Integration of ATAC-seq and RNA-seq Unravels Chromatin Accessibility during Sex Reversal in Orange-Spotted Grouper (Epinephelus coioides)

Abstract

Chromatin structure plays a pivotal role in maintaining the precise regulation of gene expression. Accessible chromatin regions act as the binding sites of transcription factors (TFs) and cis-elements. Therefore, information from these open regions will enhance our understanding of the relationship between TF binding, chromatin status and the regulation of gene expression. We employed an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq analyses in the gonads of protogynous hermaphroditic orange-spotted groupers during sex reversal to profile open chromatin regions and TF binding sites. We focused on several crucial TFs, including ZNF263, SPIB, and KLF9, and analyzed the networks of TF-target genes. We identified numerous transcripts exhibiting sex-preferred expression among their target genes, along with their associated open chromatin regions. We then investigated the expression patterns of sex-related genes as well as the mRNA localization of certain genes during sex reversal. We found a set of sex-related genes that—upon further study—might be identified as the sex-specific or cell-specific marker genes that trigger sex reversal. Moreover, we discovered the core genes (gnas, ccnb2, and cyp21a) of several pathways related to sex reversal that provide the guideposts for future study.

Establishment of a Spermatogonial Stem Cell Line with Potential of Meiosis in a Hermaphroditic Fish, Epinephelus coioides

“…Dnd is a vertebrate-specific germ cell marker and essential for primordial germ cell survival in orange-spotted grouper [ 61 ]. Dmrt1 plays an important part in sex determination and is restricted specifically to spermatogenic cells in orange-spotted grouper [ 62 , 63 ]. Oct4 and Gfra1 critically participate in SSC self-renewal [ 3 , 64 ], and can be detected in medaka SG3 [ 26 , 65 ] and cultured tilapia SSCs [ 66 ].…”

“…Additionally, the expression of pluripotency markers Ssea1, Nanog, gfra1 , and oct4 further indicates that GPT cells possess stem cell pluripotency. Because plzf , zbtb40 , and dmrt1 are male germ cell-specific genes [ 49 , 62 , 63 ], GPT cells are genetically masculinized and do not originate from the latent female germ cells of testis. In vitro culture leads to a significant change in gene expression pattern of SSCs.…”

RNA sequencing: new technologies and applications in cancer research

“…The integration of ATAC-seq and RNA-seq enables the reveal of TF-targeted genes and their transcripts [187,188]. Chromatin conformation capture analysis (3C) technology and its several derivatives including circular chromosome conformation capture (4C), carbon copy chromosome conformation capture (5C), ChIP-Loop, Hi-C and capture Hi-C were developed and improved to detect chromatin structure as well as unknown interacting regions [189][190][191].…”

Pou5f1 and Nanog Are Reliable Germ Cell-Specific Genes in Gonad of a Protogynous Hermaphroditic Fish, Orange-Spotted Grouper (Epinephelus coioides)

“…ISH protocol was described in our previous study [ 37 ]. Briefly, after being fixed in 4% paraformaldehyde (Sangon, Shanghai, China) at 4 °C overnight, gonads were dehydrated with gradient methanol/PBS from 20% to 100% methanol, and then stored at −20 °C until further use.…”