The research team, led by Nana Matoba, Jessica McAfee, Hyejung Won and Jason Stein at the University of North Carolina at Chapel Hill, deployed a massively parallel reporter assay, or MPRA, to interrogate 9,092 genetic variants associated with human cortical structure. The logic of the assay is elegant. Each candidate variant is synthesized as a short 150-base-pair segment of DNA, cloned into a plasmid upstream of a minimal promoter that drives a luciferase reporter gene, and tagged with a random 20-base-pair barcode. When the pooled library is introduced into cells, the amount of barcode RNA produced by each construct reflects the regulatory power of the DNA segment attached to it. By sequencing both the RNA transcripts and the plasmid DNA, researchers can calculate a transcription rate for every element, and by testing both alleles of a variant side by side, they can detect whether one version of the sequence drives expression more strongly than the other.

The cellular context matters enormously here. The team performed the assay in primary human neural progenitor cells, the fetal-derived stem cells that generate the neurons of the developing cortex. Previous work had shown that variants associated with cortical structure are enriched in regulatory regions active in these progenitors and colocalize with genetic effects on chromatin accessibility and gene expression in the same cells. This supports a model in which noncoding variants act during prenatal development, altering transcription factor binding and gene expression in progenitor cells, and thereby influencing decisions about cell proliferation and fate that ultimately determine cortical size in adulthood. The researchers transfected the library into 16 biological replicates, and after 24 hours split the cultures: half received a vehicle control, while half were treated with CHIR99021, a potent inhibitor of GSK3β that activates the Wnt signaling pathway, a key developmental signal known to influence brain size.

The results were striking in their scale and reproducibility. Regulatory activity measurements correlated between replicates with a median Pearson’s coefficient of 0.99, and the positive control promoters behaved exactly as expected. Among 17,837 elements tested under baseline conditions, 988 showed regulatory activity significantly above the negative controls, representing 918 variants drawn from 150 of the 198 cortical-structure-associated loci examined—meaning that 76 percent of the loci contained at least one variant with detectable regulatory function. More than half of the active variants also showed allelic effects, meaning one DNA letter change measurably altered transcription. The team dubbed these expression-modulating variants, or emVars, and they represent the strongest candidates for the true causal variants hiding within blocks of correlated genetic variation that have long frustrated geneticists.

Perhaps the most surprising finding came when the researchers asked what sequence features distinguished the active elements. Active elements were dramatically enriched in short interspersed nuclear elements, or SINEs—a class of retrotransposons, the so-called jumping genes that copy and paste themselves throughout the genome. Within the SINE family, the enrichment was driven almost entirely by Alu elements, primate-specific repeats that have expanded in prevalence alongside increases in brain size over human evolution. A typical Alu element carries two conserved sequences in its left arm, the A box and the B box, which function as promoters for RNA polymerase III. The most active MPRA elements sat precisely in this left arm, and elements containing both boxes showed the highest activity of all. Variants with the strongest allelic effects clustered near these promoter boxes, and even a scrambled negative control that accidentally contained a B-box-like sequence showed strong activity—a striking confirmation of the mechanism.

Skeptics might worry that such activity is an artifact of the episomal plasmid system, in which DNA is not packaged into chromatin as it would be in the native genome. The team addressed this concern with multiple lines of endogenous evidence. Fetal brain tissue shows among the lowest levels of repressive chromatin marks, such as H3K9me3 and H3K27me3, deposited on Alu elements of any tissue in the body, and the MPRA-active Alu elements are rarely repressed in fetal brain specifically. The active elements were also enriched for enhancer RNAs and promoter upstream antisense RNAs, noncoding transcripts previously shown to mediate enhancer-promoter communication through Alu sequences. Finally, using CUT&RUN profiling, the researchers showed that MPRA-active Alu elements are significantly enriched within binding sites of TFIIIC, a transcription factor complex associated with polymerase III and Alu activity. Together, these data suggest that the reporter activity reflects genuine regulatory potential in the developing brain.

The evolutionary implications are tantalizing. Alu elements can be dated by their accumulated mutations, measured as sequence divergence from the ancestral consensus. The younger Alu elements—those with fewer than 100 mismatches per thousand bases—showed significantly higher regulatory activity than older ones, particularly when located in the left arm of the element. Because Alu insertions have proliferated in the primate lineage in step with brain size expansion, the findings suggest that relatively recent retrotransposon insertions created new regulatory elements that influenced neurodevelopmental genes and may have contributed to the expansion of the human cortex. The authors caution that a neutral decay of regulatory potential in older elements over evolutionary time could also explain the pattern, but the convergence with known primate-specific biology makes the adaptive interpretation compelling.

The study also connected individual variants to specific transcription factors and, remarkably, to specific brain regions. Active elements were enriched for binding motifs of three transcription factor clusters—ZNF135/ZNF460, MEF2A-D and Zfx—and the magnitude of a variant’s allelic effect correlated strongly with how severely it disrupted a predicted motif. Disruption of RARA, RARG, ZNF135 and NR2F1 motifs produced the largest impacts. Intriguingly, the consensus sequences of recently evolved Alu subfamilies contain retinoic acid receptor binding sites, linking the transposon story to developmental signaling. Even more strikingly, when the team compared the regional pattern of a variant’s effect on cortical structure with the expression of the disrupted transcription factor across prenatal brain regions from the BrainSpan Atlas, they found significant correlations for seven variants. A variant that disrupts the RARG binding motif, for example, reduced surface area most strongly in regions where RARG expression was highest—suggesting that the regional specificity of genetic effects is governed by where the relevant transcription factors are actually present.

Wnt stimulation added another layer of complexity. Under CHIR treatment, the team detected a similar number of active elements, but 32 elements gained activity specifically under stimulation while seven lost it, and 12 variants showed allelic effects that differed significantly between conditions. One example, rs4670759, associated with the surface area of the inferior temporal cortex, showed enhanced activity and increased chromatin accessibility under Wnt stimulation; it sits in the promoter of CDC42EP3, a gene involved in actin cytoskeleton formation whose expression rises with Wnt activation, and the risk allele is predicted to disrupt a PLAGL2 motif that is itself upregulated by the pathway. Another variant, rs2802295 within an intron of FOXO3, showed an allelic effect only under stimulation near a Wnt-responsive element. The team then used CRISPR interference to silence that element in progenitor cells and confirmed that FOXO3 expression dropped—experimentally tying a cortical-structure-associated variant to its target gene in a condition-specific manner.

The study is not without limitations. The MPRA measures regulatory potential on episomal plasmids, and the overlap with endogenous chromatin accessibility quantitative trait loci was limited, indicating that native chromatin context modifies some effects. The analysis also focused on variants tested primarily in populations of European ancestry and did not address sex-dependent effects. Nevertheless, by experimentally separating correlated variants, identifying the transcription factors they disrupt, and demonstrating condition-dependent function, the work delivers a genome-scale functional framework for one of the most complex traits in human neuroimaging genetics. It transforms a catalog of statistical associations into a mechanistic hypothesis space—one in which ancient jumping genes, developmental signaling pathways and spatially patterned transcription factors jointly sculpt the most distinctive feature of the human brain.

Subject of Research: Functional characterization of noncoding genetic variants associated with human cortical structure using massively parallel reporter assays in neural progenitor cells

Article Title: Massively parallel assessment of gene regulatory activity at human cortical-structure-associated variants

Article References: Matoba, N., McAfee, J. C., Krupa, O., Beltran, A. A., Bell, J. L., Le, B. D., Valone, J. M., Min, H., Crawford, G. E., Raab, J. R., Won, H., & Stein, J. L. (2026). Massively parallel assessment of gene regulatory activity at human cortical-structure-associated variants. Nature Neuroscience. https://doi.org/10.1038/s41593-026-02454-2

Image Credits: AI Generated

DOI: 10.1038/s41593-026-02454-2

Keywords: cortical structure, massively parallel reporter assay, noncoding variants, Alu elements, neural progenitor cells, Wnt signaling, transcription factors, ENIGMA, gene regulation, brain development, retrotransposons, CRISPRi