure 1 with 1 supplement
Lnc-Rewind is a conserved chromatin-associated lncRNA expressed in satellite cells.
(A) UCSC visualization showing the chromosome position and the genomic coordinates of Lnc-Rewind (red shade) in the mm9 mouse genome. Mmu_Lnc-Rewind reads coverage and quantification (FPKM) from …
-
Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Lnc-Rewind is a conserved chromatin-associated lncRNA expressed in satellite cells.
(A) Table represents the values obtained by analysing the local sequence alignment between the human and murine Lnc-Rewind transcripts. Data were produced by using the implementation of the …
-
Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
Lnc-Rewind regulates muscle system processes and MuSC expansion.
(A) Heatmap representing hierarchical clustering performed on the final list of genes differentially expressed in MuSC-derived myoblasts upon Lnc-Rewind depletion (p-value threshold<0.05; see also Fi…
-
Figure 2—source data 1
List of the differentially expressed genes (DEGs) between GAP-SCR and GAP-REW transfected MuSCs.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig2-data1-v2.xlsx
-
Figure 2—source data 2
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Lnc-Rewind regulates muscle system processes and MuSC expansion.
(A) Upper part: Experimental timeline used for the transfection of MuSC-derived myoblasts with GAP-SCR or GAP-REW. Lower part: qRT-PCR quantification of Lnc-Rewind spliced and unspliced transcript …
-
Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Lnc-Rewind and Wnt7b genes display opposite pattern of expression and a functional interplay.
(A) Schematic UCSC visualization showing the Lnc-Rewind chromosome position and its neighbouring genes. (B) TSS usage analyses of Wnt7b, Lnc-Rewind, Atxn10, and Cdpf1 performed by using FANTOM5 …
-
Figure 3—source data 1
5'CAGE TSS expression profile of Wnt7b, Lnc-Rewind, Atxn10 and Cdpf1 genes.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig3-data1-v2.xlsx
-
Figure 3—source data 2
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Lnc-Rewind and Wnt7b genes display opposite pattern of expression and a functional interplay.
(A) qRT-PCR (left panel) and Western blot (right panel) analyses performed on extracts from MuSC-derived myoblasts, treated with GAP-SCR or GAP-REW. Gapdh mRNA and protein were used as endogenous …
-
Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
Lnc-Rewind directly interacts with the methyltransferase G9a and mediates specific in cis repression of Wnt7b.
(A) G9a crosslinked RNA immunoprecipitation assay (CLIP) performed on nuclear extracts from C2C12 proliferating myoblasts. Western blot analysis of G9a (left panel) and qRT-PCR quantification of …
-
Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Lnc-Rewind directly interacts with the methyltransferase G9a and mediates specific in cis repression of Wnt7b.
(A) Western blot analysis performed on protein extracted from WT MuSC-derived myoblasts cultured in growth (GM) and differentiated (DM) conditions. GAPDH protein was used as endogenous control. (B) …
-
Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/54782/elife-54782-fig4-figsupp1-data1-v2.xlsx
Figure 5
Lnc-Rewind and G9a colocalize on Wnt7b gene locus.
(A) Top panel: RNA/DNA-FISH combined with immunofluorescence performed on MuSC-derived myoblasts; Lnc-Rewind RNA (red), Wnt7b DNA locus (green), and G9a protein (grey) are shown. Bottom panel: …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Strain, strain background (mouse) | C57Bl/10 (WT) | JAX | Stock# 000665 RRID:MGI:5656893 | ||
| Cell line (mouse) | C2C12 | ATCC | C3H RRID:CVCL_UR38 | ||
| Cell line (human) | Male myoblast (WT) | Telethon Biobank | N/A | ||
| Antibody | Anti-CD31-PE (cell line monoclonal) | MiltenyBiotec | Cat# 130111540 RRID:AB_2657296 | (1:25) | |
| Antibody | Anti-CD45-PE (cell line monoclonal) | MiltenyBiotec | Cat# 130110797 RRID:AB_2658218 | (1:25) | |
| Antibody | Anti-Ter119-PE (cell line monoclonal) | MiltenyBiotec | Cat# 130112909 RRID:AB_2654115 | (1:25) | |
| Antibody | Anti-Sca1-FITC (cell line monoclonal) | MiltenyBiotec | Cat# 130116490 RRID:AB_2751322 | (1:50) | |
| Antibody | Anti-α7Integrin-APCVio770 (cell line monoclonal) | MiltenyBiotec | Cat# 130095212 Custom | (1:20) | |
| Antibody | Anti-G9a (rabbit monoclonal) | Abcam | Cat# ab185050 RRID:AB_2792982 | (CLIP, RIP: 10 ug; ChIP: 5 ug; WB: 1:1000; IF: 1:100) | |
| Antibody | Anti-EZH2 (mouse monoclonal) | Cell Signaling | Cat# 3147 RRID:AB_10694383 | (RIP: 5 ug; WB: 1:1000) | |
| Antibody | Anti-GAPDH (rabbit polyclonal) | Sigma–Aldrich | Cat# G9545 RRID:AB_796208 | (1:10000) | |
| Antibody | Anti-MyHC (mouse monoclonal) | DSHB | Cat# Mf20-s RRID:AB_2147781 | (1:20) | |
| Antibody | Anti-CyclinD3 (rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-182 RRID:AB_2259653 | (1:200) | |
| Antibody | Anti-Wnt7b (rabbit polyclonal) | Abcam | Cat#ab94915 RRID:AB_10675749 | (1:200) | |
| Antibody | Anti-H3K9me2(mouse monoclonal) | Abcam | Cat# ab1220 RRID:AB_449854 | (ChIP: 5 ug) | |
| Antibody | Anti-Pax7 (mouse monoclonal) | DSHB | Cat# Pax7-s RRID:AB_2299243 | (1:10) | |
| Antibody | IgG Anti-Rabbit | Invitrogen | Cat# 14-4616-82 RRID:AB_2865072 | (CLIP, RIP:10 ug; ChIP: 5 ug) | |
| Antibody | Donkey anti-rabbit IgG Alexa Fluor Plus 647 | Invitrogen | Cat# A32795 RRID:AB_2762835 | (1:300) | |
| Antibody | Anti-Ki67 (rabbit polyclonal) | Abcam | Cat# ab15580 RRID:AB_443209 | (1:100) | |
| Sequence-based reagent | siRNA: nontargeting control | Sigma–Aldrich | Cat# SIC007 | ||
| Sequence-based reagent | LNA Gapmer: nontargeting control | Qiagen | Cat# 300610 | ||
| Sequence-based reagent | Mir-Let7-b | Qiagen | Cat# MS00003122 | ||
| Sequence-based reagent | Mir-Let7-c | Qiagen | Cat# 18300 | ||
| Sequence-based reagent | siG9a | Sigma–Aldrich | SASI_Mm01_00136174 SASI_Mm_00136174_AS SASI_Mm01_00136175 SASI_Mm01_00136175_AS | ||
| Sequence-based reagent | siWnt7b | Sigma–Aldrich | SASI_MM02_00316332 SASI_MM02_00316332_AS SASI_MM01_00033690 SASI_MM01_00033690_AS | ||
| Sequence-based reagent | HS-DLC1 mRNA probe set | Advanced Cell Diagnostics, Inc | Ref. 716041 | ||
| Sequence-based reagent | Wnt7b BAC probe | Invitrogen clones | RP23-272K17 | ||
| Sequence-based reagent | NCTC BAC probe | Invitrogen clones | RP23-352B6 | ||
| Sequence-based reagent | Mm-Lnc-Rewind probe set | Advanced Cell Diagnostics, Inc | Ref. 722581 | ||
| Chemical compound, drug | TriReagent | Sigma | Cat#T9424 | ||
| Chemical compound, drug | DAPI | Sigma | Cat# 28718-90-3 | (1:10000) | |
| Chemical compound, drug | DSG (di-succinimidyl glutarate) | Santa Cruz Biotechnology | CAS 79642-50-5 | ||
| Commercial assay or kit | SYBR Green Master mix | ThermoFisher Scientific | Cat# A25742 | ||
| Commercial assay or kit | Paris Kit | Thermo fisher scientific | Cat# AM1921 | ||
| Commercial assay or kit | Direct-Zol RNA MiniPrep Kit | Zymo Research | Cat# R2050 | ||
| Commercial assay or kit | MAGnify ChIP | ThermoFisher Scientific | Cat# 492024 | ||
| Commercial assay or kit | MyTaq DNA polymerase | Bioline | Cat# bio-21105 | ||
| Commercial assay or kit | SuperScript RT Vilo Master Mix | ThermoFisher Scientific | Cat# 11754050 | ||
| Commercial assay or kit | BaseScope Reagent Kit v2-RED | Advanced Cell Diagnostics, Inc | Ref. 323900 | ||
| Commercial assay or kit | MiScript II RT kit | Qiagen | Cat# 218160 | ||
| Commercial assay or kit | Lipofectamine 2000 | Invitrogen | Cat# 11668019 | ||
| Commercial assay or kit | Click-iT EdU Alexa Flour 594 HCS Assay | Invitrogen | Cat# C10354 | ||
| Commercial assay or kit | Pierce ChIP-grade Protein A/G Magnetic Beads | ThermoFisher scientific | Cat# 26162 | ||
| Peptide, recombinant protein | Dispase II | Roche | 4942078001 | ||
| Peptide, recombinant protein | Proteinase K | Roche | EO0491 | ||
| Peptide, recombinant protein | Collagenase A | Roche | 10103578001 | ||
| Peptide, recombinant protein | DNase I | Roche | 10104159001 | ||
| Peptide, recombinant protein | DNase I | ThermoFisher scientific | #EN0521 | ||
| Peptide, recombinant protein | Collagenase I | Sigma | C0130 | ||
| Software, algorithm | FACSDiva | BD Biosciences | Version 6.1.3. | ||
| Software, algorithm | FlowJo | Tree Star | Version 9.3.2 RRID:SCR_008520 | ||
| Software, algorithm | Fiji image processing package | Open-source software (OSS) projects | https://imagej.net/Fiji RRID:SCR_002285 | ||
| Software, algorithm | MetaMorph | Molecular Devices | https://www.moleculardevices.com/ RRID:SCR_002368 | ||
| Software, algorithm | FV10-ASW Viewer software | Olympus | https://www.olympus-lifescience.com/ RRID:SCR_014215 | ||
| Software, algorithm | ZEN 3.0 Blue edition | ZEISS | |||
Additional files
-
Supplementary file 1
List and sequences of the oligonucleotides, LNA gapmers, and siRNAs used.
- https://cdn.elifesciences.org/articles/54782/elife-54782-supp1-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/54782/elife-54782-transrepform-v2.docx
