Effects of a chromosomal inversion on gene regulation in a major malaria vector

The National Institute of Allergy and Infectious Diseases awarded The Medical College of Wisconsin, Inc. $213,088 on December 26, 2025, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to study how a chromosomal inversion in Anopheles coluzzii mosquitoes affects gene regulation and intrinsic malaria resistance.

The research will biochemically and functionally characterize a candidate enhancer (EN1) that contains an inversion-specific single nucleotide polymorphism associated with natural malaria resistance in the mosquito vector. Aim 1 studies will use electrophoretic mobility shift assays with antibody super-shift to identify differentially bound transcription factors, and luciferase assays on cell lines with transcription factors silenced by RNA interference to determine if the transcription factor is required for EN1 activity. Aim 2 will functionally characterize EN1 by identifying its target genes and their role in the malaria resistance phenotype. The work builds on preliminary micro-C and bulk RNA-sequencing showing differential chromatin interactions and gene expression between alternate inversion forms.

Performance is based in Wauwatosa, Wisconsin, with a period of performance through June 30, 2029. This is a Project Grant assistance type.

Abstract

Malaria is a potentially lethal vector-borne disease caused by the plasmodium parasite and transmitted by anopheles mosquitoes. Because malaria continues to be a major threat to global health, newer and more effective vector control strategies are needed. Anopheles coluzzii mosquitoes are primary vectors of malaria and segregate for a large, stable chromosomal inversion. This paracentric inversion is associated with an intrinsic resistance to malaria infection, among other vector competence phenotypes. The mechanism(s) of how the inversion altered enhancer-mediated gene regulation related to intrinsic malaria resistance are unknown. Preliminary micro-c and bulk RNA-sequencing show there are differential chromatin interactions and differentially expressed genes between alternate inversion forms. I have identified a candidate enhancer (EN1) that (i) contains the genetic marker to which natural malaria resistance was most significantly mapped and (II) contains an inversion-specific single nucleotide polymorphism (SNP) that is responsible for the observed difference in enhancer activity between the two inversion forms. The central hypothesis is that due to genetic variation distinctively fixed between alternate inversion forms, EN1 differentially modulates expression of its target genes in the two inversion forms and underlies the natural malaria resistance phenotype. Studies in aim 1 will biochemically characterize the differential interaction(s) of EN1 and its transcription factor(s) in alternate inversion forms. Electrophoretic mobility shift assays with antibody super-shift will identify the differentially bound transcription factor. Luciferase assays on cell lines with the transcription factor silenced by RNA interference will determine if the transcription factor is required for EN1 activity. Studies in aim 2 will functionally characterize EN1 by identifying EN1 target genes, elucidating EN1's role in plasmodium infection outcome in mosquitoes, and determining EN1 chromatin interactions under immune stimulation. Collectively, these data will provide a functional understanding of enhancer-promoter interactions in a medically important insect vector, especially in the context of a large chromosomal inversion. Findings from this proposal can help inform the development of better vector control strategies, such as evolutionarily informed genetically modified mosquitoes, to prevent malaria transmission to humans. Specific training activities planned as part of my fellowship training include presenting my research yearly at mstp and departmental research in progress meetings, attending and presenting at national tropical medicine conferences, and presenting my data at weekly lab meetings. The meetings organized by MCW's mstp and the department of microbiology and immunology allow me to improve my communication skills in sharing scientific data, practice my presentation skills, and network with experts in the field of vector-borne diseases.