General population ZBTB18 missense variants influence DNA binding and transcriptional regulation

Abstract

Genetic variation of the multi‐zinc finger BTB domain transcription factor ZBTB18 can cause a spectrum of human neurodevelopmental disorders, but the underlying mechanisms are not well understood. Recently, we reported that pathogenic, de novo ZBTB18 missense mutations alter its DNA‐binding specificity and gene regulatory functions, leading to human neurodevelopmental disease. However, the functional impact of the general population ZBTB18 missense variants is unknown. Here, we investigated such variants documented in the Genome Aggregation Database (gnomAD) to discover that ZBTB gene family members are intolerant to loss‐of‐function and missense mutations, but not synonymous mutations. We studied ZBTB18 as a protein–DNA complex to find that general population missense variants are rare, and disproportionately map to non‐DNA‐contact residues, in contrast to the majority of disease‐associated variants that map to DNA‐contact residues, essential to motif binding. We studied a selection of variants (n = 12), which spans the multi‐zinc finger region to find 58.3% (7/12) of variants displayed altered DNA binding, 41.6% (5/12) exhibited altered transcriptional activity in a luciferase reporter assay, 33.3% (4/12) exhibited altered DNA binding and transcriptional activity, whereas 33.3% (4/12) displayed a negligible functional impact. Our results demonstrate that general population variants, while rare, can influence ZBTB18 function, with potential consequences for neurodevelopment, homeostasis, and disease.

A novel heterozygous ZBTB18 missense mutation in a family with non-syndromic intellectual disability and structural brain abnormalities

“…Thirdly, our molecular modelling studies reveal that His441 is critical as a zinc-coordinating reside in the second zincnger motif. As all four zinc ngers are essential for ZBTB18 to bind DNA [31,55], a His441Gln mutation could destabilize the protein, disrupt sequence-speci c binding, alter transcriptional regulation in cells, or all of the above. Indeed, recent studies of a p.Asn461Ser mutation detected in a child with ID [46] demonstrated that such a variant not only resulted in reduced steady-state levels of the mutant protein, but also compromised sequence-speci c DNA binding and transcriptional repression [31].…”

Understanding the impact of ZBTB18 missense variation on transcription factor function in neurodevelopment and disease

“…More broadly, it is tempting to speculate that general population missense variants within conserved domains function as modifier alleles to influence TF signalling within cells. Nevertheless, it is relevant to consider that the weak yet measurable functional impact of general population missense ZBTB18 variants stands in stark contrast to disease-associated variants, the latter of which are endowed with strong effects on transcriptional regulation (Hemming et al, 2020).…”

“…luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSAs), in utero electroporation (IUE) studies; as well as as well as more recently reported screening methods (e.g. multi-plex reporter assays (MPRAs) (Mulvey, Lagunas, & Dougherty, 2021), mammalian targeted damID (MaTaDa) (Cheetham et al, 2018), Cut&Run (Meers, Bryson, Henikoff, & Henikoff, 2019)) and, where feasible, binding free energy calculations (Blake et al, 2021;Hemming et al, 2019;Hemming et al, 2020)) in order to study the DNA-binding, protein-protein interaction and transcriptional regulatory signalling properties of TFs as well as their query variants ( N/A Note: The severity of functional missense variants are defined by their impact on TF proteins to signal via co-operation, competition as well as combination. Category 0 variants show negligible functional impact, based upon these three mechanistic criteria.…”

“…Given that disease‐associated variants to conserved domains for TF genes such as to FOXG1 (Han et al, 2019) and ZBTB18 (Hemming et al, 2019) can disrupt protein function, does it follow that general population missense variants to such domains are functionally benign? In the case of ZBTB18, where the majority of disease‐associated missense variants map to the C‐terminal, zinc finger‐containing DNA‐binding region (Hemming, Blake, Agostino, & Heng, 2020), we recently investigated a subset of general population ZBTB18 missense variants within this region to find that the majority of these (8 out of 12) influence DNA‐binding, transcriptional regulation, or both (Hemming et al, 2020). Given such evidence for functional impact, it would be noteworthy to determine to what extent functional general population ZBTB18 missense variants influence TF combination , competition and co‐operation .…”

“…More broadly, it is tempting to speculate that general population missense variants within conserved domains function as modifier alleles to influence TF signalling within cells. Nevertheless, it is relevant to consider that the weak yet measurable functional impact of general population missense ZBTB18 variants stands in stark contrast to disease‐associated variants, the latter of which are endowed with strong effects on transcriptional regulation (Hemming et al, 2020). Furthermore, we recently conducted a series of molecular modelling studies of ZBTB18 and four native DNA binding sequences to find that disease‐associated (clinical) missense variants and general population missense variants could be delineated on the basis of their capacity to disrupt DNA binding (Blake et al, 2021).…”