Categories: NATURE

Clonal inactivation of TERT impairs stem cell competition

Animals

All animal experiments were approved by the Administrative Panel on Laboratory Animal Care (APLAC) in protocol APLAC-12684, and all experiments were in compliance with the ethical regulations of Stanford University. Mice were housed at an ambient temperature of 22 °C and at 40% humidity, with a 12-h light–dark cycle (07:00–19:00). Tert-CreER, TetO-Tert, TetO-Tertci and Tert-Tdtomato mice were previously reported^3,16,27. Rosa-lslTdtomato⁴⁵, Rosa-lsl-tTA⁴⁶, TetO-hMYC⁴⁷, Terc-KO⁴⁸ and Trp53-flox⁴⁹ mice were purchased from The Jackson Laboratory. Tamoxifen (Cayman) was dissolved in corn oil (Sigma-Aldrich) at 5–20 mg ml⁻¹ by incubating at 50 °C for 30 min with mixing every 5 min. Two- to four-month-old mice were administered with 0.25–4 mg tamoxifen per 25 g body weight by oral gavage or intra-peritoneal injection. Doxycycline (Sigma-Aldrich) was dissolved in drinking water in light-protected bottles at 1 or 3 μg ml⁻¹ and changed every three or four days. BrdU (Sigma-Aldrich) was dissolved in PBS at 10 mg ml⁻¹ and intraperitoneally injected at 1.25 mg per 25 g body weight two hours before euthanasia.

Generation of Tert-flox mice

A 9-kb fragment of the Tert locus was subcloned and a Lox-Puro-lox cassette from the pBS.DAT-LoxStop plasmid (a gift from D. Tuveson) was inserted at the BsiWI site in the second intron. Another loxP sequence and NdeI site were inserted at the KasI site in the sixth intron. The targeting vector was linearized and electroporated into J1 mouse ES cells. After positive selection with puromycin, correctly targeted ES clones were selected by long-range PCR and Southern blotting, and then injected into C57BL6 blastocysts to generate the knock-in line. To remove floxed puro cassette, the knock-in line was crossed with CMV-cre mice⁵⁰ and puro-negative Tert-floxed mice were selected by PCR and Southern blotting using genomic DNA from tail tips. TERT^flox/+ mice were born at normal Mendelian frequency. Original uncropped images of Southern blots are provided in Supplementary Fig. 1.

Lineage-tracing assay

After tamoxifen injection, testes were detunicated and seminiferous tubules were untangled using fine forceps in PBS containing 1 mg ml⁻¹ collagenase IV (Worthington) for 10 min, and placed in cold PBS. Images were captured with a fluorescent dissection microscope and the patch number and length were measured with ImageJ. Total patch length was calculated by multiplying the patch number by the average patch length.

Sperm count

Cauda epididymides were dissected into small pieces and incubated in potassium simplex optimized medium (KSOM) at 37 °C for one hour under 5% CO₂ to allow sperm to exude. The collected sperm were then fixed with 4% PFA and counted with a haemacytometer.

Whole-mount immunofluorescence of seminiferous tubules

Seminiferous tubules were dissociated using fine forceps in PBS containing 1 mg ml⁻¹ collagenase IV for 10 min, fixed with 4% PFA at 4 °C for two hours, cleared with 0.1% Igepal CA-630 (Sigma-Aldrich) in PBST and dehydrated and rehydrated by immersing in a gradient of methanol diluted with PBST (25%, 50%, 75%, 100%, 75%, 50%, 25%) at 4 °C for 5 min each. After washing in PBST, tubules were incubated in blocking buffer (0.5% BSA in PBST), followed by incubation with antibodies in Immuno Shot Immunostaining, Mild (Cosmo Bio) at 4 °C for two days. After extensive washing with PBST, tubules were incubated with secondary antibodies in blocking buffer at room temperature for 90 min, washed with PBST and then mounted in Vectashield with DAPI (Vector Laboratories). Images were captured on a Leica SP5 confocal microscope and processed in Photoshop CC or later versions. Syncytium of US (A_s–A₁₆) were visually judged on the basis of continuous E-cadherin, GFRA1 or tdTomato staining. The following antibodies were used: anti-RFP (Abcam, ab124754, rabbit polyclonal, 1:500 dilution); anti-RFP (MBL, M208-3, mouse monoclonal 1G9 and 3G5, 1:200 dilution); anti-E-cadherin (R&D systems, AF748, goat polyclonal, 1:200 dilution); anti-GFRA1 (R&D systems, AF560, goat polyclonal, 1:200 dilution); and anti-KIT (Cell Signaling Technology, 3074, rabbit monoclonal D13A2, 1:200 dilution).

Section immunostaining

Testes were detunicated, fixed with 4% PFA at 4 °C overnight, incubated in a gradient of ethanol and xylen, embedded in paraffin and cut into 5-μm sections. After rehydration, antigen retrieval was performed using either citric acid or tris-based antigen retrieval solution (Vector Laboratories) for 5 min in a pressure cooker. Sections were blocked with 0.5% BSA in PBST and incubated with primary antibody at 4 °C overnight. After washing with PBST, sections were incubated with secondary antibodies at room temperature for 1 h and mounted in Vectashield with DAPI. For BrdU detection, slides were treated with 2 M HCl for 20 min, blocked with 0.5% BSA in PBST and incubated with rabbit anti-PLZF and rat anti-BrdU antibodies at 4 °C overnight, and signals were detected by Alexa 488-conjugated anti-rat IgG and Cy5-conjugated anti-rabbit IgG antibodies. For co-staining using rabbit anti-RFP antibodies and rabbit anti-PLZF antibodies, sections were antigen retrieved, blocked with 0.5% BSA in PBST, incubated with anti-RFP antibody, then with HRP-conjugated anti-rabbit secondary antibody as described above, and signals were detected with the TSA Plus Cyanine 3 system (Akoya Biosciences). After signal detection, the antibodies were stripped off by antigen retrieval, and sections were further stained with other antibodies. For triple staining using rabbit anti-RFP, rabbit anti-PLZF and rabbit anti-MYC antibodies, sections were stained with anti-RFP antibody using the TSA Plus Cyanine 3 system, and antibodies were stripped off with antigen retrieval. Then, those sections were stained with anti-MYC antibody with the TSA Plus Fluorescein system (Akoya Biosciences), followed by antigen retrieval to remove antibodies. Finally, the sections were further stained with anti-PLZF antibody and Cy5-conjugated anti-rabbit IgG. Slides were mounted in Vectashield with DAPI. For chromogenic staining for tdTomato, sections were incubated with anti-RFP antibody followed by HRP-conjugated antiboy. Signals were detected with a DAB substrate kit (Vector Laboratories). Sections were counterstained with haematoxylin, dehydrated with ethanol and xylene and then mounted in Clearmount (American MasterTech). To quantify PLZF⁺ cells, seminiferous tubules in stage VII–VIII were excluded from the analyses to prevent the inclusion of PLZF⁺ early DS cells. Images were captured on a fluorescent microscope and processed in Photoshop. The signal intensities of MYC and PLZF were quantified with ImageJ. Immunofluorescence data were captured using Leica Application Suite AF and immunohistochemistry data were captured using Leica LAS 4.2. The following antibodies were used: anti-RFP (Abcam, ab124754, rabbit polyclonal, 1:500 dilution for immunofluorescence without TSA or 1:3,000 dilution for immunofluorescence with TSA); anti-BrdU (Bio-Rad, MCA2483, mouse monoclonal Bu201, 1:500 dilution); anti-GFRA1 (R&D systems, AF560, goat polyclonal, 1:200 dilution); anti-PLZF (Santa Cruz, sc-22839, rabbit monoclonal H-300, 1:200 dilution for immunofluorescence without TSA or 1:5,000 dilution for immunofluorescence with TSA); anti-cleaved PARP (Cell Signaling Technology, 9548, mouse monoclonal 7C9, 1:500 dilution); anti-γH2AX (EMD Millipore, 05-636, mouse monoclonal JBW301, 1:2,000 dilution); and anti-MYC (Cell Signaling Technology, 13987, rabbit monoclonal D3N8F, 1:500 dilution).

TRAP assays

A two-step TRAP (telomeric repeat amplification protocol) procedure was performed as previously reported⁵¹. Extracted fractions from whole testis at three weeks or fluorescence-activated cell sorting (FACS)-sorted US were incubated with telomeric primers for a 30-min initial extension step at 30 °C in a PCR machine, followed by 5 min inactivation at 72 °C. Without purification, 1 μl of the extended reaction was PCR amplified (cycles of 30 s at 94 °C, followed by 30 s at 59 °C) in the presence of ³²P end-labelled telomeric primers that had been purified using a micro-spin G-25 column (GE Healthcare). PCR reactions were resolved by 9% polyacrylamide gel electrophoresis at room temperature, and the gel was exposed to a phosphor imager and scanned by a Typhoon scanner. The scanned image was quantified using the TotalLab Quant software. Representative gel images were presented among at least two repeats. Original uncropped images are provided in Supplementary Figs. 1 and 2.

FACS analysis

Testes were detunicated, lightly dissociated in PBS and incubated in PBS containing 1 mM EDTA, 1 mg ml⁻¹ collagenase I (Worthington) and DNase I (Worthington) at 32 °C for 8 min. Cells were centrifuged at 250g for 5 min and the supernatant was removed. After repeating the collagenase I treatment, testicular cells were further digested with TrypLE Express (Gibco) at 32 °C for 15 min. During enzymatic digestions, seminiferous tubules were mechanically fragmented with vigorous pipetting every 5 min. Cells were sequentially filtered with 70-μm and 40-μm strainers, resuspended in cold FACS buffer (2% FBS and 1 mM EDTA in PBS) and incubated with antibodies on ice for 30 min. After washing with PBS, cells were resuspended in cold FACS buffer containing DAPI, and analysed and sorted with a BD Aria II (BD Biosciences). Flow cytometry data were acquired using BD FACSDiva software v.8.0. Data were analysed with FlowJo v.9 software. The following antibodies were used; anti-α6 integrin with Pe/Cy7 (Biolegend, 313622, rat monoclonal GoH3, 1:150 dilution); anti-MCAM with APC (Biolegend, 134712, rat monoclonal ME-9F1, 1:200 dilution); and anti-KIT with BB515 (BD Biosciences, 564481, mouse monoclonal 2B8, 1:200 dilution). The gating strategy is provided in Supplementary Fig. 3.

Telomere FISH

For preparing cytospin slides of testicular cells, single-cell suspensions were prepared as described above and resuspended in PBS. Cells were then fixed with 4% PFA at room temperature for 10 min. After washing, cells were resuspended in PBS and cytospun at 250g for 5 min. Slides were stored in 70% ethanol at 4 °C. For telomere FISH combined with antibody staining against tdTomato, slides were hydrated in PBS, blocked with PBST containing 0.5% BSA and then incubated with anti-RFP antibody at 4 °C overnight. After washing with PBST, slides were incubated with HRP-conjugated anti-rabbit secondary antibody at room temperature for 30 min and washed with PBST. Signals were detected using the TSA Plus Cyanine 3 system. For telomere FISH, slides after antibody staining were washed with PBS, and dehydrated by immersing in 70%, 90% and 100% ethanol. After drying, slides were incubated in hybridization buffer (70% formamide, 10 mM Tris-HCl pH 7.4 and 0.5% blocking reagent (Roche)) containing 0.2 μM Alexa 488-conjugated telomere probes (PNA Bio) and 0.2 μM Cy5-conjugated centromere probes (PNA Bio) at 80 °C for 10 min and then at 4 °C overnight. Slides were washed with 70% formamide containing 10 mM Tris-HCl pH 7.4 for 15 min twice, then with PBS, and were then mounted in Vectashield with DAPI. Images were captured on a fluorescent microscope and processed in Photoshop. Telomere sum signals and centromere sum signals were determined using Telometer v.3.0.5. Anti-RFP antibody (Abcam, ab124754, rabbit polyclonal, 1:500 dilution) was used.

RNA in situ hybridization

At five days after labelling, testes were collected and tdTomato⁺ US were FACS-sorted, and cytospun at 200g for 5 min onto slides. Slides were fixed in 4% PFA for 30 min at room temperature and processed for single-molecule RNA FISH using the RNAscope 2.5 HD Reagent KIT—RED (Advanced Cell Diagnostics) and probes against mouse Tert or Trp53 (Advanced Cell Diagnostics) according to the manufacturer’s instructions.

qRT–PCR

For qRT–PCR, cells were directly sorted into Trizol LS (Thermo Fisher Scientific) by FACS. RNA was purified using the Direct-zol RNA Microprep kit (Zymo Research) and cDNA was synthesized using oligo-dT and the SuperScript IV First-Strand Synthesis system (Thermo Fisher Scientific). For qRT–PCR of Tert exon 2, Tert exon 6 and mouse Myc, TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) was used, along with Universal Probe Library Probes: no. 66 for Tert exon 2, no. 93 for Tert exon 6 and no. 77 for mouse Myc (Roche). For other qRT–PCR, the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) was used according to the product manual. PCR analysis was done with a 7900HT Fast Real-Time PCR System machine (ABI). Primer information is available in Supplementary Table 2.

RNA-seq

US-h cells were directly sorted into Trizol LS by FACS and RNA was purified using the Direct-zol RNA Microprep kit. Genomic DNA was digested with on-column DNase treatment. RNA quality was checked by Bioanalyzer 2100 (Agilent). RNA-seq libraries were constructed using the SMARTer Stranded Total RNA-seq Kit v2—Pico Input Mammalian (Clontech), starting from 5 ng total RNA. cDNA was synthesized and amplified according to the manual. After the rRNA removal step, cDNA was amplified with 13 cycles of PCR reactions. The quality of purified cDNA libraries was confirmed by Bioanalyzer 2100. Libraries were sequenced on the Illumina NextSeq platform, generating about 16 million–24 million 75-bp paired-end reads per library. Four biological replicates per sample were analysed. Raw reads were trimmed by TrimGalore v.0.4.0 (Babraham Bioinformatics), mapped to mm10 by TopHat v.2.0.13 and analysed by DESeq2.

ATAC-seq

ATAC-seq libraries were made as described previously⁵² using the Omni-ATAC protocol. Adjustments to the protocol were made to reflect two main features of the cell types profiled in this work. First, the amount of Tn5 transposase added to each reaction was modulated to maintain proportionality with the number of cells assayed. For example, a normal reaction uses 50,000 cells and 2.5 μl of Tn5 transposase in a 50-μl reaction. In the case of rarer SSCs, only 5,000 cells could be obtained so only 0.25 μl of Tn5 transposase was used in a 50-μl reaction. The difference in volume was adjusted using water. Second, the ploidy of each cell type was taken into account and the amount of Tn5 was adjusted on the basis of ploidy as well. For example, round spermatid cells are haploid, so the transposition of 50,000 cells would require 1.25 μl of Tn5 transposase in a 50-μl reaction. Similarly, spermatocytes are 4N meiotic cells so the amount of Tn5 transposase was increased proportionately and the amount of water in the reaction was reduced. In all cases, regardless of cell number or ploidy, the reaction volume of the transposition reaction was kept constant at 50 μl. All ATAC-seq reactions were performed using homemade Tn5 transposase and Tagment DNA buffer⁵³. Downstream amplification and purification of libraries was performed as described previously^34,52,54.

Preprocessing of ATAC-seq data was completed using the PEPATAC pipeline (https://pepatac.databio.org/). The mm10 genome build (https://github.com/databio/refgenie) was used for alignment. In brief, all fastq files were first trimmed to remove the Illumina Nextera adapter sequence using Skewer with “-f sanger –t 20 –m pe –x” options. FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to validate proper trimming and check overall sequence data quality. Bowtie2 was then used for pre-alignments to remove reads that would map to chrM (revised Cambridge Reference Sequence), alpha satellite repeats, Alu repeats, ribosomal DNA repeats and other repeat regions with “-k 1 -D 20 -R 3 -N 1 -L 20 -i S,1,0.50 -X 2000 –rg-id” options. Bowtie2 was then used to align to the mm10 reference genome using “–very-sensitive -X 2000 –rg-id” options. SAMtools was used to sort and isolate uniquely mapped reads using “-f 2 -q 10 -b -@ 20” options. Picard (http://broadinstitute.github.io/picard/) was used to remove duplicates. Then the bam files were merged by conditions, and MACS2 was used to call peaks with parameter “-q 0.05 –nomodel –shift 0”. The narrow peaks were then filtered by the ENCODE 7 hg19 blacklist, as well as peaks that extend beyond the ends of chromosomes. Bedtools was used to retrieve the reads of the called peaks for each sample with the multicov module. All of the samples have a similar sequencing depth, mitochondrial rate and duplication rate. The spermatocyte and the round spermatid samples have a similar sequencing depth compared with all other samples, but a slightly higher mitochondrial rate and lower duplication rate, so have more final reads after initial processing and filtering. To make all the samples comparable for the statistical analysis, we used final reads as the normalization factor. The R package DESeq2 was used for statistical analysis to identify significant peaks between different conditions. The differential peaks were called between US-h CreER/+ and US-h CreER/flox samples. Peaks with FDR < 0.01 and a fold change larger than 2 or smaller than −2 were considered significant. The R package ChIPseeker was used for peak annotation. The R package ngsplot was used for visualizing the cumulated peak signal.

Statistics and reproducibility

No statistical methods were used to predetermine sample sizes. All of the experiments were replicated more than twice and were statistically analysed and presented in the paper. When comparing two groups, P values were determined by two-sided unpaired t-test. When comparing more than two groups, P values were determined by one-way ANOVA with Tukey’s test. Values are presented as mean ± s.e.m. The mice were randomly assigned to each experimental or control group. Statistics and plots were generated by ggplot2 in R and GraphPad Prism 8.