Regulatory sequences and transposable elements (TEs) account for a large proportion of the genomic sequences of species; however, their roles in gene transcription, especially tissue-specific expression, remain largely unknown. Pigs serve as an excellent animal model for studying genomic sequence biology due to the extensive diversity among their wild and domesticated populations. Here, we conducted an integrated analysis using H3K27ac ChIP-seq, H3K4me3 ChIP-seq, and RNA-seq data from 10 different tissues of seven fetuses and eight closely related adult pigs. We aimed to annotate the regulatory elements and TEs to elucidate their associations with histone modifications and mRNA expression across different tissues and developmental stages. Based on correlation analysis between mRNA expression and H3K27ac and H3K4me3 peak activity, results indicated that H3K27ac exhibited stronger associations with gene expression than H3K4me3. Furthermore, 1.45% of TEs overlapped with either the H3K27ac or H3K4me3 peaks, with the majority displaying tissue-specific activity. Notably, a TE subfamily (LTR4C_SS), containing binding motifs for SIX1 and SIX4, showed specific enrichment in the H3K27ac peaks of the adult and fetal ovaries. RNA-seq analysis also revealed widespread expression of TEs in the exons or promoters of genes, including 4 688 TE-containing transcripts with distinct development stage-specific and tissue-specific expression. Of note, 1 967 TE-containing transcripts were enriched in the testes. We identified a long terminal repeat (LTR), MLT1F1, acting as a testis-specific alternative promoter in SRPK2 (a cell cycle-related protein kinase) in our pig dataset. This element was also conserved in humans and mice, suggesting either an ancient integration of TEs in genes specifically expressed in the testes or parallel evolutionary patterns. Collectively, our findings demonstrate that TEs are deeply embedded in the genome and exhibit important tissue-specific biological functions, particularly in the reproductive organs.
猪作为人类主要的蛋白质来源以及优良医学生物模型，目前的功能基因组注释远远落后于小鼠、斑马鱼等模式生物。该研究以全同胞胚胎及半同胞成年个体为实验材料，采集了2个不同品种、不同性别、7头胚胎阶段和8头成年阶段猪的10种组织（大脑、心脏、肝脏、肺、肌肉、垂体、小肠、胃、卵巢和睾丸）样本，分别开展了以H3K4me3和H3K27ac为抗体的染色质免疫共沉淀测序（ChIP-seq）实验以及转录组测序（RNA-seq）实验。通过分析ChIP-seq数据，我们一共鉴定到115 181个H3K4me3 peaks、271 376个H3K27ac peaks，显著扩充了猪调控元件数据库。我们发现与H3K27ac peaks重叠的转座元件（TEs）存在组织特异性的调控活性，例如，内含SIX1和SIX4结合位点的转座元件亚家族（LTR4C_SS）偏好在卵巢组织中受到H3K27ac修饰，进一步分析发现该转座元件可能与雌性性腺发育的功能相关。通过分析RNA-seq数据，本研究还鉴定了作为外显子或启动子参与的基因表达调控的TEs，包括4 688个阶段-组织特异性表达TE-gene嵌合体（TE-gene junctions），其中睾丸组织中具有功能作用的TEs数量明显多于其他组织类型，说明睾丸组织为TEs转录激活提供了适宜的组织细胞环境，同时也暗示TEs与睾丸组织的生物学功能密切相关。此外，我们在人和小鼠的对应组织中也证实了TE-gene junctions的存在，并发现多个组织特异性表达的TEs具有物种保守性，表明这些TEs的插入是一类古老事件，可能存在于物种分化之前。该工作丰富了猪调控元件资源文库的同时，也证实了TEs可能通过调控基因表达、剪接等方式发挥重要的组织特异性生物学功能。.
Keywords: Alternative promoter; Histone modification; Porcine; TE-containing transcript; Transposable elements.