bamSliceR: cross-cohort variant and allelic bias analysis for rare variants and rare diseases.

Rare diseases and conditions create unique challenges for genetic epidemiologists precisely because cases and samples are scarce. In recent years, whole-genome and whole-transcriptome sequencing (WGS/WTS) have eased the study of rare genetic variants. Paired WGS and WTS data are ideal, but logistical and financial constraints often preclude generating paired WGS and WTS data. Thus, many databases contain a patchwork of specimens with either WGS or WTS data, but only a minority of samples have both. The NCI Genomic Data Commons facilitates controlled access to genomic and transcriptomic data for thousands of subjects, many with unpaired sequencing results. Local reanalysis of expressed variants across whole transcriptomes requires significant data storage, compute, and expertise. We developed the bamSliceR package to facilitate swift transition from aligned sequence reads to expressed variant characterization. bamSliceR leverages the NCI Genomic Data Commons API to query genomic sub-regions of aligned sequence reads from specimens identified through the robust Bioconductor ecosystem. We demonstrate how population-scale targeted genomic analysis can be completed using orders of magnitude fewer resources in this fashion, with minimal compute burden. We demonstrate pilot results from bamSliceR for the TARGET pediatric AML and BEAT-AML projects, where identification of rare but recurrent somatic variants directly yields biologically testable hypotheses. bamSliceR and its documentation are freely available on GitHub at https://github.com/trichelab/bamSliceR.

DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles.

Mechanoreceptor cells develop specialized mechanosensory organelles (MOs), where force-sensitive channels and supporting structures are organized in an orderly manner to detect forces. It is intriguing how MOs are formed. Here, we address this issue by studying the MOs of fly ciliated mechanoreceptors. We show that the main structure of the MOs is a compound cytoskeleton formed of short microtubules and electron-dense materials (EDMs). In a knock-out mutant of DCX-EMAP, this cytoskeleton is nearly absent, suggesting that DCX-EMAP is required for the formation of the MOs and in turn fly mechanotransduction. Further analysis reveals that DCX-EMAP expresses in fly ciliated mechanoreceptors and localizes to the MOs. Moreover, it plays dual roles by promoting the assembly/stabilization of the microtubules and the accumulation of the EDMs in the MOs. Therefore, DCX-EMAP serves as a core ultrastructural organizer of the MOs, and this finding provides novel molecular insights as to how fly MOs are formed.

Prenatal diagnosis in a fetuses with a clenched hands, overlapping fingers, and clubfoot due to MED12 deficiency in three affected siblings: A case report.

A fetal clenched hand with overlapping fingers is more common in aneuploidy syndrome and was not well-documented in MED12 deficiency. This study reports the clinical and genetic findings of three affected siblings from a Chinese family. The chromosome karyotype analysis diagram shows that karyotypes of the three children were normal. Trio whole-exome sequencing and Sanger sequencing verification found that there was a MED12 R296Q variant in normal mothers and their two offspring. A pattern of clenched hand with overlapping fingers (clinodactyly) and clubfoot was found in all the three affected siblings by three-dimensional ultrasound. The discovery of this case shows that even if the chromosome karyotype is normal, comprehensive prenatal genetic diagnosis is required when the ultrasound results show a clenched hand with clinodactyly and clubfoot symptoms.

Histone benzoylation serves as an epigenetic mark for DPF and YEATS family proteins.

Histone modifications and their functional readout serve as an important mechanism for gene regulation. Lysine benzoylation (Kbz) on histones is a recently identified acylation mark associated with active transcription. However, it remains to be explored whether putative readers exist to recognize this epigenetic mark. Here, our systematic binding studies demonstrated that the DPF and YEATS, but not the Bromodomain family members, are readers for histone Kbz. Co-crystal structural analyses revealed a 'hydrophobic encapsulation' and a 'tip-sensor' mechanism for Kbz readout by DPF and YEATS, respectively. Moreover, the DPF and YEATS family members display subtle yet unique features to create somewhat flexible engagements of different acylation marks. For instance, YEATS2 but not the other YEATS proteins exhibits best preference for Kbz than lysine acetylation and crotonylation due to its wider 'tip-sensor' pocket. The levels of histone benzoylation in cultured cells or in mice are upregulated upon sodium benzoate treatment, highlighting its dynamic regulation. In summary, our work identifies the first readers for histone Kbz and reveals the molecular basis underlying Kbz recognition, thus paving the way for further functional dissections of histone benzoylation.

Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia.

Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.

Protocol for Biochemical Analysis and Structure Determination of the ZZ Domain of the E3 Ubiquitin Ligase HERC2.

Since its discovery, several ligands of the ZZ domain have been identified; however, molecular and structural information underlying binding of these ligands remains limited. Here, we describe a protocol for biochemical and structural analysis of the ZZ domain of human E3 ubiquitin ligase HERC2 (HERC2ZZ) and its interaction with its ligands: the N-terminal tails of histone H3 and SUMO1. This methodology could be applied for characterization of binding activities of other histone readers. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).

Structural Insight into Binding of the ZZ Domain of HERC2 to Histone H3 and SUMO1.

Human ubiquitin ligase HERC2, a component of the DNA repair machinery, has been linked to neurological diseases and cancer. Here, we show that the ZZ domain of HERC2 (HERC2ZZ) binds to histone H3 tail and tolerates posttranslational modifications commonly present in H3. The crystal structure of the HERC2ZZ:H3 complex provides the molecular basis for this interaction and highlights a critical role of the negatively charged site of HERC2ZZ in capturing of A1 of H3. NMR, mutagenesis, and fluorescence data reveal that HERC2ZZ binds to H3 and the N-terminal tail of SUMO1, a previously reported ligand of HERC2ZZ, with comparable affinities. Like H3, the N-terminal tail of SUMO1 occupies the same negatively charged site of HERC2ZZ in the crystal structure of the complex, although in contrast to H3 it adopts an α-helical conformation. Our data suggest that HERC2ZZ may play a role in mediating the association of HERC2 with chromatin.

Probing the Function of Metazoan Histones with a Systematic Library of H3 and H4 Mutants.

Replication-dependent histone genes often reside in tandemly arrayed gene clusters, hindering systematic loss-of-function analyses. Here, we used CRISPR/Cas9 and the attP/attB double-integration system to alter numbers and sequences of histone genes in their original genomic context in Drosophila melanogaster. As few as 8 copies of the histone gene unit supported embryo development and adult viability, whereas flies with 20 copies were indistinguishable from wild-types. By hierarchical assembly, 40 alanine-substitution mutations (covering all known modified residues in histones H3 and H4) were introduced and characterized. Mutations at multiple residues compromised viability, fertility, and DNA-damage responses. In particular, H4K16 was necessary for expression of male X-linked genes, male viability, and maintenance of ovarian germline stem cells, whereas H3K27 was essential for late embryogenesis. Simplified mosaic analysis showed that H3R26 is required for H3K27 trimethylation. We have developed a powerful strategy and valuable reagents to systematically probe histone functions in D. melanogaster.

Critical roles of long noncoding RNAs in Drosophila spermatogenesis.

Long noncoding RNAs (lncRNAs), a recently discovered class of cellular RNAs, play important roles in the regulation of many cellular developmental processes. Although lncRNAs have been systematically identified in various systems, most of them have not been functionally characterized in vivo in animal models. In this study, we identified 128 testis-specific Drosophila lncRNAs and knocked out 105 of them using an optimized three-component CRISPR/Cas9 system. Among the lncRNA knockouts, 33 (31%) exhibited a partial or complete loss of male fertility, accompanied by visual developmental defects in late spermatogenesis. In addition, six knockouts were fully or partially rescued by transgenes in a trans configuration, indicating that those lncRNAs primarily work in trans Furthermore, gene expression profiles for five lncRNA mutants revealed that testis-specific lncRNAs regulate global gene expression, orchestrating late male germ cell differentiation. Compared with coding genes, the testis-specific lncRNAs evolved much faster. Moreover, lncRNAs of greater functional importance exhibited higher sequence conservation, suggesting that they are under constant evolutionary selection. Collectively, our results reveal critical functions of rapidly evolving testis-specific lncRNAs in late Drosophila spermatogenesis.

Ack promotes tissue growth via phosphorylation and suppression of the Hippo pathway component Expanded.

Non-receptor tyrosine kinase activated cdc42 kinase was reported to participate in several types of cancers in mammals. It is also believed to have an anti-apoptotic function in Drosophila. Here, we report the identification of Drosophila activated cdc42 kinase as a growth promoter and a novel Hippo signaling pathway regulator. We find that activated cdc42 kinase promotes tissue growth through modulating Yorkie activity. Furthermore, we demonstrate that activated cdc42 kinase interacts with Expanded and induces tyrosine phosphorylation of Expanded on multiple sites. We propose a model that activated cdc42 kinase negatively regulates Expanded by changing its phosphorylation status to promote tissue growth. Moreover, we show that ack genetically interacts with merlin and expanded. Thus, we identify Drosophila activated cdc42 kinase as a Hippo pathway regulator.

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach.

Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.