Flipped DNA segments rewire mosquito chromosomes and malaria immunity genes

Deep inside the genome of the malaria mosquito, chunks of DNA sit in reverse orientation, and those flipped segments turn out to do far more than shuffle gene order. A new study published in BMC Biology shows that chromosomal inversions in Anopheles gambiae and its close relative Anopheles coluzzii physically reshape the three-dimensional folding of chromosomes, rewiring the regulatory connections between genes and the enhancers that switch them on. The work, led by Kathryn Taquet, Cameron Anderson, Kenneth Vernick, and Michelle Riehle, provides the highest-resolution view yet of how large structural variants remodel chromatin architecture in a disease vector, and it pinpoints new regulatory elements near genes that mosquitoes use to fight Plasmodium parasites.

The team focused on the 2La inversion, a roughly 20-million-base-pair paracentric inversion on chromosome arm 2L that segregates in the major African malaria vectors. The 2La arrangement is the ancestral form, while the 2L+a arrangement represents the derived, standard-order configuration. This inversion has long been associated with natural variation in susceptibility to malaria parasites, aridity tolerance, and habitat partitioning between Anopheles coluzzii and Anopheles gambiae, but the molecular mechanisms connecting the rearranged DNA to parasite susceptibility have remained obscure. The researchers asked a direct question: does flipping this enormous segment of chromosome change how the genome folds in the nucleus, and if so, does that folding change alter gene regulation?

To answer it, the group used two Anopheles coluzzii hemocyte-like cell lines, Ag55 and Sua4.0, which are homozygous for different 2La karyotypes. Hemocyte-like cells are a sensible choice because mosquito immune responses to Plasmodium depend on blood-cell-derived signaling pathways, so regulatory differences in these cells are relevant to infection outcomes. The team first confirmed the karyotypes of the cell lines with molecular assays for the 2Rb and 2Rc inversions as well, then mapped the 2La inversion breakpoints precisely. With matched pairs of cells differing mainly in inversion status, they could attribute differences in chromatin contact maps and gene expression to the structural variant itself rather than to unrelated genetic background effects.

The core technique was Micro-C, a proximity ligation sequencing method that captures chromatin contacts at nucleosome-level resolution, far finer than the original Hi-C approach. In Micro-C, DNA in the nucleus is crosslinked, cut with an enzyme, and ligated so that pieces of DNA lying close together in three-dimensional space are joined into single molecules that can then be sequenced. The resulting contact matrices reveal which regions of the genome physically touch each other, including topologically associating domains, or TADs, which are self-contacting neighborhoods of chromatin where genes and their regulatory elements tend to be confined. The researchers combined Micro-C with bulk RNA sequencing to measure gene expression and ATAC sequencing to profile open, accessible chromatin where transcription factors and enhancers are active.

The results were striking. Comparing the two inversion forms, the team detected hundreds of differential interactions spanning the 2La breakpoints, meaning that the physical flip changed which distant DNA segments come into contact. Crucially, they found transcriptional enhancers and genes that were reciprocally rewired by the inversion: enhancers that contacted and presumably activated certain genes in one karyotype contacted different genes in the other, and the expression of those genes shifted accordingly. This reciprocal rewiring provides a concrete mechanism for how an inversion can alter phenotype without changing the sequence of any gene itself. The effect is a matter of geometry: an enhancer stranded on one side of a breakpoint in the ancestral arrangement may be brought within reach of a new target gene in the inverted arrangement, or vice versa.

To test whether this was a general property of inversions, the researchers repeated the analysis on the 2Rbc inversion, a compound arrangement involving the 2Rb and 2Rc inversions on chromosome arm 2R. Here they also observed enhancer rewiring across the breakpoints, but the pattern was not reciprocal between the alternate forms. The authors attribute this asymmetry to the distinct evolutionary histories of the two inversions. The 2La inversion is an ancient polymorphism shared across the species complex, so both arrangements have had long evolutionary time to accumulate compensatory regulatory changes, whereas the 2Rbc arrangement has a different origin and demographic history. The comparison suggests that the regulatory consequences of inversions depend on how long the alternative arrangements have coexisted and what sequence changes they carry with them.

Beyond documenting rewiring, the study introduced a new analytical tool: a distance-normalized interaction frequency analysis for Micro-C data. Raw contact counts in proximity ligation experiments are strongly biased by genomic distance, since nearby sequences naturally ligate more often than distant ones. By normalizing interaction frequencies against this distance expectation, the method highlights contacts that are unusually frequent or rare for their separation, allowing fine-scale detection of regulatory interactions that would otherwise be buried in the distance decay curve. The authors emphasize that this approach is broadly applicable across species, not just to mosquitoes, which could make it a standard tool for dissecting chromatin architecture in non-model organisms where genome annotation is still coarse.

Applying this method to the 2La region paid off immediately. The team identified a novel common enhancer for the APL1 gene family, a cluster of leucine-rich repeat immune genes that are among the most important antagonists of Plasmodium parasites in the mosquito. They also identified a novel candidate enhancer for LRIM1, another leucine-rich repeat immune protein with a well-established role in parasite killing. All of these genes sit within the 2La inversion, which means the inversion that modulates malaria susceptibility physically contains, and reshapes the regulation of, some of the mosquito’s most potent anti-parasite weapons. The discovery of shared and candidate enhancers for these genes gives vector biologists specific regulatory elements to manipulate, rather than entire genes, when trying to alter immune output.

The broader significance lies in connecting a classic piece of population genetics to modern genome biology. Inversions in Anopheles have been studied for decades as markers of ecological adaptation, used to distinguish sibling species and track their distributions across Africa. This study shows that the same inversions are not passive markers but active modulators of the regulatory genome, changing chromatin contacts, enhancer-promoter wiring, chromatin accessibility, and gene expression. Because the affected genes include major parasite antagonists, the 2La inversion plausibly influences malaria transmission partly through these architectural effects, offering a mechanistic explanation for the long-observed association between karyotype and infection phenotype.

The work also refines the mosquito regulatory genome itself. By integrating Micro-C contact maps, ATAC-seq accessibility profiles, RNA-seq expression data, and previously characterized enhancers within the Plasmodium susceptibility locus, the researchers assembled a high-resolution catalog of cis-regulatory architecture in Anopheles, including the placement of inversion breakpoints relative to TAD boundaries and enhancer clusters. For a vector that transmits a parasite responsible for hundreds of thousands of deaths each year, this level of regulatory detail matters. It highlights concrete candidates for future exploration in malaria control, whether through gene drive strategies that manipulate enhancer activity, through screens for natural variation in regulatory elements, or through studies of how chromatin architecture shapes the evolutionary arms race between mosquito immunity and Plasmodium infection. The methods developed here, from the cell-line karyotyping to the distance-normalized interaction analysis, give the field a toolkit for asking the same questions in other vectors and, indeed, in any species where structural variants shape the folded genome.

Subject of Research: How chromosomal inversions alter 3D chromatin architecture and gene regulation in Anopheles malaria mosquitoes

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

Article References: Taquet, K. S., Anderson, C. E., Vernick, K. D., & Riehle, M. M. (2026). Chromosomal inversions modulate 3D chromatin interactions and regulatory architecture in Anopheles gambiae. BMC Biology. https://doi.org/10.1186/s12915-026-02753-x

Image Credits: AI Generated

DOI: 10.1186/s12915-026-02753-x

Keywords: Anopheles gambiae, chromosomal inversion, 2La inversion, Micro-C, chromatin architecture, enhancer rewiring, gene regulation, malaria, Plasmodium, APL1, LRIM1, topologically associating domain