Identification of GPR1-AS orthologs from public placental transcriptomes

UCSC Genome browser screenshots of the GPR1-ZDBF2 locus in humans (A), rhesus macaques (B) and mice (C). Predicted transcripts were generated using public directional placental RNA-seq datasets (accession numbers: SRR12363247 for humans, SRR1236168 for rhesus macaques, and SRR943345 for mice) using the Hisat2-StringTie2 programs. Genes annotated from GENCODE or RefSeq databases and LTR retrotransposon positions are also displayed. Among the gene lists, only the human reference genome includes an annotation for GPR1-AS (highlighted in green). GPR1-AS-like transcripts and MER21C retrotransposons are highlighted in red.

Identification of GPR1-AS orthologs from original placental and extra-embryonic transcriptomes

Predicted transcripts were generated from placental and extra-embryonic directional RNA-seq datasets of chimpanzee (A), rabbit (B), pig (C), cow (D), and opossum (E) with the Hisat2-StringTie2 programs. Genes annotated from RefSeq or Ensembl databases and their LTR positions are also shown. GPR1-AS-like transcripts and MER21C retrotransposons are highlighted in red.

Allele-specific RT-PCR sequencing of ZDBF2 in tammar wallabies and cattle

One and three heterozygous genotypes were used to distinguish between parental alleles in adult tissues form tammar wallabies (A) and fetal/embryonic tissues from cattle (B), respectively. Primers were designed to amplify the 3’-UTR regions of ZDBF2 orthologs and detect SNPs. Each SNP position are highlighted in red. Reverse primers were also used for Sanger sequencing.

Multi-species comparison of LTR retrotransposon locations at GPR1 locus

A total of 24 mammalian genomes were compared, including six primates (human, chimpanzee, rhesus macaque, marmoset, tarsier, and gray mouse lemur), one colugo (flying lemur), one treeshrew (Chinese treeshrew), two rabbits (rabbit and pika), eight rodents (squirrel, guinea pig, lesser jerboa, blind mole rat, giant pouched rat, mouse, rat, and golden hamster), and six eutherians (pig, cow, horse, dog, elephant, and armadillo). The locations of MER21C and MER21B retrotransposons, which exhibited 88% similarity between their consensus sequences through pairwise alignment, are highlighted in red and purple, respectively.

Comparison of MER21C-derived sequences overlapping the first exon of GPR1-AS orthologs

(A) Phylogenetic tree of MER21C-derived sequences estimated by multiple sequence alignment (MSA) using multiple sequence comparison by log-expectation (MUSCLE) program. (B) Positions of common and unique cis-acting elements at each sequence. (C) Motif structures of the common region that contains E74-like factor 1 and 2 (ELF1 and ELF2) binding motifs. (D) Motif structures of transcription factor AP-2 gamma (TFAP2C) and Zinc finger and SCAN domain containing 4 (ZSCAN4).

Initiation of GPR1-AS transcription before implantation

Genome browser screenshots of the GPR1-ZDBF2 locus in humans during preimplantation stages: MII oocyte, zygote, 2-, 4-, and 8-cell, inner cell mass (ICM) and trophectoderm (TE) from blastocyst. Predicted transcripts were generated using publicly available placental full-length RNA-seq datasets.