The accumulation of gene regulation through time

Gene expression is governed by an intricate combination of transcription factors (TFs), microRNAs (miRNAs), splicing factors, and other regulators. Genes cannot support infinitely complex regulation due to sequence constraints and the increased likelihood of harmful errors. However, the upper limit of regulatory complexity in the genome is not known. Here, we provide evidence that human genes are currently not operating at their maximum capacity in terms of gene regulation. We analyze genes spanning the full spectrum of eukaryote evolution, from primate-specific genes to genes present in the eukaryote ancestor, and show that older genes tend to be bound by more TFs, have more conserved upstream sequences, generate more alternative isoforms, house more miRNA targets, and are more likely to be affected by nonsense-mediated decay and RNA editing. These results cannot be explained by overrepresentation of certain functional categories among younger or older genes. Furthermore, the increase in complexity is continuous over evolutionary time, without signs of saturation, leading to the conclusion that most genes, at least in the human genome, have the capacity to evolve even more complex gene regulation in the future.