The Anopheles gambiae 2La chromosomal inversion influences chromatin organization and 3D landscape of genes related to malaria transmission

Background Malaria parasite transmission by Anopheles 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, Anopheles coluzzii and gambiae , 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. 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 A. coluzzii hemocyte-like cell lines for the allelic 2La inversion karyotypes. Utilizing a novel combination of Micro-C and bulk RNA-sequencing, our results detected transcriptional enhancers and genes that are rewired by the physical rearrangement caused by the inversion. Through development and application of novel distance-normalized interaction frequency analysis on Micro-C data, we identify a novel candidate enhancer for LRIM1, a major parasite antagonist immune gene within the 2La inversion. Our genome-wide analysis examines the distribution of all chromatin interactions across the genome and identifies chromatin interaction hubs that are positively associated with enhancers. Conclusions This multifaceted approach yields high resolution characterization of gene cis -regulation within Anopheles mosquitoes, and specifically within the context of a malaria-associated paracentric inversion. Additionally, development and validation of analytical methods for proximity ligation data allow fine scale exploration of mosquito chromatin interactions and are broadly applicable across species.