Regulation of BACH1/FANCJ Function in DNA Damage Repair: A Dissertation

The DNA damage response (DDR) pathway is a complicated network of interacting proteins that function to sense and remove DNA damage. Upon exposure to DNA damage, a signaling cascade is generated. The damage is either removed, restoring the original genetic sequence, or apoptosis is activated. In the absence of DDR, cells are unable to effectively process DNA damage. Unprocessed DNA damage can lead to chromosomal changes, gene mutations, and malignant transformation. Thus, the proteins involved in DDR are critical for maintaining genomic stability. One essential DDR protein is the BRCA1 Associated C-terminal Helicase, BACH1. BACH1 was initially identified through its direct association with the BRCT domain of the Breast Cancer Associated Gene, BRCA1. Similar to BRCA1, germline mutations in BACH1were identified in patients with early onset breast cancer. Interestingly, the disease-associated mutations in BACH1 were shown to have altered helicase activity in vitro, providing a direct link between BACH1 helicase activity and disease development. The correlation between BACH1 and cancer predisposition was further confirmed by the identification of BACH1 as the cancer syndrome Fanconi anemia (FA) gene product, FANCJ. Similar to other FA proteins, suppression of FANCJ leads to decreased homologous recombination, enhanced sensitivity to DNA interstrand crosslinking (ICL) agents, and chromosomal instability. In an effort to further understand the function of FANCJ in DDR, FANCJ was shown to directly associate with the mismatch repair (MMR) protein MLH1. This interaction is facilitated by lysines 141 and 142 within the helicase domain of FANCJ. Importantly, the FANCJ/MLH1 interaction is critical for ICL repair. Furthermore, in an attempt to dissect the binding site of FANCJ on MLH1, we discovered an HNPCC associated MLH1 mutation (L607H) that has intact mismatch repair, but lacks FANCJ interaction. In contrast to the MLH1 interaction, the FANCJ/BRCA1 interaction was not required for correcting the cellular defects in FANCJ null cells. Thus, in an effort to understand the functional significance of the FANCJ/BRCA1 interaction, we discovered that FANCJ promotes Pol η dependent translesion synthesis (TLS) bypass when uncoupled from BRCA1. In this thesis, we provide evidence suggesting that FANCJ and MLH1 are functionally linked and that the interaction of these proteins is critical for repair choice

Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses

Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways

FANCJ/BACH1 Acetylation at Lysine 1249 Regulates the DNA Damage Response

BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance

Seasonal changes in patterns of gene expression in avian song control brain regions.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity

Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal

The canonical Wnt/β-catenin signaling pathway governs diverse developmental, homeostatic and pathologic processes. Palmitoylated Wnt ligands engage cell surface Frizzled (Fzd) receptors and Lrp5/6 co-receptors enabling β-catenin nuclear translocation and Tcf/Lef-dependent gene transactivation1–3. Mutations in Wnt downstream signaling components have revealed diverse functions presumptively attributed to Wnt ligands themselves, although direct attribution remains elusive, as complicated by redundancy between 19 mammalian Wnts and 10 Fzds1 and Wnt hydrophobicity2,3. For example, individual Wnt ligand mutations have not revealed homeostatic phenotypes in the intestinal epithelium4, an archetypal canonical Wnt pathway-dependent rapidly self-renewing tissue whose regeneration is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs)5–9. R-spondin ligands (Rspo1–4) engage distinct Lgr4-6 and Rnf43/Znrf3 receptor classes10–13, markedly potentiate canonical Wnt/β-catenin signaling and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo8,14–17. However, the interchangeability, functional cooperation and relative contributions of Wnt versus Rspo ligands to in vivo canonical Wnt signaling and ISC biology remain unknown. Here, we deconstructed functional roles of Wnt versus Rspo ligands in the intestinal crypt stem cell niche. We demonstrate that the default fate of Lgr5+ ISCs is lineage commitment, escape from which requires both Rspo and Wnt ligands. However, gain-of-function studies using Rspo versus a novel non-lipidated Wnt analog reveal qualitatively distinct, non-interchangeable roles for these ligands in ISCs. Wnts are insufficient to induce Lgr5+ ISC self-renewal, but rather confer a basal competency by maintaining Rspo receptor expression that enables Rspo to actively drive and specify the extent of stem cell expansion. This functionally non-equivalent yet cooperative interplay between Wnt and Rspo ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precision control of tissue regeneration

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

TLR activation synergizes with Kilham rat virus infection to induce diabetes in BBDR rats

Virus infection is hypothesized to be an important environmental trigger of type 1 diabetes in humans. We used the BBDR rat model to investigate the relationship between viral infection and autoimmune diabetes. BBDR rats are diabetes-free in viral Ab-free housing, but the disease develops in approximately 30% of BBDR rats infected with Kilham rat virus (KRV) through a process that does not involve infection of pancreatic beta cells. Pretreatment with polyinosinic-polycytidylic (poly(I:C)), a ligand of TLR3, acts synergistically to induce diabetes in 100% of KRV-infected rats. The mechanisms by which KRV induces diabetes and TLR3 ligation facilitates this process are not clear. In this study, we demonstrate that activation of the innate immune system plays a crucial role in diabetes induction. We report that multiple TLR agonists synergize with KRV infection to induce diabetes in BBDR rats, as do heat-killed Escherichia coli or Staphylococcus aureus (natural TLR agonists). KRV infection increases serum IL-12 p40 in a strain-specific manner, and increases IL-12 p40, IFN-gamma-inducible protein-10, and IFN-gamma mRNA transcript levels, particularly in the pancreatic lymph nodes of BBDR rats. Infection with vaccinia virus or H-1 parvovirus induced less stimulation of the innate immune system and failed to induce diabetes in BBDR rats. Our results suggest that the degree to which the innate immune system is activated by TLRs is important for expression of virus-induced diabetes in genetically susceptible hosts