Biology subjects

Hamilton, B. A.

Publications and source records attributed to Hamilton, B. A..

9 recordsLinked to original sources

bioRxiv2026

Mysm1 mutations in meander tail mice cause anterior-selective cerebellum malformation

Mouse meander tail (mea) mutations produce kinked tails and selective malformation of the cerebellum anterior compartment. The anterior cerebellum defects are cell autonomous with respect to granule cell precursors, but the molecular basis has not been known. Myb-like, SWIRM, and MPN domain containing protein 1 (MYSM1) is a chromatin-associated deubiquitinase that promotes gene expression by removing monoubiquitin from histone H2A, among other targets. Loss of MYSM1 function in mice or humans results in bone marrow failure with defective maturation of B cell lineages. Here we show that extant mea alleles have mutations in Mysm1 and cause both neurological and hematological phenotypes, as do new non-complementing endonuclease-mediated mutations. Multimodal single-nucleus assays show Mysm1 effects on gene expression in several lineages and on the proportion of granule cell precursors by E14.5. Intriguingly, Mysm1 orthologs have been independently lost in several animal and fungal lineages, including yeast, flies, and nematodes. These results unite previously disconnected literature and demonstrate a requirement for MYSM1 activity in compartment-specific development of the cerebellum and suggest potential for compensatory pathways. Significance StatementPerturbations to core regulatory machinery often produce pleiotropic effects and even intensively studied systems can have significant phenotypic effects that were not assessed in models developed for a different purpose. Here we show that classical meander tail mice, characterized by ankylosing spondylitis in tail vertebrae and a cerebellum malformation that defined the anterior-posterior compartment boundary, have mutations in Mysm1, encoding a histone 2A deubiquitinase. We show pigmentation defects and hematopoietic abnormalities that model human disease. While Mysm1 mutations change gene expression patterns in many cerebellar cell types, they selectively decrease the proportion of granule cell lineages. Recurrent loss of Mysm1 orthologs across fungal and animal phylogenies suggests the potential for bypass mechanisms.

genetics↗

bioRxiv2025

Nonequivalence of Zfp423 premature termination codons in mice.

Genetic variants that introduce a premature termination codon (PTC) are often assumed equivalent and functionally null. Exceptions depend on the specific architectures of the affected mRNA and protein. Here we address phenotypic differences among early truncating variants of mouse Zfp423, whose phenotypes resemble Joubert Syndrome and Related Disorders (JSRD). We replicate quantitative differences previously seen between presumptive null PTC variants based on their position in the coding sequence. We show with reciprocal congenic strains that large phenotype differences between two PTC variants with the same predicted stop and reinitiation codons is due to the specific allele rather than different strain backgrounds, with no evidence for induced exon skipping. Differences in RNA structure, however, could influence translation rate across the affected exon. Using a reporter assay, we find differences in translational reinitiation between two deletion variants that corelate with predicted RNA structure rather than distance from the canonical initiation codon. These results confirm and extend earlier evidence for differences among Zfp423 PTC variants, identify parameters for translational reinitiation after an early termination codon, and reinforce caution in the null interpretation of early PTC variants.

genetics↗

bioRxiv2025

Tulp3 quantitative alleles titrate requirements for viability, brain development, and kidney homeostasis but do not suppress Zfp423 mutations in mice.

Tubby-like protein 3 (TULP3) regulates receptor trafficking in primary cilia and antagonizes SHH signaling. Tulp3 knockout mice are embryonic lethal with developmental abnormalities in multiple organs, while tissue-specific knockouts and viable missense alleles cause polycystic kidney disease. Human patients with TULP3 mutations present with variable, but often multi-organ fibrotic disease. We previously showed that mouse and human Tulp3 expression is negatively regulated by ZNF423, which is required for SHH sensitivity in some progenitor cell models. The level of TULP3 function required to prevent mutant phenotypes has not been known. Here we report a Tulp3 quantitative allelic series, designed by targeting the polypyrimidine tract 5 to the splice acceptor of a critical exon, that shows distinct dose-response effects on viability, brain overgrowth, weight gain, and cystic kidney disease. We find limited evidence for genetic interaction with Zfp423 null or hypomorphic mutations. Together, these results establish an approach to developing quantitative allelic series by exon exclusion, rank-order dose-sensitivity of Tulp3 phenotypes, and model thresholds for TULP3 function to prevent severe outcomes. Author SummaryTULP3 protein plays critical roles in regulating receptor trafficking and signaling in the primary cilium. Mutations in the TULP3 gene can cause severe, multi-organ disorders in both mice and humans, yet the amount of TULP3 activity needed to avoid these outcomes has been unclear. In this study, we used precise genome editing in mice to create a set of new Tulp3 gene variants that reduce TULP3 expression to varying degrees. This allowed us to test how much TULP3 is required for survival, normal brain and kidney development, and weight regulation. We found that as little as 5% of normal TULP3 levels is enough to avoid lethal birth defects, but still leads to obesity, mild brain overgrowth, and progressive kidney cysts preceded by reductions in cilium frequency and length in situ. The severity of these effects was related to TULP3 protein levels, highlighting a dose-dependent response. We also investigated whether reducing TULP3 levels would suppress brain abnormalities in Zfp423 mutant mice, based on prior evidence of a genetic interaction, but did not find evidence to support this effect. Our work provides a framework for understanding how varying levels of TULP3 affect various organ systems and offers a general strategy for creating quantitative genetic models of human disease.

genetics↗

bioRxiv2021

Ankfn1 vestibular defects in zebrafish require mutations in both ancestral and derived paralogs.

How and to what degree gene duplication events create regulatory innovation, redundancy, or neofunctionalization remain important questions in animal evolution and comparative genetics. Ankfn1 genes are single copy in most invertebrates, partially duplicated in jawed vertebrates, and only the derived copy retained in most mammals. Null mutations in the single mouse homolog have vestibular and neurological abnormalities. Null mutation of the single Drosophila homolog is typically lethal with severe sensorimotor deficits in rare survivors. The functions and potential redundancy of paralogs in species with two copies is not known. Here we define a vestibular role for Ankfn1 homologs in zebrafish based on simultaneous disruption of each locus. Zebrafish with both paralogs disrupted showed vestibular defects and early lethality from swim bladder inflation failure. One intact copy at either locus was sufficient to prevent major phenotypes. Our results show that vertebrate Ankfn1 genes are required for vestibular-related functions, with at least partial redundancy between ancestral and derived paralogs.

genetics↗

bioRxiv2021

Inducible Yeast Two-Hybrid with Quantitative Measures

The yeast two-hybrid (Y2H) assay has long been used to identify new protein-protein interaction pairs and to compare relative interaction strengths. Traditional Y2H formats may be limited, however, by use of constitutive strong promoters if expressed proteins have toxic effects or post-transcriptional expression differences in yeast among a comparison group. As a step toward more quantitative Y2H assays, we modified a common vector to use an inducible CUP1 promoter, which showed quantitative induction of several "bait" proteins with increasing copper concentration. Using mouse Nxf1 (homologous to yeast Mex67p) as a model bait, copper titration achieved levels that bracket levels obtained with the constitutive ADH1 promoter. Using a liquid growth assay for an auxotrophic reporter in multiwell plates allowed log-phase growth rate to be used as a measure of interaction strength. These data demonstrate the potential for quantitative comparisons of protein-protein interactions using the Y2H system.

genetics↗

bioRxiv2020

Strain-dependent modifier genes determine survival in Zfp423 mice

Zfp423 encodes a transcriptional regulatory protein that interacts with canonical signaling and lineage pathways. Mutations in mouse Zfp423 or its human ortholog ZNF423 are associated with a range of developmental abnormalities reminiscent of ciliopathies, including cerebellar vermis hypoplasia and other midline brain defects. Null mice have reduced viability in most strain backgrounds. Here we show complete lethality on a C57BL/6J background, dominant rescue in backcrosses to any of 13 partner strains, with strain-dependent survival frequencies, and evidence for a BALB/c-derived survival modifier locus on chromosome 5. Survival data indicate both perinatal and postnatal periods of lethality. Anatomical data from a hypomorphic gene trap allele observed on both C57BL/6J and BALB/c congenic backgrounds shows an aggregate effect of background on sensitivity to Zfp423 loss rather than a binary effect on viability.

genetics↗

bioRxiv2020

ZNF423 patient variants, truncations, and in-frame deletions in mice define an allele-dependent range of midline brain abnormalities

Interpreting rare variants remains a challenge in personal genomics, especially for disorders with several causal genes and for genes that cause multiple disorders. ZNF423 encodes a transcriptional regulatory protein that intersects several developmental pathways. ZNF423 has been implicated in rare neurodevelopmental disorders, consistent with midline brain defects in Zfp423-mutant mice, but pathogenic potential of most patient variants remains uncertain. We engineered ~50 patient-derived and small deletion variants into the highly-conserved mouse ortholog and examined neuroanatomical measures for 791 littermate pairs. Three substitutions previously asserted pathogenic appeared benign, while a fourth was effectively null. Heterozygous premature termination codon (PTC) variants showed mild haploabnormality, consistent with loss-of-function intolerance inferred from human population data. In-frame deletions of specific zinc fingers showed mild to moderate abnormalities, as did low-expression variants. These results affirm the need for functional validation of rare variants in biological context and demonstrate cost-effective modeling of neuroanatomical abnormalities in mice. AUTHOR SUMMARYGene identification in rare disorders is typically supported by finding different mutations of the same gene in multiple families with the same disorder. However, causal evidence for any specific mutation found in one or a few related individuals is weaker, especially if the disorder can be caused by any of several genes and the functional effect of the mutation is not certain. Experimental models can be helpful in testing causal effects, but only to the extent that the model is validated to recapitulate one or more aspects of the disorder. We used CRISPR/Cas9-based genome engineering to create a wide range of mutations in mouse Zfp423, whose human cognate is implicated in neurodevelopmental disorders, especially cerebellar vermis hypoplasia and Joubert syndrome. This large collection of animal models shows that both reduced Zfp423 expression, including heterozygosity for loss-of-function mutations, and normally-expressed domain deletions, including specific zinc finger domains, produce measureable abnormalities in midline development. Despite this high level of validation, most patient-derived amino acid substitution variants tested do not produce measureable effects. The single exception is a substitution, H1277Y, that destroys a structural element in the last zinc finger domain and results in dramatic loss of steady-state Zfp423 protein level.

genetics↗

bioRxiv2020

ZNF423 orthologs are highly constrained in vertebrates but show domain-level plasticity across invertebrate lineages

ZNF423 encodes 30 C2H2 zinc fingers that bind DNA and a variety of lineage- and signal-dependent transcription factors. ZNF423 genetic variants are proposed to cause neurodevelopmental and ciliopathy-related disorders in humans. Mouse models show midline brain defects, including cerebellar vermis hypoplasia, and defects in adipogenesis. Here I show strong protein sequence constraint among 165 vertebrate orthologs. In contrast, orthologs from invertebrate lineages, spanning larger time intervals, show substantial differences in zinc finger number, arrangement, and identity. A terminal zinc finger cluster common among other lineages was independently lost in vertebrates and insects. Surprisingly, a moderately-constrained non-C2H2 sequence with potential to form a C4-class zinc finger is a previously-unrecognized conserved feature of nearly all identified homologs. These results highlight evolutionary dynamics of a likely signal integration node across species with distinct developmental strategies and body plans. Functions of the newly identified C4-like sequence and lineage-specific fingers remain to be studied.

genetics↗

bioRxiv2019

The Lipid Elongation Enzyme ELOVL2 is a molecular regulator of aging in the retina

Methylation of the regulatory region of the Elongation Of Very Long Chain Fatty Acids-Like 2 (ELOVL2) gene, an enzyme involved in elongation of long-chain polyunsaturated fatty acids, is one of the most robust biomarkers of human age, but the critical question of whether ELOVL2 plays a functional role in molecular aging has not been resolved. Here, we report that Elovl2 regulates age-associated functional and anatomical aging in vivo, focusing on mouse retina, with direct relevance to age-related eye diseases. We show that an age-related decrease in Elovl2 expression is associated with increased DNA methylation of its promoter. Reversal of Elovl2 promoter hypermethylation in vivo through intravitreal injection of 5-Aza-2-deoxycytidine (5-aza-dc) leads to increased Elovl2 expression and rescue of age-related decline in visual function. Mice carrying a point mutation C234W that disrupts Elovl2-specific enzymatic activity show electrophysiological characteristics of premature visual decline, as well as early appearance of autofluorescent deposits, well-established markers of aging in the mouse retina. Finally, we find deposits underneath the retinal pigment epithelium in Elovl2 mutant mice, containing components of complement system and lipid metabolism. These findings indicate that ELOVL2 activity regulates aging in mouse retina, provide a molecular link between polyunsaturated fatty acids elongation and visual functions, and suggest novel therapeutic strategies for treatment of age-related eye diseases.

molecular biology↗