Gene regulatory landscapes are highly complex and evolutionarily unstable. Understanding how genetic and regulatory variations affect gene expression evolution is paramount to gaining insight into phenotypic divergence across species. Although gene expression programs are broadly maintained during mammalian evolution, the collection of cis-regulatory elements and transcription factor (TF) binding events has diverged at different rates. In this thesis, I investigated several layers of gene regulatory divergence across mammalian species.

In the first part of the thesis, I investigated the coevolution of DNA methylation patterns and transcription factor binding. Mammalian regulatory architecture evolves through widespread rewiring of TF binding events. Reportedly, TF binding is the fastest evolving layer of cis-gene regulation. However, whether DNA methylation patterns are reshaped during TF binding evolution has not been extensively studied. To fill this gap, I analyzed whole-genome bisulfite sequencing (WGBS) and matched chromatin immunoprecipitation assays followed by sequencing (ChIP-seq) of five TFs from the livers of five mammals. I first characterized DNA methylation patterns at transcription factor binding regions and found four prototypical methylation profiles that resolve alternative functional and chromatin contexts. These profiles were highly conserved across species and consistent across TFs. However, CTCF was an exception, with different methylation profiles likely underlying its various roles in both gene regulation and chromatin organization. I then investigated the relationship between DNA methylation patterns and TF binding divergence. Not only do genomic regions with evolutionary loss of binding regain their hypermethylated state, but distinct methylation profiles also follow species conservation patterns that reflect the turnover rate of the genomic context they correspond to (i.e., promoters and enhancers). Therefore, these results demonstrate coordinated evolution between DNA methylation patterns and transcription factor binding turnover.

In the second part of my thesis, I explored how the three-dimensional organization of the genome is reshaped during mammalian evolution. Technological advancements in the field of 3D genomics now enable the investigation of spatial chromatin interactions underlying gene regulation and genome organization. Thus, in the third chapter of the thesis, I described several strategies for creating custom capture-HiC probe sets to investigate distinct aspects of gene regulatory evolution. Two designs focus on transposable elements and the rewiring of chromatin interactions that underlie their repression and evolutionary co-option into gene regulatory networks. An additional design targets one-to-one orthologous genes across five mammals and cis-regulatory elements (CREs) that have various functional and evolutionary constraints, such as deeply conserved and tissue-specific CREs, as well as CRE sequences that switch activity (promoter-enhancer switchers) either between species or between tissues. This last capture system was designed to conduct a comparative genomic study of five mammals and three tissues (brain, liver, and testis), thus to investigate how gene regulatory landscapes differ between tissues and throughout evolution.

The final results chapter of the thesis focuses on bioloigical insights resulting from an experiment using the capture system described above targeting one-to-one orthologous genes across five mammals and three tissues. This study shows that orthologous promoters confirm established concepts, such as enhancers having more chromatin contacts with genes that have higher expression levels, and cis-regulatory sequences being more frequently distributed around 250 kbp away. However, they also exhibit distinctive, tissue-specific patterns of chromatin interactions. Interestingly, while the brain has the highest number of interactions per gene, the testis shows significantly fewer 3D contacts than somatic tissues and an increase in short-range interactions. I present a set of analyses that further investigate these results in the testis and their potential link to the restructuring of the genome that occurs in a large subpopulation cells going through spermatogenesis. In addition, I provide evidence of pervasive hubs of chromatin interactions, which often result in promoter-promoter networks that connect both active and inactive genes together. Finally, the investigation of the tissue-specificity of the promoter-centered chromatin organization shows only modest correlation across species, likely reflecting the evolutionary dynamics of regulatory landscapes. Therefore, I define a framework for studying how chromatin looping rewires at orthologous genes across different species.