Here we report islands of GGC and GCC STRs of extraordinary density and periodicity in stretches of 1-2 kb of genomic DNA, which were specific to the human species or revealed directional trend in human versus other species. The occurrence of these islands was exceedingly significant in statistical terms, even considering the fact that GGC and GCC STRs are enriched in genic regions 14,15. Several of the genes containing those islands revealed significant gene expression divergence in human versus numerous other species. Based on the above observations, we propose that these islands contribute to the evolution of the human species. The segmental duplications resulting in these islands were also phenomenal with respect to the number of those events in the genomic lengths that they occurred. The reported human segmental duplications involve lengths of ≥40 kb of genomic DNA 22.
Although largely speculative at this time, it is possible that a number of the identified islands, such as in RGPD4, RGPD3, NOC4L, and RGPD1 function as genomic centers for regulating other GGC and CCG-repeats at local and distant parts of the genome by forming complementary sequences and loops. Intriguingly, the RGPD4 (GGC)3 and NOC4L (GCC)3 islands are reverses of one another and almost identical with respect to the number of the STRs. Although hypothetical, this could suggest a long-distance correlation or interaction that could be non-local in origin, especially that the two islands are the reverse sequences of each other. Evidence on the algebraic distribution of segmental duplication lengths in whole-genome sequence self-alignments 23 lends support to the above hypothesis.
The human RGPD family has eight members 24, which resulted from duplications of a region of chromosome 2 containing the RANBP2 (RAN binding protein 2) and GCC2 (GRIP and coiled-coil domain containing 2) genes. RANBP2 (NUP388) is a component of the nuclear pore complex and plays a role in facilitation of protein import and export, sumoylation of protein cargoes, intracellular trafficking, and energy maintenance 25. GCC2 is a sub compartment of the trans-Golgi network 26. By genomic sequence analysis, Ciccarelli and co-workers mapped the eight RGPD genes to chromosome 2p11.2-q13 24. The RGPD genes are rapidly evolving and under positive selection 27. The macaque genome, as well as the mouse and other mammalian genomes, does not contain duplicates, suggesting that expansion occurred within the great apes after the separation from old world monkeys. The RGPD family is the largest in human, with eight members. It is likely that the positive selection of the family members in human paralleled the selection of the identified GGC STR islands in those members. In line with the above, across numerous primate species, RGPD1, RGPD2, RGPD4, and RGPD8 reach maximum level of expression in the human brain (https://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/) 28.
The human FOXK2 (GGC)3 island is a prime instance, in which there is a directional link between STR density at a particular genomic region and human evolution. Across numerous primate species, the brain expression of FOXK2 reaches the maximum quantile in human (https://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/) 28. FOXK2 is likely to act as an important regulator that reprograms cellular metabolism to induce aerobic glycolysis, which is important in adaptation to the environment and extraction of energy 29. Another rare instance of directional abundance and density of STRs at a specific locus in human is a CT-repeat complex encompassing the core promoter and 5′ untranslated region of PAXBP1 (OMIM: 617621) 30.
Although the literature on NOC4L is sparse, it has been shown that NOC4L-mediated ribosome biogenesis controls activation of regulatory and conventional T cells 31, and targeted disruption of Noc4l leads to preimplantation embryonic lethality in mice 32.
Mixed islands of GGC and GCC repeats were also detected, such as in USP7-AS1 and F8A2. Of note, among numerous primate species, the expression of F8A2 in various tissues is exceptionally human-specific (https://www.ncbi.nlm.nih.gov). While targets of USP7-AS1 have not been identified at this time, lnRNAs are stimulating large interest due to their versatile roles in fine-tuning numerous signaling pathways 33.
As opposed to CpG islands, which are mainly located in gene promoters, and include random occurrence of CpG dinucleotides in a stretch of between 200 to 500+ bp 34,35, several of the GGC and GCC STR islands were intronic. For a subset of genes, the most important sequences that are of regulatory importance are situated not in the promoters, but rather are located within introns 36-38.
Remarkably, proteins encoded by the genes containing human-specific islands interact directly or through only a few intermediary proteins in a physical interaction network (Fig. 9). The consequence of this interaction may be of relevance in human-specific characteristics and phenotypes, such as the higher order brain functions.
The identified GGC and GCC STR islands fractionally overlap with G quadraplex (G4) structures 4,15,39. G4 structures are DNA tetraplexes that typically form in guanine-rich regions of genomes. Four guanine bases associate with each other through Hoogsteen hydrogen bonds to form a guanine tetrad plane (G-quartet), and then two or more G-quartet planes stack on top of each other to form a G4 structure 40. Organisms may have evolutionarily developed G4 into a novel and elaborate transcriptional regulatory mechanism benefiting multiple physiological activities of higher organisms 41,42.
While largely overlooked, (GGC)3 and (GCC)3 STRs form unprecedented structures that only fractionally overlap with the sequences that conform to G4 structures reported to date. The biological implications of those islands are yet to be identified. The supplementary data (Additional File 1) will also enable studying a vast number of other colonies, the occurrence of each one of which is statistically significant, and may add to the knowledge of how GGC and GCC STRs may link to the evolution of human and other species.