Early vertebrate origin of CTCFL, a CTCF paralog, revealed by proximity-guided shark genome scaffolding

Abstract

the nuclear protein ccctc-binding factor (ctcf) contributes as an insulator to chromatin organization in diverse animals. The gene encoding this protein has a paralog which was first identified to be expressed exclusively in the testis in mammals and designated as CTCFL (also called BORIS). CTCFL orthologs were reported only among amniotes, and thus CTCFL was once thought to have arisen in the amniote lineage. In this study, we identified elasmobranch CTCFL orthologs, and investigated its origin with the aid of a shark genome assembly improved by proximity-guided scaffolding. Our analysis employing evolutionary interpretation of syntenic gene location suggested an earlier timing of the gene duplication between CTCF and CTCFL than previously thought, that is, around the common ancestor of extant vertebrates. Also, our transcriptomic sequencing revealed a biased expression of the catshark CTCFL in the testis, suggesting the origin of the tissue-specific localization in mammals more than 400 million years ago. To understand the historical process of the functional consolidation of the long-standing chromatin regulator ctcf, its additional paralogs remaining in some of the descendant lineages for spatially restricted transcript distribution should be taken into consideration. The CCCTC-binding factor (CTCF) contains the C2H2 Zn finger-type DNA binding domains and plays a pivotal role in chromatin organization as an insulator in diverse metazoans 1,2. In vertebrates, the genome-wide binding landscape of the CTCF protein has been characterized for mammals 3,4 , sharks 5 , and the lamprey 6 , but the property of its paralog, CTCFL (also called BORIS, brother of the regulator of imprinted sites), has not been well characterized in a molecular phylogenetic context. CTCFL was first identified in human and mouse as a protein that functions in the testis and binds to the known target DNA of the CTCF protein in vitro 7. Their differential functions have been intensively investigated in germ cells and cancer cells mainly from epigenetic viewpoints 8-11. Comparison of amino acid sequences between CTCF and CTCFL exhibits a high similarity in the Zn finger DNA binding domain while the homology was low in other regions 7 , i.e., in the C-terminal region indispensable for the insulator function of CTCF 12. In contrast to the ubiquitously expressed CTCF 7 , the expression of CTCFL is restricted to the male testis, more specifically in the spermatocyte and the spermatogonia 7,13. Concordantly, while the mice lacking CTCF are embryonically lethal as early as E4.5 14 , mice lacking CTCFL are viable with phenotypes only in the testis, showing the marked reduction of its size caused in part by the increased rate of apoptosis during spermatogenesis 15. The report of CTCFL orthologs was long confined to mammals and lizards 16 but more recently the orthologs were identified in birds, turtle, snakes, and crocodiles 6,17. The gene expression patterns of CTCFL has been documented for only amniote species, and it is hypothesized that the testis...

The combined action of CTCF and its testis-specific paralog BORIS is essential for spermatogenesis

“…Thus, our data provide convincing evidence of functional cooperation between CTCF and BORIS in male germ cell development. Taking together the male germline restricted pattern of BORIS expression from sharks to mammals 26,28 and the functional cooperation of CTCF and BORIS at the promoters of germ-cell specific genes, we would suggest that CTCF and BORIS heterodimerization is evolutionary more adapted in regulation of many germ-cell specific transcripts compared to CTCF homodimerization.…”

“…The Ctcf gene has a close paralog known as Boris (Brother Of the Regulator of Imprinted Sites) or Ctcfl (CCCTC-binding Factor-Like) 26,27 that appeared later in evolution as a duplication of Ctcf in early vertebrates 28 . The two genes share a high degree of homology in the central 11-Zinc finger DNA binding domain.…”

BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites

“…Among CTA genes, BORIS occupies a truly exceptional position, as it is the sole paralogue of CTCF (CCCTC-Binding Factor) [ 16 , 27 , 28 ], a global regulator of genome organization and gene expression [ 29 – 33 ]. CTCF divides genomes into topologically associated domains (TADs) by restricting cohesin-mediated extrusion of chromatin loops owing to the activity of its N-terminal domain [ 30 , 34 – 36 ].…”

“…As a result of high homology in the DNA-binding domains, CTCF and BORIS can recognize and bind to similar DNA sequences; however, the functional outcome of their binding differs dramatically [ 22 , 34 , 36 , 43 ]. In contrast to the ubiquitously expressed CTCF, BORIS expression is strictly restricted to male germ cells in virtually all vertebrates [ 15 , 27 , 44 ]. When CTCF and BORIS are co-expressed, essentially only in germline and cancer cells, they tend to form a heterodimer at clustered (double) CTCF binding sites, which encompass two or more CTCF binding consensus sequences (2xCTSes) [ 22 , 43 ].…”