Disease‐associated missense variants in ZBTB18 disrupt DNA binding and impair the development of neurons within the embryonic cerebral cortex

Abstract

The activities of DNA‐binding transcription factors, such as the multi‐zinc‐finger protein ZBTB18 (also known as RP58, or ZNF238), are essential to coordinate mammalian neurodevelopment, including the birth and radial migration of newborn neurons within the fetal brain. In humans, the majority of disease‐associated missense mutations in ZBTB18 lie within the DNA‐binding zinc‐finger domain and are associated with brain developmental disorder, yet the molecular mechanisms explaining their role in disease remain unclear. To address this, we developed in silico models of ZBTB18, bound to DNA, and discovered that half of the missense variants map to residues (Asn461, Arg464, Glu486) predicted to be essential to sequence‐specific DNA contact, whereas others map to residues (Leu434, Tyr447, Arg495) with limited contributions to DNA binding. We studied pathogenic variants to residues with close (N461S) and limited (R495G) DNA contact and found that each bound DNA promiscuously, displayed altered transcriptional regulatory activity in vitro, and influenced the radial migration of newborn neurons in vivo in different ways. Taken together, our results suggest that altered transcriptional regulation could represent an important pathological mechanism for ZBTB18 missense variants in brain developmental disease.

General population ZBTB18 missense variants influence DNA binding and transcriptional regulation

“…Firefly luciferase expression constructs were utilized for luciferase assays (Four separate Rnd2 3′-enhancer constructs comprising E1 and E2 motifs; E1 mut and E2 motifs; E1 and E2 mut motifs; and E1 mut and E2 mut motifs were previously reported (Hemming et al, 2019;Heng et al, 2008Heng et al, , 2013 undertaken with this previously reported pIDT-AMP-WT construct.…”

“…The ZBTB18 gene encodes a polypeptide comprising an N-terminal BTB domain for protein-protein interaction, as well as four Cys2-His2-like (C2H2) ZFs at its C terminus for DNA binding to a consensus DNA motif, defined as 5′-[AC]ACANCTG [GT][AC]-3′ (Aoki et al, 1998;Hirai et al, 2012). Recently, we and others have reported that disease-associated ZBTB18 missense variants cluster within the C-terminal ZF DNA-binding domain (Farwell et al, 2015;Hemming et al, 2019;Landrum et al, 2018;Rauch et al, 2012;van der Schoot et al, 2018). Furthermore, we characterized two disease-associated ZBTB18 missense variants (N461S (Farwell et al, 2015) and R495G (Rauch et al, 2012)) and found that each disrupted its DNA-binding specificity, transcriptional regulatory function and neurodevelopmental signaling capacity in different ways (Hemming et al, 2019).…”

“…Recently, we and others have reported that disease-associated ZBTB18 missense variants cluster within the C-terminal ZF DNA-binding domain (Farwell et al, 2015;Hemming et al, 2019;Landrum et al, 2018;Rauch et al, 2012;van der Schoot et al, 2018). Furthermore, we characterized two disease-associated ZBTB18 missense variants (N461S (Farwell et al, 2015) and R495G (Rauch et al, 2012)) and found that each disrupted its DNA-binding specificity, transcriptional regulatory function and neurodevelopmental signaling capacity in different ways (Hemming et al, 2019). However, despite the detection of general population missense variants within the DNA-binding domain of ZBTB18, their functional impact remains poorly understood.…”

“…K435), as well as in ZBTB18-E2 (S390 and R403) models (Figure 2b and (Figure 2c and Table S10 and data not shown for ZBTB18-E2). Also, these residues (11 of 14, 78.57% [95% CI, 73.67-81.98) predominantly lie within the third and fourth ZF motifs which significantly contribute to DNAbinding energies within both complexes (Hemming et al, 2019).…”

“…We next performed binding energy decomposition on models of missense variants bound to native DNA motifs E1 and E2, so that we could better understand the site-specific impact of altered protein-DNA contacts for each species. We recently reported that energetic contributions from three key residues, Tyr458, Asn461, and His493, were critical to explain binding affinity and sequence specificity of ZBTB18 to E1 and E2 motifs (Hemming et al, 2019).…”