Diverse processes drive the origination and maturation of super-enhancers and super-silencers during a vast evolutionary timescale of the bicistronic gene SMIM45

A central question in molecular genetics concerns how transcriptional regulatory sequences and de novo genes originate and reach evolutionary fixation. In this study, we utilize the human bicistronic gene SMIM45 as a model to analyze the evolutionary trajectories of gene development. This locus comprises several functional units: three enhancers (one featuring an embedded silencer), an exonic silencer that partially overlaps an ORF, a highly conserved ancestral sequence encoding a 68-aa microprotein, and a human-specific de novo gene encoding a 107-aa protein expressed spatiotemporally in embryonic brain tissues. We identify significant disparities in formation mechanisms; for example, the NANOG hESC enhancer originated simply via two Alu insertions that constitute the regulatory element. In contrast, the exonic silencer (ATAC-STARR-seq lymphoblastoid silent region 13815) originated by a combination of diverse mechanisms, including a "cultivator gene" process of base pair fixation consistent with the Cultivator Model proposed by Li Zhao and coworkers. Consequently, SMIM45 exemplifies novel mechanisms of regulatory element development that occurred over several hundred million years, culminating in the birth of a human-specific de novo 107-aa cistron. The properties of these super-enhancers and super-silencers suggest a complex, intricate regulation of the 107-aa protein in fetal tissues. Author SummarySMIM45 is one of a small number of known human bicistronic genes that encode two different proteins. It also contains a complex of super-enhancers and super-silencers, regulatory elements that likely control the expression of one of the cistrons, a human-specific, de novo protein cistron expressed in human embryonic brain tissues. The presence of these regulators raises questions about how these multifaceted components developed evolutionarily. We show that enhancers and silencers originated by very dissimilar processes, indicating a versatility of evolutionary mechanisms taking part in gene development. The exonic silencer stands out, having itself developed through a series of diverse processes. It appears unique; excluding the exonic splicing silencers, no other silencers are currently known to fully or partially overlap coding regions. Finally, we discuss the properties of known enhancers and silencers with respect to the spatiotemporal control of the de novo protein cistron, using these comparisons to gain insights into regulation of this cistron.