(89) Understanding MT2 Retrotransposon Activation in Preimplantation Development by CRISPRi and Multi-omic Analyses

Zhiyuan Chen^1,2, Jian Yang^1,2, Lauryn Cook^1,2,

1. Reproductive Sciences Center, Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA;

2. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA;

Abstract Text:

Accumulating evidence indicates that retrotransposons, especially long terminal repeats (LTRs), can act as cis-regulatory elements, such as promoters and enhancers, to regulate host gene expression. After fertilization, the mammalian zygote activates its own genome in a process called zygotic genome activation (ZGA). Hundreds of retrotransposon subfamilies – which are repetitive elements that constitute ~40% of mammalian genomes – become activated as well. Whereas retrotransposon expression is strongly repressed in somatic tissues, this wave of retrotransposon activation is believed critical for ZGA and embryogenesis with its disruption leading to embryonic arrest. Thus, understanding the functions of retrotransposon activation during ZGA has important implications for both basic biology and reproductive medicine, but how specific retrotransposon subfamilies regulate ZGA, and preimplantation development is not known.

Genetical manipulation of retrotransposon is technically challenging because each subfamily has hundreds to thousands of copies interspersed throughout the genome. Indeed, to date only a single LTR-derived promoter (i.e., the MT2B2 for Cdk2ap1 gene) has been characterized in mouse preimplantation embryos. Here, we applied CRISPR interference (CRISPRi) to perturb the LTR MT2_Mm, a well-known ZGA and totipotency marker that exists in ∼2,667 insertions throughout the mouse genome. CRISPRi robustly perturbed 2,485 (∼93%) MT2_Mm insertions and 1,090 (∼55%) insertions of the closely related MT2C_Mm in 2-cell embryos. Remarkably, such perturbation caused downregulation of hundreds of ZGA genes and embryonic arrest mostly at the morula stage.

To systematically define cis-acting functions of MT2 retrotransposon, we re-analyzed H3K4me3 (a chromatin marker for promoters), ATAC (open chromatin), and H3K27ac (enhancer) public datasets at the late 2-cell stage. Strikingly, MT2 retrotransposon were globally enriched for ATAC and H3K27ac signals. These observations were not artifacts due to the challenge of mapping reads at repeats because the enrichments were not observed in embryonic stem cells. We also determined MT2-chimeric transcription by identifying spliced RNA-seq reads. In sum, MT2 retrotransposon were enriched for active chromatin signatures and the majority of MT2 copies formed MT2-chimeric transcripts.

We also compared how transcription factors OBOX and DUX may regulate MT2 retrotransposon. Re-analyses of publicly available data revealed that 2,278 (85.4%) and 2,464 (92.3%) MT2_Mm insertions were bound by DUX and at least one OBOX protein, respectievely. This suggests that OBOX and DUX binding are enriched at MT2 in a genome-wide manner. We next analyzed MT2 expression in Obox and Dux maternal and zygotic KO (mzKO) embryos. MT2 repeats were downregulated in both Obox and Dux mutants. Notably, these repeats in general were more affected upon Obox depletion than Dux KO at the early 2-cell stage (when minor ZGA completes). This suggests that OBOX proteins play a major role in activating these repeats during minor ZGA. Lastly, we identified hundreds of ZGA genes that were regulated by MT2, OBOX, and DUX, suggesting that OBOX and DUX activate these genes at the 2-cell stage through regulating MT2 retrotransposon.  

In sum, we not only provided direct evidence to support the functional importance of MT2 activation in development, but also systematically define cis-regulatory function of MT2 in embryos by integrating functional perturbation and multi-omic analyses.