The APE2 nuclease is essential for DNA double-strand break repair by microhomology-mediated end joining.

Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.

Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

Saul-Wilson Syndrome Missense Allele Does Not Show Obvious Golgi Defects in a C. elegans Model.

Saul-Wilson Syndrome is an ultra-rare skeletal syndrome caused by a mutation in the COG4 gene resulting in a glycine-to-arginine substitution at amino acid position 516. The COG4 gene encodes one of 8 subunits of the conserved oligomeric Golgi complex. Using CRISPR-Cas9, our lab generated a C. elegans model for Saul-Wilson Syndrome by recreating the same glycine-to-arginine substitution in the worm ortholog cogc-4. Upon observation, the cogc-4(av107) worms did not display any obvious differences compared to wild-type worms. We used a variety of assays including stressing the worms using heat and Paraquat, as well as RNAi against the 7 other COG complex subunit genes in an attempt to uncover a phenotype. Our data suggest that this mutation in cogc-4(av107) worms does not lead to a detectable phenotype. Further studies should aim at more directly assessing Golgi function in this disease model.

Loss of the seipin gene perturbs eggshell formation in Caenorhabditiselegans.

Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.

Aurora B functions at the apical surface after specialized cytokinesis during morphogenesis in C. elegans.

Although cytokinesis has been intensely studied, the way it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant Caenorhabditis elegans embryonic divisions and found several parameters that are altered at different stages in a reproducible manner. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we found several unexpected alterations to cytokinesis, including apical midbody migration in polarizing epithelial cells of the gut, pharynx and sensory neurons. Aurora B kinase, which is essential for several aspects of cytokinesis, remains apically localized in each of these tissues after internalization of midbody ring components. Aurora B inactivation disrupts cytokinesis and causes defects in apical structures, even if inactivated post-mitotically. Therefore, we demonstrate that cytokinesis is implemented in a specialized way during epithelial polarization and that Aurora B has a role in the formation of the apical surface.

Lipid droplet and peroxisome biogenesis occur at the same ER subdomains.

Nascent lipid droplet (LD) formation occurs in the endoplasmic reticulum (ER) membrane but it is not known how sites of biogenesis are determined. We previously identified ER domains in S. cerevisiae containing the reticulon homology domain (RHD) protein Pex30 that are regions where preperoxisomal vesicles (PPVs) form. Here, we show that Pex30 domains are also sites where most nascent LDs form. Mature LDs usually remain associated with Pex30 subdomains, and the same Pex30 subdomain can simultaneously associate with a LD and a PPV or peroxisome. We find that in higher eukaryotes multiple C2 domain containing transmembrane protein (MCTP2) is similar to Pex30: it contains an RHD and resides in ER domains where most nascent LD biogenesis occurs and that often associate with peroxisomes. Together, these findings indicate that most LDs and PPVs form and remain associated with conserved ER subdomains, and suggest a link between LD and peroxisome biogenesis.