Single-cell profiling of lncRNAs in human germ cells and molecular analysis reveals transcriptional regulation of LNC1845 on LHX8
- Center for Stem Cell Biology and Regenerative Medicine, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, China
- Key Laboratory of Intelligent Information Processing, Advanced Computer Research Center, Institute of Computing Technology, Chinese Academy of Sciences, China
- Biodynamic Optical Imaging Center & Department of Obstetrics and Gynecology, College of Life Sciences, Third Hospital, Peking University, China
- Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, China
Figures
Figure 1 with 1 supplement
Figure numerous.
(A) Long non-coding RNAs (lncRNAs) are differentially expressed during human germ cell (hGC) development. (B) Two unannotated lncRNAs and their neighboring protein-coding genes with their genomic …
Figure 1—figure supplement 1
Numerous long non-coding RNAs (lncRNAs) are differentially expressed during human germ cell development.
(A) Numbers of protein-coding genes expressed in individual female (upper) and male (bottom) germ cells or somatic cells from different developmental week. Student’s t-test was used for the …
Figure 2 with 1 supplement
Integrating long non-coding RNA (lncRNA) expression enhances cell-type classifications of human female germ cells.
(A) tSNE plot of female germ cells and somatic cells colored by identified cell types. Left: cluster according to protein-coding genes. Right: cluster according to protein-coding genes and lncRNAs. …
Figure 2—figure supplement 1
Integrating long non-coding RNA (lncRNA) expression enhances cell-type classifications of human female germ cells.
(A) Gene ontology biological processes enrichment of mitotic-4 cells feature genes. (B) Single-cell trajectories of female germ cell states through the pseudotime according to protein-coding genes …
Figure 3 with 1 supplement
Genomic distributions and biotypes of the long non-coding RNAs (lncRNAs) expressed during human gonadal development.
(A) The genomic position distribution of lncRNAs expressed in human germ cells (hGCs) and gonadal somatic cells. (B) The distribution and percentage of the six locus biotypes of each developmental …
Figure 3—figure supplement 1
Genomic distributions and biotypes of the long non-coding RNAs (lncRNAs) expressed during human gonadal development.
(A) The genomic position distribution of annotated and unannotated lncRNAs expressed in human germ cells (hGCs) and gonadal somatic cells. (B) Expression correlations of lncRNA–mRNA pairs of the six …
Figure 4 with 2 supplements
Characterization of a divergent long non-coding RNA (lncRNA), LNC1845, expressed during the meiotic stage and is required for normal expression of LHX8.
(A) Genomic locus and relative position of LNC1845 (in green) and LHX8 (in blue). (B) Expression analysis by RT-qPCR of PA-1 cells, undifferentiated H9 cells, and RNA sample extracted from human …
Figure 4—figure supplement 1
Characterization of a divergent long non-coding RNA (lncRNA), LNC1845, expressed during the meiotic stage and is required for normal expression of LHX8.
(A) LNC1845 was predicted to be non-coding RNA. The RNA sequences of LNC1845 were put into the Coding Potential Calculator (CPC) program and the Coding Potential Assessment Tool. (B) In vitro …
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Figure 4—figure supplement 1—source data 1
Characterization of a divergent long non-coding RNA (lncRNA), LNC1845, expressed during the meiotic stage and is required for normal expression of LHX8.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
Upregulated XH long non-coding RNA (lncRNA)–mRNA pairs in hPGCLCs.
Relative position of upregulated divergent lncRNA–mRNA pairs and their relative expression in primordial germ cell-like cells (PGCLCs) and control cells.
Figure 5 with 1 supplement
LNC1845 RNA transcripts are required for normal expression of LHX8.
(A) Schematic diagram of knockout strategies at the LNC1845 loci. (B) Expression analysis by RT-qPCR of WT primordial germ cell-like cells (PGCLCs) (n = 3) and LNC1845 KO PGCLCs (n = 3). (C) Western …
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Figure 5—source data 1
Western blot analysis of LHX8 in PGCLCs from WT, LNC1845 KO, or RNA interfering LNC1845 (KD) hESCs. SD, spontaneous differentiation cells as the negative control.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig5-data1-v2.zip
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Figure 5—source data 2
Western blot analysis of GAPDH in PGCLCs from WT, LNC1845 KO, or RNA interfering LNC1845 (KD) hESCs. SD, spontaneous differentiation cells as the negative control.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig5-data2-v2.zip
Figure 5—figure supplement 1
LNC1845 RNA transcripts are required for normal expression of LHX8.
(A) Genotyping and sequencing of LNC1845 KO single colonies to confirm the deletion of LNC1845. (B) Principle component analysis (PCA) of RNA-seq datasets from ESCs, LNC1845 KO primordial germ …
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Figure 5—figure supplement 1—source data 1
Genotyping of LNC1845 KO single colonies to confirm the deletion of LNC1845.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
Genotyping of LNC1845-upstream 3×polyA insertion single colonies.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig5-figsupp1-data2-v2.zip
Figure 6 with 2 supplements
LNC1845 regulates LHX8 expression in cis by changing chromatin modifications.
(A) Schematic diagram of LNC1845-upstream EF1α promoter knock-in and the targeted allele after EF1α promoter insertion. (B) Expression analysis by RT-qPCR of LNC1845 and LHX8 in WT primordial germ …
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Figure 6—source data 1
Western blot analysis of LHX8 in PGCLCs from WT, LNC1845-upstream 3×polyA knock-in, or LNC1845-upstream EF1α promoter knock-in cells.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig6-data1-v2.zip
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Figure 6—source data 2
Western blot analysis of GAPDH in PGCLCs from WT, LNC1845-upstream 3×polyA knock-in, or LNC1845-upstream EF1α promoter knock-in cells.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig6-data2-v2.zip
Figure 6—figure supplement 1
LNC1845 regulates LHX8 expression in cis by changing chromatin modifications.
(A) Genotyping and sequencing of LNC1845-upstream EF1α promoter insertion single colonies. (B) Karyotype analysis of LNC1845 KO human embryonic stem cell (hESC) clone F11, LNC1845 3xpolyA knock in …
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Figure 6—figure supplement 1—source data 1
Genotyping of LNC1845-upstream EF1α promoter insertion single colonies.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig6-figsupp1-data1-v2.zip
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Figure 6—figure supplement 1—source data 2
Agarose gel analysis of LNC1845 transcripts by in vitro transcription.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig6-figsupp1-data2-v2.zip
Figure 6—figure supplement 2
LNC1845 regulates LHX8 expression in cis by physically interacting with WDR5.
(A) WDR5 expression level in single hPGC or gonadal somatic cells at different developmental stages. (B) Immunofluorescence of WDR5 (green) co-stained with DAZL (red) in 14-week ovary section …
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Figure 6—figure supplement 2—source data 1
LNC1845 binding for WDR5 was verified by CHIRP-western blot.
- https://cdn.elifesciences.org/articles/78421/elife-78421-fig6-figsupp2-data1-v2.zip
Figure 7 with 1 supplement
FOXP3 upregulates expression of LNC1845 and other long non-coding RNAs (lncRNAs).
(A) RT-qPCR analysis of LNC1845 and LHX8 in primordial germ cell-like cells (PGCLCs) overexpresses FOXP3. (B) Heatmap of differentially expressed ms-lncRNAs in control or OE FOXP3 groups, rep1 and …
Figure 7—figure supplement 1
FOXP3 upregulates expression of LNC1845 and other long non-coding RNAs (lncRNAs).
(A) Schematic diagram of input features for the computational prediction used to predict transcription factors involved in female meiotic stage-specific lncRNAs upregulation. (B) Bar chart …
Author response image 1
Integrative genomic viewer images showing FOXP3 binding sites at the LC1845/LHX8 locus.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | LIM homebox 8(LHX8) | Human genome informatics | Ensembl:ENSG 00000162624 | |
| Gene (Homo sapiens) | LNC1845(AC099786.3) | Human genome informatics | NONHSAG001845. 2/ENSG00000261213.1 | |
| Cell line (Homo sapiens) | H9 LNC1845 KO stem cells | This paper | LNC1845 KO cell line generated by CRISPR/Cas9 | |
| Cell line (Homo sapiens) | H9 LNC1845 polyA KI stem cells | This paper | LNC1845 polyA KI cell line generated by CRISPR/Cas9 | |
| Cell line (Homo sapiens) | H9 LNC1845 EF1α KI stem cells | This paper | LNC1845 EF1α KI cell line generated by CRISPR/Cas9 | |
| Cell line (Homo sapiens) | PA-1 cells | This paper | Human ovary teratocarcinoma Cell Line | |
| Cell line (Homo sapiens) | 293 FT cells | Thermo Fisher Scientific | Cat# R70007 | |
| Cell line (Mus musculus) | Mouse embryonic fibroblasts | This paper | Mouse embryonic fibroblasts isolated from embryonic day 13.5 | |
| Recombinant DNA reagent | P2k7-EF1α-DAZL2- P2A-mCherry | This study | Methods | |
| Recombinant DNA reagent | P2k7-EF1α-mCherry | This study | Methods | |
| Recombinant DNA reagent | P2k7-EF1α-1845 | This study | Methods | |
| Recombinant DNA reagent | P2k7-EF1α-FOXP3 | This study | Methods | |
| Recombinant DNA reagent | P2k7-EF1α-TFAP2A | This study | Methods | |
| Recombinant DNA reagent | P2k7-EF1α-FOXP3- P2A-mCherry | This study | Methods | |
| Recombinant DNA reagent | P2k7-EF1α-FOXP3- 3xFLAG-P2A-mCherry | This study | Methods | |
| Recombinant DNA reagent | pEASY-T7 promoter | This study | Methods | |
| Recombinant DNA reagent | pEASY-T7-1845 | This study | Methods | |
| Recombinant DNA reagent | pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) | Addgene | # 42335 | |
| Recombinant DNA reagent | PGK-puro-1845KO donor | This study | Methods | |
| Recombinant DNA reagent | SV40-puro-3xpolyA 1845 KI donor | This study | Methods | |
| Recombinant DNA reagent | SV40-puro-EF1α 1845 KI donor | This study | Methods | |
| Recombinant DNA reagent | H1-shLACZ-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-sh1845①-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-sh1845②-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-sh15266①-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-sh15266②-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-sh3346①-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-sh3346②-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-shFOXP3①-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-shFOXP3②-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-shWDR5①-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-shWDR5②-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | H1-shWDR5③-Ubc-GFP | This study | Methods | |
| Recombinant DNA reagent | Lenti-dCAS-VP64-Blast | Addgene | # 61425 | |
| Recombinant DNA reagent | Lenti-sgRNA(MS2)-zeo backbone | Addgene | # 61427 | |
| Recombinant DNA reagent | Lenti MS2-P65-HSF1_Hygro | Addgene | # 61426 | |
| Recombinant DNA reagent | Lenti-sgRNA(MS2)–1845 gRNA 1 | This study | Methods | |
| Recombinant DNA reagent | Lenti-sgRNA(MS2)–1845 gRNA 2 | This study | Methods | |
| Recombinant DNA reagent | Lenti-sgRNA(MS2)–1845 gRNA 3 | This study | Methods | |
| Biological sample (Homo sapiens) | Human 17 w ovary RNA | BIOPIKE | ||
| Antibody | anti-DAZL (Mouse monoclonal) | Bio-Rad | Cat# MCA2336, RRID:AB_2292585 | (IF 1:50) |
| Antibody | anti-LHX8 (Rabbit polyclonal) | abcam | Cat# ab221882 | (IF 1:50; WB 1:1000) |
| Antibody | anti-WDR5 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 13105S, RRID:AB_2620133 | (IF 1:200; WB 1:2000) |
| Antibody | anti-GAPDH (Mouse monoclonal) | Cwbio | Cat# CW0100M, RRID:AB_2801390 | (WB 1:5000) |
| Antibody | anti-FLAG (Rabbit monoclonal) | Sigma | Cat# F3165, RRID:AB_259529 | (IP 3 μg) |
| Antibody | anti-H3K27Ac (Rabbit monoclonal) | Cell Signaling Technology | Cat# 8173, RRID:AB_10949503 | (ChIP 1 μg/rxn) |
| Antibody | anti-H3K4me3 (Rabbit monoclonal) | Millipore | Cat# 04-745, RRID:AB_1163444 | (ChIP 1 μg/rxn) |
| Antibody | Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-11008, RRID:AB_143165 | (WB 1:5000) |
| Antibody | Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A32742, RRID:AB_2762825 | (WB 1:5000) |
| Antibody | Horseradish-labeled goat anti-rabbit IgG (H + L) (Goat polyclonal) | ZSGB-Bio | Cat# ZB-2301, RRID:AB_2747412 | (WB 1:5000) |
| Antibody | Peroxidase-Conjugated Goat anti-Mouse IgG (H + L) (Goat polyclonal) | ZSGB-Bio | Cat# ZB-2305, RRID:AB_2747415 | (WB 1:5000) |
| Chemical compound, drug | Puromycin | Sigma | Cat# P9620 | |
| Chemical compound, drug | Geneticin | Thermo Fisher Scientific | Cat# 10131035 | |
| Chemical compound, drug | Blasticidin | Thermo Fisher Scientific | Cat# 461120 | |
| Chemical compound, drug | ROCK inhibitor | stemRD | Cat# Y-005 | |
| Peptide, recombinant protein | Recombinant human BMP-4 | R&D | Cat# 314 BP | |
| Peptide, recombinant protein | Recombinant human BMP-8a | R&D | Cat# 1073-BPC | |
| Peptide, recombinant protein | Recombinant human FGF basic | R&D | Cat# 233-FB-001MG/CF | |
| Commercial assay or kit | Gateway LR Clonase II Kit | Thermo Fisher Scientific | Cat# 11791100 | |
| Commercial assay or kit | PrimeScript RT reagent Kit with gDNA Eraser | TaKaRa | Cat# RR047A | |
| Commercial assay or kit | Fluorescent In Situ Hybridization Kit | RIBOBIO | Cat# C10910 | |
| Commercial assay or kit | RNAmax-T7 kit | RIBOBIO | Cat# R11073 | |
| Commercial assay or kit | TnT Quick Coupled Transcription/ Translation Systems | Promega | Cat# L1170 | |
| Commercial assay or kit | FirstChoice RLM-RACE kit | Ambion | Cat# AM1700 | |
| Commercial assay or kit | AMPure XP beads | Beckman Coulter | A63881 | |
| Commercial assay or kit | NEBNext DNA Library Prep Kit | NEB | #E7645S | |
| Commercial assay or kit | NEBNext Multiplex Oligos | NEB | #E7335S | |
| Commercial assay or kit | VAHTS mRNA-seq v2 Library Prep Kit | Vazyme | NR611-02 | |
| Software, algorithm | GraphPad Prism 6.0 | GraphPad Prism | https://www.graphpad.com/ | |
| Software, algorithm | FlowJo | FlowJo | https://www.flowjo.com/ | |
| Software, algorithm | DAVID Bioinformatics Resources | Huang et al., 2009 | https://david.ncifcrf.gov | |
| Software, algorithm | Coding Potential Calculator | Kong et al., 2007 | http://cpc.cbi.pku.edu.cn/ | |
| Software, algorithm | Coding Potential Assessment Tool | Wang et al., 2013 | http://rna-cpat.sourceforge.net/ | |
| Software, algorithm | PROMO | Messeguer et al., 2002 | http://alggen.lsi.upc.es/ | |
| Software, algorithm | TopHat (v2.1.1) | Trapnell et al., 2009 | http://ccb.jhu.edu/software/tophat/index.shtml | |
| Software, algorithm | HTSeq package | Anders et al., 2015 | https://htseq.readthedocs.io/en/release_0.9.1/ | |
| Software, algorithm | GO (DAVID) | Huang et al., 2009 | https://david.ncifcrf.gov/home.jsp | |
| Software, algorithm | HISAT2 (v2.1.0) | Kim et al., 2015 | http://ccb.jhu.edu/software/hisat2/manual.shtml | |
| Software, algorithm | NONCODE (v5) | Fang et al., 2017 | http://www.noncode.org/index.php | |
| Software, algorithm | Gencode (v29) | Uszczynska-Ratajczak et al., 2018 | https://www.gencodegenes.org/ | |
| Software, algorithm | Samtools (1.3.1) | Li et al., 2009 | http://samtools.sourceforge.net/ | |
| Software, algorithm | MACS2 | Zhang et al., 2008 | https://pypi.org/project/MACS2/2.1.1.20160309/ | |
| Software, algorithm | IGV | Thorvaldsdóttir et al., 2013 | http://www.igv.org/ | |
| Software, algorithm | Treeview | Saldanha, 2004 | http://jtreeview.sourceforge.net/ | |
| Software, algorithm | R (3.5.1) | N/A | https://www.r-project.org/ | |
| Software, algorithm | pheatmap | N/A | R software | |
| Software, algorithm | DESeq2 | Love et al., 2014 | R software | |
| Software, algorithm | ggplot2 | Wickham, 2016 | R software | |
| Software, algorithm | Seurat (3.2.3) | Satija et al., 2015 | R software | |
| Software, algorithm | Monocle (2.10.1) | Qiu et al., 2017 | R software | |
| Other | Matrigel | Corning | Cat# 354277 | Matrigel Basement Membrane Matrix |
| Other | DAPI | Invitrogen | Cat# D1306 | Blue-fluorescent DNA stain |
| Other | TrypLE Express | Invitrogen | Cat# 12605010 | Trypsin substitution |
| Other | Dynabeads Protein A | Invitrogen | Cat# 10002D | Dynabeads Protein A for Immunoprecipitation |
| Other | Dynabeads Protein G | Invitrogen | Cat# 10003D | Dynabeads Protein G for Immunoprecipitation |
| Other | ProLong Diamond Antifade Mountant | Invitrogen | Cat# P10144 | Antifade Solution |
| Other | TRIzol Reagent | Invitrogen | Cat# 15596026 | RNA extraction reagents |
| Other | TransStart Top Green qPCR Mix | TransGen | Cat# AQ131-02 | qPCR reagents |
| Other | Lipofectamine 2000 | Invitrogen | Cat# 11668019 | Transfection reagents |
| Other | Lipofectamine 3000 | Invitrogen | Cat# L3000001 | Transfection reagents |
Additional files
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Supplementary file 1
Weekly expressed lncRNAs of female and male germ cells and gonadal somatic cells.
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp1-v2.xlsx
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Supplementary file 2
Human germ cells and gonadal somatic cells specific lncRNAs.
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp2-v2.xlsx
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Supplementary file 3
Human germ cell each stage-specific lncRNAs.
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp3-v2.xlsx
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Supplementary file 4
Feature protein-coding genes or lncRNAs of each cluster.
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp4-v2.xlsx
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Supplementary file 5
Female germ cell (fGC) meiotic and male germ cell (mGC) mitotic arrest lncRNA–mRNA pairs and expression correlation.
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp5-v2.xlsx
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Supplementary file 6
Upregulated lncRNA genes in primordial germ cell-like cells (PGCLCs).
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp6-v2.xlsx
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Supplementary file 7
Upregulated protein-coding genes in primordial germ cell-like cells (PGCLCs).
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp7-v2.xlsx
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Supplementary file 8
Differentially expressed protein-coding genes in LNC1845 KO primordial germ cell-like cells (PGCLCs).
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp8-v2.xlsx
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Supplementary file 9
Differentially expressed protein-coding genes in LHX8 OE primordial germ cell-like cells (PGCLCs).
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp9-v2.xlsx
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Supplementary file 10
Oligonucleotides.
- https://cdn.elifesciences.org/articles/78421/elife-78421-supp10-v2.xlsx
