MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells

The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.


Isolation of sperm
Mouse sperm were isolated from the cauda epididymis of more than 10-week-old wild type (C57Bl/6) and the MORC3 mutant mice by dissecting tissue in PBS. Sperm were allowed to swim up for 30 min in 37°C with 5% CO2. To avoid the contamination of somatic cells, the upper fraction was collected for bisulfite sequence analysis.

Northern blot analysis
Total RNA samples were prepared from testes using ISOGEN (Nippon Gene CO., LTD.,

Detection of small RNAs
The detection of small RNAs from E16.5 testes of the Morc3 homozygous mutant and wild type (control) mice using anti-MIWI2 and MILI antibodies were performed as described previously 1 .
ChIP-qPCR assay The E16.5 testes were removed and treated with 1 mg/mL collagenase type II and 0. NaCl, 1 mM EDTA, and 0.5% SDS) and incubated overnight at 65°C. After incubation, the samples were treated with 30 μg/mL RNase A for 1 h at 37°C, and 100 μg/mL proteinase K overnight at 56°C. DNA was extracted with phenol/chloroform and precipitated with ethanol and Dr. GenTLE (Takara Bio, Shiga, Japan) as a carrier.
Precipitated DNA was resuspended in 50 μL water and analyzed by qPCR using the CFX384 Real-Time PCR system (BIO-RAD) with THUNDERBIRD SYBR qPCR MIX (TOYOBO, Osaka, Japan) and specific primers mentioned below.

Sperm morphology and motility
Sperm morphology was observed and sperm motility was analyzed as described  (d) Spermatozoa from the cauda epididymis.
(e) Percentage of motile spermatozoa. No significant differences were found (by f-test).
(f) Velocity parameters of spermatozoa. There were no significant differences in all the parameters (by f-test). N=3. VAP = average path velocity, VSL = straight line velocity, VCL = curvilinear velocity. Error bars denote standard deviation (SD).
(g) The results of the mating test using wild type female mice coupled with MORC3-cKO or wild type adult male mice. The number of plug and delivery in using each MORC3-cKO adult male mouse is described. Litter size is the number of pups in each delivery.
Supplementary Figure S3 Supplementary figures of uncropped northern blots and co-immunostaining of the testes of the E17.5 MORC3-heterozygous and the mutant mice.
(d) Full-length images of Figure 5a.
(e) Length distribution of MIWI2-and MILI-associated 25-31 nt small RNAs derived from each strand (sense or antisense) in E16.5 wild type and MORC3-cKO testes.
(f) Nucleotide distribution of the 1 st or 10 th nucleotide of piRNAs from each strand in MIWI2-and MILI-associated piRNA libraries from wild type and MORC3-cKO testes.
(g) Co-immunostaining of the testes of the E16.5 MORC3-heterozygous and the mutant mice with anti-MIWI2 antibody (red), anti-MORC3 antibody (green), and DAPI (blue) for DNA are shown. The nucleus is surrounded by a white line. Scale bar, 20 μm.
Supplementary Figure S5 Expression of piRNA precursors from representative embryonic piRNA clusters (a and b) Structure of the piRNA clusters (Chr 8 and Chr 7 (2)) and positions of individual primer set (upper). The region of IAP sequence in Chr 7 (2) cluster is described with red bars. Quantitative RT-PCR for the expression analysis of piRNA precursors transcribed from embryonic piRNA clusters using E16.5 wild type and MORC3-cKO embryonic testes (bottom). Data is normalized to -actin and is shown as means and SD (Error bar) from more than triplicate PCR reactions. Significant differences ( * p < 0.05 by the t test) between wild type and MORC3-cKO data using primer sets for the Chr 8-1 and 2 positions are shown (a).
The embryonic testis lysates were chromatin-immuno-precipitated with control IgG and anti-MORC3 antibody. N=3. The promoter region of Chr 7 (1) piRNA cluster was analyzed. Error bars denote standard deviation (SD). The asterisk shows statistically significant differences (p<0.01 by t-test).
Supplementary Figure S6 A gene-targeting construct for Morc3 Exons are shown as filled boxes and loxP and FRT sites as empty arrowheads and black arrows. The resultant allele lacks exon 2 (containing a part of Histidine kinase/HSP90-like ATPase superfamily domain).
Supplementary Item S1 The DNA sequence of the targeting vector (pDTMorc3neo) The sequences of the vector (pDT), loxP, exon 2, and FRT are shown in red, blue, green, and pink, respectively.