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. 2022 Sep 15;32(9):1655-1668.
doi: 10.1101/gr.276578.122. Online ahead of print.

Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction

Kimberly N Griffin #  1 , Benjamin William Walters #  1 , Haixin Li #  1 , Huafeng Wang  2 , Giulia Biancon  3   4 , Toma Tebaldi  3   4   5 , Carolyn B Kaya  1 , Jean Kanyo  6 , TuKiet T Lam  6   7 , Andy L Cox  1 , Stephanie Halene  3   4   8   9   10 , Jean-Ju Chung  2   11 , Bluma J Lesch  1   4   11
Affiliations

Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction

Kimberly N Griffin et al. Genome Res. .

Abstract

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

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Figures

Figure 1.
Figure 1.
Nucleus-specific protein function for AGO2 in male germ cells. (A) Western blot for AGO2 protein in whole-cell lysate (WCL), cytoplasmic fractions, or nuclear fractions of meiotic (pachytene spermatocyte, SYCP3+) and postmeiotic (round spermatid, ACRV1+) male germ cells. Tubulin and histone H3 are found in cytoplasmic and nuclear fractions, respectively. Round spermatids have not yet begun histone-to-protamine exchange and therefore have similar levels of histone H3 compared with meiotic cells. (B) Immunoprecipitation of AGO2 for IP-MS in cytoplasmic and nuclear germ cell fractions validated by western blotting with anti-AGO2 antibody. One percent of total lysate was used as input. IP with mouse IgG is a negative control. (C) Common and compartment-specific AGO2 protein interactors in the nucleus and cytoplasm of wild-type postmeiotic cells. Box shows detected miRNA/siRNA pathway components along with the number of unique peptides identified in each compartment. (D) Statistically enriched Gene Ontology (GO) terms for AGO2 protein interactors in the nuclear compartment.
Figure 2.
Figure 2.
Identification of AGO2 mRNA targets in nuclei of male germ cells by eCLIP. (A) Immunoblot analysis of AGO2 immunoprecipitation. (B) Size selection of cDNA (orange and purple bars) following AGO2 IP, linker (147 nt) ligation, and PCR amplification. (C) Overlap of AGO2 targets from two eCLIP replicates from meiotic (left) and postmeiotic (center) cells, and transcripts identified in both replicates of both cell types (right). (D) GO analysis of meiotic consensus targets. (E) Genomic distribution of all AGO2 eCLIP binding regions. (F) Distribution of categories of bound transcript in both cell types (common), meiotic cells only, or postmeiotic cells only. Shuffled control distributions were generated by randomly shuffling a representative CLIP peak set across all annotated transcripts. (TEC) To be experimentally confirmed. (G) Overlap between cytoplasmic and nuclear AGO2 eCLIP targets.
Figure 3.
Figure 3.
AGO2 interacts with chromatin and binds the corresponding nuclear transcripts in a developmentally dynamic manner. (A) Genome browser tracks (mm10) showing representative AGO2 ChIP peaks in two replicates from each cell type and from Ago2 cKO mixed meiotic and postmeiotic cells. (B) Enrichment of AGO2 on chromatin in premeiotic, meiotic, and postmeiotic male germ cells. (Top) Metagenes showing average ChIP signal at AGO2 peaks, with reference point set to the peak center. “Shuffle” shows ChIP signal when the same number of length-matched peaks were randomized across the genome. (Bottom) Heatmaps showing ChIP signal enrichment at each peak. (C) Distribution of AGO2 peaks in introns, exons, and intergenic regions. (D) Metagene of H3K9me3 ChIP signal at AGO2 peaks in meiotic cells. (E) Metagene of ATAC-seq signal at AGO2 peaks in meiotic cells. (F) Top motifs enriched in AGO2 ChIP peaks in meiotic cells. (G) AGO2 ChIP enrichment signal at eCLIP peaks in meiotic cells relative to shuffled eCLIP peaks. (H) Genome browser tracks (mm10) showing representative genes associated with both ChIP signal and eCLIP peaks (pink bars). (I) Fraction of meiotic CLIP target genes for which the gene body overlaps a meiotic ChIP peak. “Extended ChIP peaks” represent the same analysis performed with ChIP peaks extended by 10 kb in both directions. (***) P < 10−15, hypergeometric test. (J) Overlap between genes associated with meiotic ChIP peaks and genes containing eCLIP peaks in both meiotic and postmeiotic cells; P-value, hypergeometric test. (K) Motifs enriched in AGO2 ChIP peaks associated with genes that also contain eCLIP peaks. Only motifs matching a transcription factor expressed in meiotic cells are shown.
Figure 4.
Figure 4.
AGO2 regulates protein and transcript expression of male reproduction-related genes. (A) Volcano plots showing protein level changes in meiotic and postmeiotic male germ cells from Ago2 cKO animals. Statistically significant differentially expressed proteins (P ≤ 0.05) are shown by a dashed line and are highlighted in red. Differentially expressed proteins belonging to the GO category “spermatogenesis” (GO:0007283) are highlighted in blue. (B) Enriched GO terms associated with down-regulated proteins in Ago2 cKO meiotic and postmeiotic cells. GO terms related to male fertility are highlighted in blue. (C) Distribution of transcripts encoding differentially expressed proteins bound by AGO2 in nucleus, cytoplasm, or both. (D) Selected enriched GO terms for the set of genes with reduced protein expression in the Ago2 cKO and transcripts bound by AGO2 in meiotic or postmeiotic nuclei. (E) Changes in protein expression of meiotic AGO2 CLIP targets between meiotic and postmeiotic stages. Statistically significantly up-regulated proteins are in red. Cartoon plot illustrates that proteins in the upper right quadrant are up-regulated at postmeiotic stages, and up-regulation is attenuated in Ago2 cKO for proteins below the diagonal. (F) Volcano plots showing global transcriptional changes in Ago2 cKO male germ cells at meiotic (left) and postmeiotic (right) stages. Transcripts with log2 fold change ≤1.5 or ≥1.5 and adjusted P-value ≤ 0.05 are demarcated by dashed lines and highlighted in blue (up-regulated) or red (down-regulated). (G–I) Gene set enrichment analysis (GSEA) for differentially expressed transcripts relative to the set of proteins differentially expressed in meiotic cells (G), the set of proteins differentially expressed in postmeiotic cells (H), or genes for which AGO2 interacts with both chromatin and nuclear transcripts (I) (as shown in Fig. 3J).
Figure 5.
Figure 5.
Loss of AGO2 impairs expression of proteins required for normal sperm production and results in oligospermia and abnormal sperm head morphology. (A) Western blot validation of spermiogenesis protein down-regulation. Bar chart shows levels of each protein relative to histone H3, quantified using densitometric analysis for three biological replicates per group. Error bars, SEM; (ns) not statistically significant, (*) P ≤ 0.05, Welch's t-test. (B, left) Percentage of Ago2 cKO spermatozoa with abnormal heads, representing n > 400 spermatozoa from each of three to four biological replicates. Points represent mean of each replicate, and bars show median and interquartile range of all replicates. (***) P < 0.001, Welch's t-test. (Right) Brightfield images of spermatozoa stained with Coomassie blue, showing representative cells collected from control and Ago2 cKO cauda epididymides. Both normal and abnormal examples of Ago2 cKO sperm are shown. (C) Representative spermatozoa stained with fluorophore-conjugated lectin-peanut agglutinin to visualize the acrosome (red) and DAPI to show DNA (blue). Both normal and abnormal examples of Ago2 cKO sperm are shown. (D) Epididymal sperm count in Ago2 cKO males and littermate controls. (E,F) Fertility in Ago2 cKO males and littermate controls, assessed as number of pups at birth (E) or at weaning (F). Error bars, SD; (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, Welch's t-test.
Figure 6.
Figure 6.
Model for RISC-independent nuclear AGO2 function in germ cells during meiotic prophase. AGO2 interacts with chromatin and binds nearby nascent transcripts. At genes important for spermatogenic development, AGO2 maintains its interaction with the nuclear transcript and helps to stabilize and shuttle these transcripts to the cytoplasm, eventually resulting in increased protein expression and promoting sperm production. Diagrams created with BioRender (https://biorender.com).
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References

    1. Alavattam KG, Maezawa S, Sakashita A, Khoury H, Barski A, Kaplan N, Namekawa SH. 2019. Attenuated chromatin compartmentalization in meiosis and its maturation in sperm development. Nat Struct Mol Biol 26: 175–184. 10.1038/s41594-019-0189-y - DOI - PMC - PubMed
    1. Alisch RS, Jin P, Epstein M, Caspary T, Warren ST. 2007. Argonaute2 is essential for mammalian gastrulation and proper mesoderm formation. PLoS Genet 3: e227. 10.1371/journal.pgen.0030227 - DOI - PMC - PubMed
    1. Ameyar-Zazoua M, Rachez C, Souidi M, Robin P, Fritsch L, Young R, Morozova N, Fenouil R, Descostes N, Andrau J-C, et al. 2012. Argonaute proteins couple chromatin silencing to alternative splicing. Nat Struct Mol Biol 19: 998–1004. 10.1038/nsmb.2373 - DOI - PubMed
    1. Baker CL, Walker M, Kajita S, Petkov PM, Paigen K. 2014. PRDM9 binding organizes hotspot nucleosomes and limits Holliday junction migration. Genome Res 24: 724–732. 10.1101/gr.170167.113 - DOI - PMC - PubMed
    1. Bellvé AR. 1993. Purification, culture, and fractionation of spermatogenic cells. Methods Enzymol 225: 84–113. 10.1016/0076-6879(93)25009-Q - DOI - PubMed

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