Human higher cognition is attributed to the evolutionary expansion and elaboration of the human cerebral cortex. However, the genetic mechanisms contributing to these developmental changes are poorly understood. We used comparative epigenetic profiling of human, rhesus macaque and mouse corticogenesis to identify promoters and enhancers that have gained activity in humans. These gains are significantly enriched in modules of co-expressed genes in the cortex that function in neuronal proliferation, migration, and cortical map organization. Gain-enriched modules also showed correlated gene expression patterns and similar transcription factor binding site enrichments in promoters and enhancers, suggesting they are connected by common regulatory mechanisms. Our results reveal coordinated patterns of potential regulatory changes associated with conserved developmental processes during corticogenesis, providing insight into human cortical evolution.

Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2264 SMC1A interactions, of which 1491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue-specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin-associated interactions in the genome and highlight their role in establishing the regulatory architecture of development.

Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of , which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the promoter and potentially redundant enhancers. enhancer null mice did not exhibit any of the characteristic morphological defects of the mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.

Gene expression patterns during development are orchestrated in part by thousands of distant-acting transcriptional enhancers. However, identifying enhancers that are essential for expression of their target genes has proven challenging. Genetic perturbation of individual enhancers in some cases results in profound molecular and developmental phenotypes, but in mild or no phenotypes in others. Topological maps of long-range regulatory interactions may provide the means to identify enhancers critical for developmental gene expression. Here, we leveraged chromatin topology to characterize and disrupt the major promoter-enhancer interaction for Pitx1, which is essential for hindlimb development. We found that Pitx1 primarily interacts with a single distal enhancer in the hindlimb. Using genome editing, we deleted this enhancer in the mouse. Although loss of the enhancer completely disrupts the predominant topological interaction in the Pitx1 locus, Pitx1 expression in the hindlimb is only reduced by ~14%, with no apparent changes in spatial distribution or evidence of regulatory compensation. Pitx1 enhancer null mice did not exhibit any of the characteristic morphological defects of the Pitx1 -/-mutant. Our results indicate that Pitx1 expression is robust to the loss of its primary enhancer interaction, suggesting disruptions of regulatory topology at essential developmental genes may have mild phenotypic effects.