Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals [CUT&TAG]

Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos in vivo and in vitro. However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, and the abnormal phenotypes could be recapitulated by overexpression of H3K18R mutation and rescued by addition of Lac-CoA. By profiling the landscape of H3K18lac in human and mouse preimplantation embryos, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and corelates with their expressions. Taken together, we demonstrate the important role for lactate in major ZGA via H3K18lac, showing a conserved metabolic mechanism underlies preimplantation development of mammalian embryos. Overall design: CUT&Tag was performed using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme Biotech). Embryos were incubated in 50 µL antibody buffer; 0.5 µg antibody was added and the mixture was incubated at 4? overnight. After washing twice with dig-wash buffer, 50 µL dig-wash buffer with 0.2 µg secondary antibody was added and the mixture was incubated at room temperature for 1 h. After washing twice with 100 µL dig-wash buffer, 1 µL pG-Tn5 and 49 µL dig-300 buffer were added, and the samples were incubated at room temperature for 1 h. Next, the samples were washed twice with 100 µL dig-wash buffer. We subsequently added 200 µL tagmentation buffer and incubated the samples at 37°C for 1 h. The reaction was stopped with 7 µL 0.5 M EDTA, 2 µL 10% SDS, and 1.7 µL 20 mg/mL proteinase K; for single-cell CUT&Tag assays, 5 µL stop buffer was used. After extraction with phenol-chloroform and ethanol precipitation, PCR was performed to amplify the libraries under the following conditions: 72°C for 3 min, 98°C for 30 s, 17 cycles of 98°C for 10 s and 60°C for 5 s, and a final extension at 72°C for 1 min with a hold at 4°C. For single-cell CUT&Tag of human embryos, CUT&Tag was performed in well of a microplate. After tagmentation, single human embryos were transferred to 200µl tubes containing 5% Proteinase K. After embryos lysis, PCR buffer and adaptors were directly added to tubes, normal PCR cycle was performed. Post-PCR clean-up was performed by adding a 1.5× volume of DNA Clean Beads (Vazyme Biotech). Libraries were sequenced on the Illumina NovaSeq 6000 platform according to the manufacturer's instructions.