Atp7a determines a hierarchy of copper metabolism essential for notochord development

SummaryThe critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects

Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

Lysine acetyltransferase 6A (KAT6A) and its paralog KAT6B form stoichiometric complexes with bromodomain- and PHD finger-containing protein 1 (BRPF1) for acetylation of histone H3 at lysine 23 (H3K23). We report that these complexes also catalyze H3K23 propionylation in vitro and in vivo. Immunofluorescence microscopy and ATAC-See revealed the association of this modification with active chromatin. Brpf1 deletion obliterates the acylation in mouse embryos and fibroblasts. Moreover, we identify BRPF1 variants in 12 previously unidentified cases of syndromic intellectual disability and demonstrate that these cases and known BRPF1 variants impair H3K23 propionylation. Cardiac anomalies are present in a subset of the cases. H3K23 acylation is also impaired by cancer-derived somatic BRPF1 mutations. Valproate, vorinostat, propionate and butyrate promote H3K23 acylation. These results reveal the dual functionality of BRPF1-KAT6 complexes, shed light on mechanisms underlying related developmental disorders and various cancers, and suggest mutation-based therapy for medical conditions with deficient histone acylation

Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

Lysine acetyltransferase 6A (KAT6A) and its paralog KAT6B form stoichiometric complexes with bromodomain- and PHD finger-containing protein 1 (BRPF1) for acetylation of histone H3 at lysine 23 (H3K23). We report that these complexes also catalyze H3K23 propionylati

SLC35A2â CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals

Pathogenic de novo variants in the Xâ linked gene SLC35A2 encoding the major Golgiâ localized UDPâ galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2â congenital disorders of glycosylation (CDG; formerly CDGâ IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin Nâ glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2â CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2â dependent UDPâ galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wildâ type to mutant alleles in fibroblasts from affected individuals.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/1/humu23731_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/2/humu23731-sup-0001-Supp_Mat__2019.2.10_.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/3/humu23731.pd

Promotion of Cell Viability and Histone Gene Expression by the Acetyltransferase Gcn5 and the Protein Phosphatase PP2A in Saccharomyces cerevisiae

Histone modifications direct chromatin-templated events in the genome and regulate access to DNA sequence information. There are multiple types of modifications, and a common feature is their dynamic nature. An essential step for understanding their regulation, therefore, lies in characterizing the enzymes responsible for adding and removing histone modifications. Starting with a dosage-suppressor screen in Saccharomyces cerevisiae, we have discovered a functional interaction between the acetyltransferase Gcn5 and the protein phosphatase 2A (PP2A) complex, two factors that regulate post-translational modifications. We find that RTS1, one of two genes encoding PP2A regulatory subunits, is a robust and specific high-copy suppressor of temperature sensitivity of gcn5∆ and a subset of other gcn5∆ phenotypes. Conversely, loss of both PP2A(Rts1) and Gcn5 function in the SAGA and SLIK/SALSA complexes is lethal. RTS1 does not restore global transcriptional defects in gcn5∆; however, histone gene expression is restored, suggesting that the mechanism of RTS1 rescue includes restoration of specific cell cycle transcripts. Pointing to new mechanisms of acetylation-phosphorylation cross-talk, RTS1 high-copy rescue of gcn5∆ growth requires two residues of H2B that are phosphorylated in human cells. These data highlight the potential significance of dynamic phosphorylation and dephosphorylation of these deeply conserved histone residues for cell viability

Promotion of Cell Viability and Histone Gene Expression by the Acetyltransferase Gcn5 and the Protein Phosphatase PP2A in Saccharomyces cerevisiae

Histone modifications direct chromatin-templated events in the genome and regulate access to DNA sequence information. There are multiple types of modifications, and a common feature is their dynamic nature. An essential step for understanding their regulation, therefore, lies in characterizing the enzymes responsible for adding and removing histone modifications. Starting with a dosage-suppressor screen in Saccharomyces cerevisiae, we have discovered a functional interaction between the acetyltransferase Gcn5 and the protein phosphatase 2A (PP2A) complex, two factors that regulate post-translational modifications. We find that RTS1, one of two genes encoding PP2A regulatory subunits, is a robust and specific high-copy suppressor of temperature sensitivity of gcn5∆ and a subset of other gcn5∆ phenotypes. Conversely, loss of both PP2A(Rts1) and Gcn5 function in the SAGA and SLIK/SALSA complexes is lethal. RTS1 does not restore global transcriptional defects in gcn5∆; however, histone gene expression is restored, suggesting that the mechanism of RTS1 rescue includes restoration of specific cell cycle transcripts. Pointing to new mechanisms of acetylation–phosphorylation cross-talk, RTS1 high-copy rescue of gcn5∆ growth requires two residues of H2B that are phosphorylated in human cells. These data highlight the potential significance of dynamic phosphorylation and dephosphorylation of these deeply conserved histone residues for cell viability

Genetic and biochemical interactions between SCP160 and EAP1 in yeast

Scp160p is a multiple KH-domain RNA-binding protein in yeast known to associate with polyribosomes as an mRNP component, although its biological role remains unclear. As a genetic approach to examine Scp160p function, we applied an ethyl methanesulfonate (EMS) screen for loci synthetically lethal with scp160 loss, and identified a single candidate gene, EAP1, whose protein product functions in translation as an eIF4E-binding protein, with additional uncharacterized spindle pole body functions. To reconfirm scp160/eap1 synthetic lethality, we constructed a strain null for both genes, supported by an SCP160 maintenance plasmid. We used this strain to establish a quantitative assay for both Scp160p and Eap1p functions in vivo, and applied this assay to demonstrate that Y109A EAP1, a previously described allele of EAP1 that cannot bind eIF4E, is markedly impaired with regard to its SCP160-related activity. In addition, we explored the possibility of physical interaction between Eap1p and Scp160p, and discovered that Eap1p associates with Scp160p-containing complexes in an RNA-dependent manner. Finally, we probed the impact of EAP1 loss on Scp160p, and vice versa, and found that loss of each gene resulted in a significant change in either the complex associations or subcellular distribution of the other protein. These results clearly support the hypothesis that Scp160p plays a role in translation, demonstrate that the interaction of SCP160 and EAP1 is biologically significant, and provide important tools for future studies of the in vivo functions of both genes