Faculty Bibliography

[This corrects the article DOI: 10.1016/j.csbj.2018.09.003.].

Purpose: Heterozygous pathogenic variants in various FOXP genes cause specific developmental disorders. The phenotype associated with heterozygous variants in FOXP4 has not been previously described.
Method(s): We assembled a cohort of eight individuals with heterozygous and mostly de novo variants in FOXP4: seven individuals with six different missense variants and one individual with a frameshift variant. We collected clinical data to delineate the phenotypic spectrum, and used in silico analyses and functional cell-based assays to assess pathogenicity of the variants.
Result(s): We collected clinical data for six individuals: five individuals with a missense variant in the forkhead box DNA-binding domain of FOXP4, and one individual with a truncating variant. Overlapping features included speech and language delays, growth abnormalities, congenital diaphragmatic hernia, cervical spine abnormalities, and ptosis. Luciferase assays showed loss-of-function effects for all these variants, and aberrant subcellular localization patterns were seen in a subset. The remaining two missense variants were located outside the functional domains of FOXP4, and showed transcriptional repressor capacities and localization patterns similar to the wild-type protein.
Conclusion(s): Collectively, our findings show that heterozygous loss-of-function variants in FOXP4 are associated with an autosomal dominant neurodevelopmental disorder with speech/language delays, growth defects, and variable congenital abnormalities.
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Long-noncoding RNAs (lncRNAs) are emerging as regulators of development and disease. lncRNAs are expressed in exquisitely precise expression patterns in vivo and many interact with chromatin to regulate gene expression. However, the limited sensitivity of RNA-purification techniques has precluded the identification of genomic targets of cell-type specific lncRNAs. RNA-DamID is a powerful new approach to understand the mechanisms by which lncRNAs act in vivo. RNA-DamID is highly sensitive and accurate, and can resolve cell-type-specific chromatin binding patterns without cell isolation. The determinants of RNA-chromatin interactions can be identified with RNA-DamID by analyzing RNA and protein cofactor mutants. Here we describe how to implement RNA-DamID and the design considerations to take into account to accurately identify lncRNA-chromatin interactions in vivo.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction¹. A unique GPCR that is known to require heterodimerization for function^2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands^7,8, while GABAB2 couples with G proteins^9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail¹⁵. Although the VFT heterodimer structure has been resolved¹⁶, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.
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We write to introduce our novel group formed to confront some of the issues raised by the COVID-19 pandemic. Information about the group, which we named "cure COVid for Ever and for All" (RxCOVEA), its dynamic membership (changing regularly), and some of its activities-described in more technical detail for expert perusal and commentary-are available upon request.

The zona pellucida (ZP) is an extracellular matrix (ECM) that surrounds all mammalian oocytes, eggs, and embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse and human ZP is composed of three or four unique proteins, respectively, called ZP1-4, that are synthesized, processed, and secreted by oocytes during their growth phase. All ZP proteins have a zona pellucida domain (ZPD) that consists of ≈270 amino acids and has 8 conserved Cys residues present as four intramolecular disulfides. Secreted ZP proteins assemble into long fibrils around growing oocytes with ZP2-ZP3 dimers located periodically along the fibrils. The fibrils are cross-linked by ZP1 to form a thick, transparent ECM to which sperm must first bind and then penetrate during fertilization of eggs. Inactivation of mouse ZP1, ZP2, or ZP3 by gene targeting affects both ZP formation around oocytes and fertility. Female mice with eggs that lack a ZP due to inactivation of either ZP2 or ZP3 are completely infertile, whereas inactivation of ZP1 results in construction of an abnormal ZP and reduced fertility. Results of a large number of studies of infertile female patients strongly suggest that gene sequence variations (GSV) in human ZP1, ZP2, or ZP3 due to point, missense, or frameshift mutations have similar deleterious effects on ZP formation and female fertility. These findings are discussed in light of our current knowledge of ZP protein synthesis, processing, secretion, and assembly.

Mitochondria and their associated membranes actively participate in biosynthesis, trafficking, and degradation of cellular phospholipids. Two crucial lipid biosynthetic activities of mitochondria include (i) the decarboxylation of phosphatidylserine to phosphatidylethanolamine and (ii) the de novo synthesis of cardiolipin. Here we describe protocols to measure these two activities, applying isotope-labeled or exogenous substrates in combination with thin-layer chromatography or mass spectrometry.