Biology subjects

Richardson, C. D.

Publications and source records attributed to Richardson, C. D..

12 recordsLinked to original sources

bioRxiv2026

Autophagy dependent HIF1α proteostasis is compromised in models of PEX1 deficiencies

Peroxisomes, along with mitochondria, coordinate and compartmentalize oxidative metabolism in eukaryotic cells. Rare genetic disorders caused by mutations in PEX genes impair peroxisome function and cause Peroxisome Biogenesis Disorders, which are characterized by liver and neurological dysfunction, hearing and vision loss, and metabolic abnormalities. The majority of Peroxisome Biogenesis Disorders (PBDs) are caused by mutations in the gene encoding PEX1, which together with PEX6 forms a hetero-hexameric AAA-ATPase complex that drives the import of enzymes into the peroxisome lumen. One particular destabilizing mutation - PEX1G843D - results in almost 30% of all cases. Here we show that deficiencies in PEX1 lead to increased levels of the oxygen-responsive transcription factor HIF1 in normoxia, as well as a HIF1 transcriptional signature. The increase in HIF1 protein was rescued by overexpression of PEX1WT, suggesting PEX1 deficiencies modulate HIF1 signaling. The increased levels of HIF1 were not explained by defects in the oxygen responsive degradation pathway of HIF1 that relies on proline hydroxylase domain enzyme-dependent hydroxylation and von Hippel Lindau tumor suppressor protein-dependent ubiquitination. Instead, we found that PEX1 deficiency alters HIF1 proteostasis by reducing degradation through a hydroxylation- and autophagy- dependent mechanism. Notably, enhancing autophagic capacity by ULK1 agonism was sufficient to reduce HIF1 levels in PEX1 deficient cells. Lastly, we demonstrate that upon hypoxia-reoxygenation, PEX1 deficient cells are slower to reset HIF1 levels. Our results suggest that PBD patients with PEX1 deficiency may be susceptible to dysregulation of the HIF1 pathway, particularly in tissues where oxygen gradients are physiological or developmentally required.

cell biology↗

bioRxiv2026

POCKET-seq enables genome-wide profiling of on- and off-target transcriptional regulation events by dCas9-KRAB during CRISPR interference experiments

CRISPR interference screens use catalytically inactive dCas9 fused to a repressor domain to enable genetic perturbations at the transcriptomic level. Interpretation of results involves identification of guide RNAs associated with the screen phenotype, followed by secondary analysis. During validation of a genetic screen, we observed different phenotypes from non-overlapping guide RNAs targeting one gene. Here, we developed POCKET-seq to map the binding of dCas9 genome-wide. We show that off-target binding occurs frequently and can generate false-positive interactions when it occurs near the promoter of genes associated with the screen phenotype. POCKET-seq classifies these false-positive and true-positive interactions using gene ontology.

genetics↗

bioRxiv2025

The FANCD2-FANCI heterodimer coordinates chromatin openness and cell cycle progression throughout DNA double-strand break repair.

The FANCD2-FANCI heterodimer contributes to DNA repair at interstrand crosslinks and sites of replication stress. This complex has been physically and mechanistically linked to double-strand break (DSB) repair, but its role in that process remains undefined. Here we show that the FANCD2-FANCI heterodimer dynamically interacts with open chromatin regions, including transient, DSB-induced open chromatin, where it can be stabilized by co-activation by the DNA repair kinase ATM and the Fanconi anemia core ubiquitin ligase. The loaded FANCD2-FANCI heterodimer stabilizes open chromatin and promotes resection and loading of RPA through increased association of BRCA1 and BLM. Chromatin-loaded FANCD2-FANCI has a second distinct function promoting a G2 arrest that is dependent on the ATR-CHK1-WEE1 axis. Our results support a two-step genome surveillance model in which FANCD2-FANCI monitors open chromatin sites and is stably loaded to coordinate DNA repair activities in response to signaling from a DNA repair kinase.

cell biology↗

bioRxiv2025

Proximity determines donor candidacy during DNA double-stranded break homology directed repair

DNA double-stranded breaks (DSBs) are especially toxic events that can be reversed by homology-directed repair (HDR), wherein information is copied from an intact template molecule. RAD51 mediates initial DSB/template pairing during homology search. A major challenge in understanding homology search in cells is the lack of tools to monitor this process. We developed RAD51 proximity identification sequencing (RaPID-seq), a sensitive method that marks all candidate templates searched by RAD51. We find that HDR is hierarchical, such that DSB proximity determines template candidacy and subsequent recombination is unlocked by DSB/template homology. Sequences that lie outside the proximal window are not efficiently searched, even if identical in sequence. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.

molecular biology↗

bioRxiv2024

PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome

The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of Peroxisome Biogenesis Disorders (PBDs). The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here we demonstrate that in vitro the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1s active D2 ATPase domain and impairs assembly with Pex6, but can still form an active AAA-ATPase motor. In vivo, ScPex1G700D exhibits only a slight defect in peroxisome import. We generated model human HsPEX1G843D cell lines and show that PEX1G843D is rapidly degraded by the proteasome, but that induced overexpression of PEX1G843D can restore peroxisome import. Additionally, we found that the G843D mutation reduces PEX1s affinity for PEX6, and that impaired assembly is sufficient to induce degradation of PEX1WT. Lastly, we found that fusing a deubiquitinase to PEX1G843D significantly hinders its degradation in mammalian cells. Altogether, our findings suggest a novel regulatory mechanism for PEX1/PEX6 hexamer assembly and highlight the potential of protein stabilization as a therapeutic strategy for PBDs arising from the G843D mutation and other PEX1 hypomorphs.

biochemistry↗

bioRxiv2024

A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and tankyrase

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerase tankyrase, which binds the peroxisomal membrane protein PEX14. We propose that RNF146 and tankyrase regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased tankyrase and RNF146-dependent degradation of non-peroxisomal substrates, including the beta-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of beta-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation, but also a novel role in bridging peroxisome function with Wnt/beta-catenin signaling during development.

cell biology↗

bioRxiv2023

The Pex6 N1 domain is required for Pex15 binding and proper assembly with Pex1

The heterohexameric AAA-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motors N-terminal domains or N-terminally bound co-factors. Here we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, S. cerevisiae. We found that although Pex1/{Lambda}N1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N terminal domains of PEX1, CDC48, or NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of co-factors and substrates with Pex1/Pex6 ATPase activity.

biochemistry↗

bioRxiv2019

The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a proximity labeling system for unbiased detection of transient protein interactions in cell-free Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 unloading and converts Cas9s activity from multi-turnover to single-turnover. In human cells, depletion of FACT delays genome editing and alters the balance between indel formation and homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-based transcriptional effectors with concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

cell biology↗

bioRxiv2019

Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

CRISPR/Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report off-target sites can be shielded from the active Cas9*single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating \"scarless\" editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a novel and flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

molecular biology↗

bioRxiv2018

Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Repair of double strand DNA breaks (DSBs) can result in gene disruption or precise gene modification via homology directed repair (HDR) from a templating donor DNA. During genome editing, altering cellular responses to DSBs may be an effective strategy to rebalance editing outcomes towards HDR and away from other repair pathways. To identify factors that regulate HDR from a double-stranded DNA donor (dsDonor), we utilized a pooled screen to define the consequences of thousands of individual gene knockdowns during Cas9-initiated HDR from a double strand plasmid donor. We find that templated dsDonor repair pathways are mostly genetically distinct from single strand donor DNA (ssDonor) repair but share aspects that include dependency upon the Fanconi Anemia (FA) pathway. We also identified several factors whose knockdown increases HDR and thus act as repressors of gene modification. Screening available small molecule inhibitors of these repressors revealed that the cell division cycle 7-related protein kinase (CDC7) inhibitor XL413 increases the efficiency of HDR by 2-3 fold in many contexts, including primary T-cells. XL413 stimulates HDR through cell cycle regulation, inducing an early S-phase cell cycle arrest that, to the best of our knowledge, is uncharacterized for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for boosting the efficiency of gene modification.

molecular biology↗

bioRxiv2018

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

molecular biology↗

bioRxiv2017

CRISPR-Cas9 Genome Editing In Human Cells Works Via The Fanconi Anemia Pathway

CRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

cell biology↗