Chromosomal inversions modulate 3D chromatin interactions and regulatory architecture in Anopheles gambiae

<title>Abstract</title> <p> Background Malaria parasite transmission by <italic>Anopheles</italic> mosquitoes remains a worldwide health burden, and understanding factors underlying natural vector susceptibility would inform rational design of vector control. The 2La chromosomal inversion segregates in the major vectors of human malaria, <italic>Anopheles coluzzii</italic> and <italic>Anopheles gambiae</italic> , and is associated with natural variation for malaria susceptibility, though underlying mechanisms are unknown. Here, we characterize alterations of chromatin conformation and gene expression induced by the two 2La inversion allelic forms, the ancestral 2La and the derived 2L +  ^a forms as well as by the two 2Rbc inversion allelic forms. We employ several novel applications of proximity ligation sequencing to refine the mosquito regulatory genome to a new level of resolution. Results We analyzed the 2La inversion breakpoints in <italic>A. coluzzii</italic> hemocyte-like cell lines for the allelic 2La inversion karyotypes. Utilizing a novel combination of Micro-C, bulk RNA-sequencing, and ATAC-sequencing, our results detected transcriptional enhancers and genes that are reciprocally rewired by the physical rearrangement caused by the inversion. Similar analyses on the 2Rbc inversion revealed enhancer rewiring without reciprocity between alternate inversion forms, likely due to the distinct evolutionary histories between the 2La and 2Rbc inversions. Through development and application of novel distance-normalized interaction frequency analysis on Micro-C data, we identify a novel common enhancer for the APL1 gene family and a novel candidate enhancer for LRIM1, all of which are major parasite antagonist immune genes within the 2La inversion. Conclusions This multifaceted approach yields high resolution characterization of gene <italic>cis</italic> -regulation within <italic>Anopheles</italic> mosquitoes, and specifically within the context of a malaria-associated paracentric inversion. Additionally, development of analytical methods for proximity ligation data allows fine scale exploration of mosquito chromatin interactions and are broadly applicable across species. Finally, the newly identified regulatory architecture of an inversion associated with <italic>Plasmodium</italic> infection, and of multiple parasite-antagonistic immune genes, highlights candidates for potential further development of a novel vector control toolkit. </p>