Mouse strains and timed matings
Mice carrying the MommeD41 mutation were produced on the FVB/NJ background homozygous for the Line3 GFP transgene, as described previously for other MommeD mutations [25, 36,37,38,39]. Maintenance of the MommeD41 allele was carried out on the Line3 background. For timed matings heterozygous males were set up with heterozygous females and the detection of a vaginal plug was counted as 0.5 dpc. Genotyping was carried out using genomic DNA extracted from embryonic tissue.
One drop of tail blood of 3 week old mice was collected FACSFlow Sheath Fluid (BD Biosciences) and flow cytometry analysis was carried out and analyzed on a Guava easyCyte HT (Merck/Millipore, Darmstadt, Germany) using Guava InCyte software, respectively. Red blood cell green fluorescence (525 nm) was recorded using a GFP-positive gate that was set to exclude 99% of WT erythrocytes, as described previously .
Mapping of the MommeD41 mutation and linkage analysis
The Line3C  was used for mapping and linkage analysis. MommeD41 heterozygous mice were backcrossed to Line3C twice and phenotyped using flow cytometry. DNA collected from tail tissue of WT (n = 12) and MommeD41 heterozygotes (n = 11) was used to perform linkage analysis using the Illumina GoldenGate genotyping assay exactly as described previously [25, 39]. Only samples with a call rate > 95 were accepted, and a linked interval was identified based on LOD score. A LOD score of > 5 was found for chromosome 16 (Additional file 1: Table S1).
Whole exome deep sequencing
Genomic DNA isolated from tail of one WT and one MommeD41 heterozygote were used for exome capture using the RocheNimbleGen reagents (SeqCap EZ Mouse exome, version beta 2, 110603_MM9_exome_rebal_2EZ_HX1, Madison, WI, USA) according to the Illumina optimized RocheNimbleGen SeqCap User’s guide. Libraries were sequencing using an Illumina GAIIx platform and reads were aligned to the mouse reference genome (build 37, mm9) as described previously . Varscan output was used to identify likely heterozygous mutations and the Morc3MD41 mutation was validated in additional heterozygous mice using Sanger sequencing.
Mouse embryonic stem cell (mESC) derivation and culture
mESCs were generated from WT and Morc3MD41/MD41 preimplantation embryos and were cultured on 0.1% gelatin without feeders in mESC medium [Knockout DMEM (10829-018; Gibco), 10% FBS (DE14-801F; BioWhittaker), NEAA (11140; Gibco), l-Glutamine (25030-123; Gibco), Sodium Pyruvate (11360; Gibco), 2-Mercaptoethanol (31350; Gibco) and Leukemia Inhibitory Factor (ESG1107; Millipore)] plus MEK inhibitor PD0325901 (1 mM) and GSK3 inhibitor CHIR99021 (3 mM, Axon Medchem). Cell cultures tested negative for mycoplasma on a regular basis.
Cell or tissues were incubated overnight in DNA lysis buffer (50 mM TrisHCl pH 8.0, 5 mM EDTA, 2%SDS) supplemented with Proteinase K (Invitrogen AM2548) at 55 ℃. After RNA digestion with RNAseA (Thermo scientific EN0531) 30 min at 37 ℃, genomic DNA was precipitated using 5 M NaCl, 1 volume isopropanol and 70% Ethanol. DNA was eluted in water and used subsequently as template for genotyping using DreamTaq Polymerase (Thermo scientific EP0705) and the following primers: TGTCCAGCCCTGTATGTTGG (forward) and ACATAGTGAATCCCAGCAGAGC (reverse). PCR products were then sequenced with Sanger sequencing.
RNA isolation and RT-qPCR analysis
Total RNA was isolated with QIAzol (5346994; Qiagen). About 1 mg of total RNA was used for reverse transcription using RevertAid H Minus First Strand cDNA Synthesis Kit (K1632; Thermo). RT-qPCR was performed in triplicate on a C1000TM Thermal cycler (Bio-Rad) with SYBR Green (170-8887; Bio-Rad). Expression data was normalized to b-actin. Primer sequences are provided in Additional file 6: Table S5.
Alkaline phosphatase staining
mESCs were controlled for pluripotency using the StemAb Alkaline phosphatase staining Kit II (00-0055; Stemgent). Briefly mESc cells cultured on 0.1% gelatin were washed with PBS twice and fixed for 5 min with 0.5 mL fixative solution at room temperature. Fixative solution was rinsed with PBS before incubation of the cells with staining solution for 15 min at room temperature. Staining solution was then rinsed with PBS and cells were observed for purple coloration under binocular.
Cells were lysed in Cell Lysis buffer (20 mM triethanolamine (T1377; Sigma), 0.14 M NaCl, 0.1% Sodium deoxycholate (D6750; Sigma), 0.1% SDS, 0.1% Triton X-100) with Protease Inhibitor Cocktail (27368400; Roche), Phosphatase Inhibitor Cocktail (04906837001; Roche) and 10 mM N-Ethylmaleimide (NEM) on ice. BCA kit (23225; Thermo) was used to measure protein concentration. Equal amounts of total cell extracts were loaded on a NuPAGE gel (4–12%, NP0321; Thermo), and transferred to a Nitrocellulose Blotting Membrane (10600016; Life Sciences). The following primary antibodies were used: Morc3 (Rockland; 100-401-N96S 1:1000) and Tubulin (T6199; Sigma, 1:5000). Donkey anti-Rabbit 800CW (926-32213; Li-Cor, 1:5000), Goat anti-Rabbit 800CW (926-32211; Westburg, 1:5000), Donkey anti-mouse 680RD (926-68072; Li-Cor, 1:5000) were used as secondary antibodies. Membranes were analyzed on Odyssey (Westburg).
Total RNA was isolated as described above and standard RNA-seq was performed at BGI. Sample preparation after rRNA depletion was performed using NEB Next Ultra Directional RNA Library Prep Kit for Illumina (E7420S/L; NEB) according to the protocol. Libraries were sequenced with 100 bp pair-end (PE) reads on a HiSeq 2500.
Cells were cross linked with 1% formaldehyde M134-200ML; VWR) for 8 min at room temperature and glycine (125 mM; G8790-1 KG; Sigma) was used to quench cross-linking for 5 min. Cells were washed twice with cold PBS and lysed in ChIP Buffer 1 (10 mM Tris HCl (pH 8.0), 0,25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, Protease Inhibitor Cocktail (05056489,001; Roche) and 1 mM PMSF) for 15 min on a rotator at 4 °C. After centrifugation 5 min at 1400g at 4 °C, cells were resuspended in ChIP Buffer 2 (10 mM Tris–HCl (pH 8.0), 200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, Protease Inhibitor Cocktail (05056489001; Roche) and 1 mM PMSF). After centrifugation 5 min at 1400g at 4 °C, cells were resuspended in ChIP Buffer 3 (10 mM Tris–HCl (pH 8.0), 10 mM EDTA, 0.5 mM EGTA, 0,1% SDS, Protease Inhibitor Cocktail (05056489001; Roche) and 1 mM PMSF and sonicated with Covaris S2 (Duty cycle: 10%, Intensity:5, Cycle/Burst:200 to obtain fragment between 200 and 600 bp. Sheared chromatin was centrifuged at 12,000g for 10 min at 4 °C to discard the pellets. The supernatant was then diluted 10 times with ChIP dilution buffer (16.7 mM Tris–HCl (pH8), 0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, 167 mM NaCl) and incubated overnight with 5 µg Morc3 antibody (Rockland; 100-401-N96S) or Rabbit IgG (Abcam ab37415). An input fraction for each sample was retained for downstream analysis. Samples were then incubated with Dynabeads Protein A (Invitrogen 10001D) for at least 4 h rotating at 4 °C. After immunoprecipitation beads were washed with low-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), 150 mM NaCl), high-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), 500 mM NaCl), LiCl washing buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–HCl (pH 8.1)) and TE buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA). IP DNA and Input DNA samples were extracted with phenol–chloroform–isoamyl alcohol (15593049; Fisher Scientific). Quantitative PCR was performed using the primer sequences provided in Additional file 6: Table S5.
Trim28 ChIP was performed as described above. Chromatin was immunoprecipitated with 5µL of Trim28 antibody (Abcam ab22553). DNA was subsequently used for quantitative PCR using the primer sequences provided in Additional file 6: Table S5.
Quantitative H3K9me3 ChIP-seq
Cells were cross linked with 1% formaldehyde (M134-200ML; VWR) for 8 min at room temperature and glycine (125 mM; G8790-1 KG; Sigma) was used to quench cross-linking for 5 min. Cells were washed twice with cold PBS and lysed in NP Buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, Protease Inhibitor Cocktail (05056489001; Roche)). Nuclei were sheared by sonication (Covaris). For each H3K9me3 ChIP-seq experiment, 25 µg of sample chromatin was mixed with 50 ng spike-in Drosophila chromatin (53,083; Active Motif). Mixture of experimental chromatin and spike-in chromatin was then incubated with a mix containing 4 µg of H3K9me3 antibody (abcam ab8898) and 2 µg of spike-in antibody (104,597; Active Motif) at 4 °C overnight. The next day, Protein A Sepharose beads (175280-01; GE Health Care) were first blocked with 1 mg/mL BSA (10484; Affymetrix) and then added to each chromatin-antibody mix and incubated at 4 °C for at least 3 h. After immunoprecipitation, beads were washed with low-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), 150 mM NaCl), high-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), 500 mM NaCl), LiCl washing buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–HCl (pH 8.1)) and TE buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA). DNA was extracted using phenol–chloroform–isoamylol (15,593–049; Life Technologies). Samples were sequenced at Macrogen on HiseqX with 150 bp paired-end (PE) reads.
Assay for transposase accessible chromatin
ATAC-seq libraries were prepared as previously described . In brief, 50,000 mouse embryonic stem cells were resuspended in 50 µL transposition mix (Nextera) and incubated for 30 min at 37 °C. Libraries were amplified by PCR with barcoded Nextera primers and sequenced on BGISEQ-500 with 150 bp pair-end (PE) reads.
V6.5 mESC culture
WT V6.5 mESCs and Morc3 null mutants derived from V6.5 mESCs were cultured on mouse embryonic fibroblasts (feeder cells). Cell culture media composed of KnockOut DMEM (10829-018, Invitrogen), 15% Hyclone fetal bovine serum (SH3007003, GE), 1X Penicillin–Streptomycin–Glutamine (Gibco, 10378016), 50 µg/mL primocin (ant-pm-2, Invivogen), 1X MEM non-essential amino acids (Gibco, 11140050), 1000 U/mL ESGRO mouse LIF (Millipore, ESG1106) and 55 µM beta-Mercaptoethanol (21985-023, Invitrogen) was used. Before RNAseq or ChIPseq, cells were transitioned off feeders and onto gelatin for two passages.
CRISPR/Cas9 genome editing
To generate a Morc3 mutant line, a guide RNA (gRNA) targeting exon 2 of Morc3 gene was designed and cloned into PX330 vector (http://www.addgene.org/42230/) (https://pubmed.ncbi.nlm.nih.gov/23287718/). 50,000 cells were plated in one well of a 6-well plate 24 h prior to transfection. ~ 3 µg of gRNA plasmid and ~ 1 µg of pMax-GFP were cotransfected using Lipofectamine 2000. Two days after transfection, GFP + cells were sorted into a 96-well plate and allowed to grow into colonies over several days. Candidate lines were screened with the Surveyor Assay. To determine the precise mutations, genomic DNA was extracted from about 1 million cells using Quick-DNA Microprep Kit (ZYMO RESEARCH, D3021). 1µL of the genomic DNA was used as PCR template for genotyping using Phusion High-Fidelity DNA Polymerase (NEW ENGLAND BioLabs, M0530L). gRNAs and genotyping primers are listed in Additional file 6: Table S5.
Cells were lysed in NuPAGE LDS Sample Buffer with NuPAGE reducing agent and were heated at 70 °C for 10–20 min. Equal amounts of cell extracts from WT and mutants were loaded on a 4–12% NuPAGE Bis–Tris gel. The gel was ran in 1X MOPS SDS running buffer at 200 V for 50–60 min. It was then transferred to a PVDF membrane at 30 V for 60 min in 1X NuPAGE transfer buffer. NuPAGE antioxidant was added to the running and transfer buffer. An ice block was placed in the gel box to keep the transfer buffer cool. The membrane was cut accordingly and blocked with 10% goat serum for 1 h at room temperature with gentle agitation. Then, the membrane was incubated in 1:3000 dilution of Morc3 antibody in 10% goat serum for 1 h at room temp with gentle agitation. After primary antibody incubation, the membranes were washed with 1xTBST for 5 min. Total 4 washes were performed and then the membrane was incubated in 1:3000 dilution of goat–anti-rabbit antibody in 10% goat serum for 1 h at room temp with gentle agitation. The membrane was then washed 4 times with 1xTBST for 5 min each, stained with ECL (1:1 solutions A and B) for 2 min, and imaged with the "camera station" instead of film.
RNA from three independently passaged Morc3–/– lines was extracted using RNeasy Mini Kit (74104, Qiagen) and libraries were prepared using TruSeq Stranded mRNA Library Prep kit (20020595, Illumina). Libraries were then sequenced with 100 bp pair-end (PE) reads on a NovaSeq 6000.
MORC3 and H3K9me3 ChIPseq
V6.5 mouse embryonic stem cells (mESCs) were cultured on mouse embryonic fibroblasts (MEFs) with fetal bovine serum (FBS) and leukemia inhibitory factor. To fix cells for ChIP, mESCs were grown for up to two passages on gelatin and without MEFs, then grown to ~ 80% confluency. Cells were harvested with 0.25% Tryspin, quenched with FBS containing media, washed with PBS and counted. Fixation was performed with 1.5% formaldehyde in PBS for 14 min with nutation then quenched with 120 mM glycine for 5 min. Fixed cells were pelleted and washed with PBS twice, counted and 8 million cell aliquots were flash frozen. Eight million cells per replicate were thawed on ice and resuspended in 1 mL 10 mM Tris pH 8.0, 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, and Roche Complete EDTA-free Mini Protease inhibitor cocktail and then incubated with rotation for 15 min at room temperature. Nuclei were pelleted by centrifugation at 1500×g for 5 min at 4 °C. The nuclei were resuspended in 10 mM Tris pH 8.0, 200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 1 mM PMSF and protease inhibitor cocktail, incubated for 10 min at room temperature with rotation, and then centrifuged again. Nuclei were then resuspended in 10 mM Tris pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 1 mM PMSF and protease inhibitor cocktail, and disrupted by sonication at high intensity. Two replicates for MORC3 ChIP were sonicated using Bioruptor with the following parameters: 30 s on/30 s off for 20 min (10 min actual sonication and 10 min dormant). Ice was replaced every 5 min. Another two replicates for MORC3 ChIP and all replicates for H3K9me3 ChIP were sonicated using the following parameters: time: 430 s, Duty cycle: 10%, Intensity: 5, Cycle/Burst: 200. For analysis, peaks common between all replicates were used. Sonicated lysate was cleared by centrifugation at 16,000×g for 10 min at 4 °C, and the supernatant was used for ChIP. Samples were diluted with an equal volume of 10 mM Tris pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 1 mM PMSF and protease inhibitor cocktail. The samples were then precleared with 30 μL of protein A magnetic Dynabeads (Thermo Fisher Scientific), which had been washed with 16.7 mM Tris pH 8.0, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, and 167 mM NaCl before use, followed by incubation for 2 h at 4 °C. The beads were then collected on a magnet, and the supernatant was retained. Once 10% of the sample was saved for input, the remaining sample was treated with 5 μg of requisite target antibody (MORC3: anti-MORC3 antibody generated in collaboration with Rockland Immunochemicals; H3K9me3 abcam 8898). Samples were incubated overnight at 4 °C with rotation. The next day, 100 μL of protein A beads, which had been washed with 16.7 mM Tris pH 8.0, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, and 167 mM NaCl before use, were added to each sample, followed by incubation for another 2 h. The beads were washed twice for 4 min each time with rotation with 50 mM Hepes pH 7.9, 1% Triton X-100, 0.1% deoxycholate, 1 mM EDTA, and 140 mM NaCl; washed twice for 4 min each time under rotation with 50 mM Hepes pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 1 mM EDTA, and 500 mM NaCl; and then washed twice for 4 min each time under rotation with 500 μL of 10 mM Tris pH 8.0 and 1 mM EDTA. The purified DNA was eluted by incubation with elution buffer (100 μL of 50 mM Tris pH 8.0, 1 mM EDTA, and 1% SDS) at 65 °C for 10 min. Eluent was collected on a magnetic rack, and the beads were resuspended with 150 μL of elution buffer and then incubated at 65 °C for 10 min. The two eluents were pooled and de-cross-linked by incubation at 65 °C overnight, as were the inputs from day 1 after being thawed. The samples were brought to room temperature and then warmed to 37 °C and incubated with 10 μg RNase A (Qiagen). The samples were then treated with 15 μg of proteinase K and incubated for 2 h at 56 °C. Finally, after cooling, the samples were purified with Qiagen MinElute columns. Purified DNA was quantified with Qubit High-Sensitivity reagent (Thermo Fisher Scientific), and libraries were generated with the Ovation Ultralow Library System Kit (Nugen) using 10 ng of input DNA. MORC3 ChIP-seq libraries were sequenced with 50 bp single-end reads and H3K9me3 ChIP-seq libraries were sequenced with 100 bp single-end reads.