O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein–protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of the SMN1 gene. SMN protein expressed from a paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function, and adult longevity. These disease-related phenotypes are domain-specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages of Drosophila development. Due to a large maternal contribution of mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, we find that only baseline levels of SMN are required during larval stages, whereas high levels of protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Altogether, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at critical developmental timepoints that is conserved from Drosophila to humans.

Polycomb complexes regulate cell type–specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent ( H3.2 K36R ) or replication-independent ( H3.3 K36R ) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined ( H3.3 K36R H3.2 K36R ) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2 K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3 K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.

Polycomb complexes regulate cell-type specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2K36R) or -independent (H3.3K36R) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3K36RH3.2K36R) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive PREs (Polycomb Response Elements) located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.

27Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising 28 from homozygous deletion or mutation of the SMN1 gene. SMN protein expressed from a 29 paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN 30 levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that 31 regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that 32 SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a 33 pronounced temperature sensitivity that affects organismal viability, larval locomotor function, 34 and adult longevity. These disease-related phenotypes are domain-specific and result from 35 decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN 36 levels during various stages of Drosophila development. Due to a large maternal contribution of 37 mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, we 38 find that only baseline levels of SMN are required during larval stages, whereas high levels of 39 protein are required during pupation. This previously uncharacterized period of elevated SMN 40 expression, during which the majority of adult tissues are formed and differentiated, could be an 41 important and translationally relevant developmental stage in which to study SMN function. 42 Altogether, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining 43 SMN homeostasis and highlight the necessity for high SMN levels at critical developmental 44 timepoints that is conserved from Drosophila to humans. 4546 47 126 127 RESULTS 128 SMN Tudor domain mutants (TDMs) are temperature-sensitive 129 Previous work using SMA patient-derived Smn missense mutations, modeled in the fly, has 130 produced robust and reproducible findings. However, in one or two instances, we noticed 131 inconsistencies in the overall viability of a given fly line that could not be attributed to normal 132 biological noise. For example, in Praveen et al. (2014), the Smn F70S mutation line (hereafter F70S) 133 displayed a relatively mild phenotype, with an eclosion frequency similar to that of the Smn WT 134 transgene (hereafter WT). In contrast, Spring et al. (2019) reported a rather severe viability defect 135 for this same F70S line. The husbandry conditions used in each study were slightly different; the 136 experiments in the earlier work were performed at room temperature (~22°C) whereas in the 137 subsequent experiments, animals were kept at a constant 25°C. In addition, we qualitatively 138 157 SMN Tudor domain mutants display SMA-related phenotypes in response to small changes 158 in temperature 159 We next examined the effect of minor changes in temperature by raising animals at 22°C and 160 27°C. The SMN WT and T205I YG box domain mutant lines were used as controls. Although the 161 (2015). Spinal Muscular Atrophy: Review of a Child Onset Disease. Br. J. Med. Med. Res. 595 6, 647-66...