Pseudogenes contribute to the evolution of topological domains across species via forming three-dimensional genome [mESC_KI_Hi-C]

The human genome harbors 15,000 pseudogenes, except very few can transcribe non-coding RNAs or encode truncated proteins, a large number of which without transcriptional capacity are functionally unknown. Here, we proposed and verified that pseudogene DNA sequences can form chromatin contacts that act as anchors of chromatin loops and boundaries of topologically associating domains (TADs). More amazing, due to the sequence-specificity of pseudogenes, TAD boundaries containing them in the human genome were primarily species-specific, including human-specific and primate-specific ones. We found that during primate evolution, by inheritance from parent genes, certain pseudogenes can introduce additional transcription factor-binding sequences, especially CTCF, at their insertion sites to generate species-specific TAD boundaries, and due to the participation in TAD evolution, CTCF binding motifs on pseudogenes were subjected to significantly heightened selection pressure. Deleting these pseudogenes in human embryonic stem cells (hESCs) disrupted the structure of species-specific TADs, whereas inserting them in mouse embryonic stem cells (mESCs) mediated new TADs formation, and pseudogenes involved in three-dimensional (3D) genome formation were critical for maintaining hESCs self-renewal. The structural necessity and biological function of these pseudogenes demonstrated the broad significance of pseudogenes in 3D genome construction and evolution. Overall design: Two mouse embryonic stem cells (mESCs) knock-in (KI) cell lines were obtained for each of the two pseudogenes, AC106872.1 and KRT18P67. KRT18P67-KI-C4, KRT18P67-KI-B5, AC106872.1-KI-B9, AC106872.1-KI-D11, and wild-type (WT) mESCs were subjected to Hi-C sequencing. To find out how insertion of pseudogenes influence gene expression in mESCs, we also performed RNA-seq for WT, KRT18P67-KI and AC106872.1-KI mESCs, each with two replicates. We also performed Hi-C in KRT18P37 knock-out human embryonic stem cells (hESCs) with two replicates.