Biology subjects

Reid, W.

Publications and source records attributed to Reid, W..

6 recordsLinked to original sources

bioRxiv2025

A systems genetics approach identifies roles for proteasome factors in heart development and congenital heart defects

Congenital heart defects (CHDs) occur in about 1% of live births and are the leading cause of infant death due to birth defects. While there have been remarkable efforts to pursue large-scale whole-exome and genome sequencing studies on CHD patient cohorts, it is estimated that these approaches have thus far accounted for only about 50% of the genetic contribution to CHDs. We sought to take a new approach to identify genetic causes of CHDs. By combining analyses of genes that are under strong selective constraint along with published embryonic heart transcriptomes, we identified over 200 new candidate genes for CHDs. We utilized protein-protein interaction (PPI) network analysis to identify a functionally-related subnetwork consisting of known CHD genes as well as genes encoding proteasome factors, in particular POMP, PSMA6, PSMA7, PSMD3, and PSMD6. We used CRISPR screening in zebrafish embryos to preliminarily identify roles for the PPI subnetwork genes in heart development. We then used CRISPR to create new mutant zebrafish strains for two of the proteasome genes in the subnetwork: pomp and psmd6. Phenotypic analyses confirm critical roles for pomp and psmd6 in heart development. In particular, we find defects in myocardial cell shapes and in outflow tract development in pomp and psmd6 mutant zebrafish embryos, and these phenotypes have been observed in other zebrafish CHD-gene mutants. Our study provides a novel systems genetics approach to further our understanding of the genetic causes of human CHDs. Author SummaryCongenital heart defects (CHDs) are birth defects resulting in the abnormal structure and function of the heart. Genetic mutations are a significant cause of CHDs. Many studies have used genome sequencing of CHD patients and their families to gain knowledge of the mutations that cause CHDs. However, these studies have only found about 50 percent of the genes involved in CHDs. Here, we take a new approach to identifying genes that are required for heart development and that may cause CHDs, generating a list of over 200 candidate genes. Using multiple data systems, including human exome sequences, mouse transcriptomes, and protein-protein interactions, we identify a small group of related potential CHD genes that includes multiple genes encoding proteasome factors. These factors are known to be important for assembling the proteasome, a large molecular machine that eliminates unneeded or damaged proteins from the cell, but which has not been shown to contribute to CHD. We use a CRISPR-based approach in zebrafish to specifically eliminate some of these candidate genes and reveal new roles for proteasome genes in heart development. We show that loss of proteasome gene functions leads to zebrafish heart defects that resemble those seen in other zebrafish CHD-gene mutants. This study shows that a proteasome gene family contributes to heart development, advancing our understanding of the causes of CHDs. By increasing our understanding of the genetic causes of CHDs, our work should lead to better screening, more accurate diagnoses, and, ultimately, better treatments for these disorders.

genetics↗

bioRxiv2023

Conserved and novel enhancers regulate expression of Aedes aegypti single-minded in the embryonic ventral midline

Transcriptional cis-regulatory modules, e.g., enhancers, control the time and location of metazoan gene expression. While changes in enhancers can provide a powerful force for evolution, there is also significant deep conservation of enhancers for developmentally important genes, with function and sequence characteristics maintained over hundreds of millions of years of divergence. Not well understood, however, is how the overall regulatory composition of a locus evolves, with important outstanding questions such as how many enhancers are conserved vs. novel, and to what extent are the locations of conserved enhancers within a locus maintained? We begin here to address these questions with a comparison of the respective single-minded (sim) loci in the two dipteran species Drosophila melanogaster (fruit fly) and Aedes aegypti (mosquito). sim encodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify two enhancers in the A. aegypti sim locus and demonstrate that they function equivalently in both transgenic flies and transgenic mosquitoes. One A. aegypti enhancer is highly similar to known Drosophila counterparts in its activity, location, and autoregulatory capability. The other differs from any known Drosophila sim enhancers with a novel location, failure to autoregulate, and regulation of expression in a unique subset of midline cells. Our results suggest that the conserved pattern of sim expression in the two species is the result of both conserved and novel regulatory sequences. Further examination of this locus will help to illuminate how the overall regulatory landscape of a conserved developmental gene evolves. AUTHOR SUMMARYThe expression patterns and roles of genes, especially those involved in core developmental processes, are often conserved over vast evolutionary distances. Paradoxically, the DNA sequences surrounding these genes, which contain the cis-regulatory sequences (enhancers) that regulate gene expression, tend to be highly diverged. The manner and extent to which enhancers are functionally conserved, and how the overall organization of regulatory sequences within a locus is preserved or restructured, is not well understood. In this paper, we investigate these questions by identifying enhancers controlling expression of a master nervous system regulatory gene named sim in the mosquito Aedes aegypti, and comparing their functions and locations to those in the well-characterized sim locus of the fruit fly Drosophila melanogaster. Our results suggest that the two species generate identical patterns of sim expression through a mix of conserved and novel regulatory sequences. Continued exploration of the sim locus in these two species will help to build a comprehensive picture of how a regulatory locus for a master developmental regulator has evolved.

evolutionary biology↗

bioRxiv2022

SARS-CoV-2 triggers Golgi fragmentation via downregulation of GRASP55 to facilitate viral trafficking

The COVID-19 pandemic is caused by SARS-CoV-2, an enveloped RNA virus. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Disrupting Golgi function with small molecules strongly inhibits viral infection. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. TGN46 depletion and GRASP55 expression additively inhibit viral secretion, indicating that they act at different stages. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to control viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.

cell biology↗

bioRxiv2021

Golgi structural defect impairs glycosaminoglycan synthesis, sulfation, and secretion

Synthesis of glycosaminoglycans such as heparan sulfate (HS) and chondroitin sulfate (CS) occurs in the lumen of the Golgi but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNacT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans.

cell biology↗

bioRxiv2021

GRASP55 regulates mutant huntingtin unconventional secretion and aggregation

Recent studies demonstrated that the Golgi stacking proteins, GRASPs, especially GRASP55, regulate Golgi-independent unconventional secretion, but the underlying mechanism remains unknown. Here, we used mutant huntingtin (Htt-Q74) as a model system to address this question. Our results demonstrate that Htt secretion is GRASP55- and autophagy-dependent, and is enhanced under stress conditions such as starvation and ER stress. Mechanistically, GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the ERGIC lumen. Moreover, GRASP55 level is upregulated by various stresses to facilitate unconventional secretion, while inhibition of Htt-Q74 secretion by GRASP55 knockout enhances Htt aggregation and toxicity. Lastly, comprehensive secretomic analysis identified novel cargoes secreted by the same unconventional pathway, such as TAGLN, PAICS and PRDX1. This study provides important information on the role of GRASP55 in unconventional protein secretion and Huntingtons disease progression.

cell biology↗

bioRxiv2021

Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line , {Delta}aslrim1 , by embryonic genome editing using CRISPR-Cas9. {Delta}aslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of {Delta}aslrim1 and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when {Delta}aslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

microbiology↗