Biology subjects

Garzon, J.

Publications and source records attributed to Garzon, J..

6 recordsLinked to original sources

bioRxiv2024

AAV-mediated genome editing is influenced by the formation of R-loops

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3 end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop- deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

molecular biology↗

bioRxiv2022

Hypoxic response is driven by the BAF form of SWI/SNF

SWI/SNF has been shown to have important functions in hypoxia-mediated gene expression through roles of its catalytic and core subunits. Since SWI/SNF exists as three distinct assemblies, and usage of complex specific subunits of the complex can be expected to vary within a given cell under changing environmental conditions. It remains an open question as to the compositional makeup of SWI/SNF and the roles of individual complexes in gene expression and cell viability in a hypoxic environment. In our current study, we find that hypoxia regulates levels of unique subunits that define each complex. Protein levels of ARID2 and PBRM1, members of PBAF and BRD9, a member of ncBAF, are downregulated in hypoxic cells, while members of BAF complex are retained. Our studies further show that loss of ARID1A, ARID1B and DPF2, which are unique subunits of BAF, reduces induction of HIF target genes and ARID1A or DPF2 are important for cell survival during hypoxia. Collectively, our results provide evidence that levels of SWI/SNF forms are not static within cells, but can be dynamically altered as a response to environmental changes.

biochemistry↗

bioRxiv2021

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

molecular biology↗

bioRxiv2020

CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins under conditions of cellular stress and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity. Author SummaryFanconi anemia (FA) is a rare genetic disease characterized by high risk for bone marrow failure and cancer. FA has strong genetic and biochemical links to hereditary breast and ovarian cancer. The FA proteins function to repair DNA damage and to maintain genome stability. The FANCD2 protein functions at a critical stage of the FA pathway and its posttranslational modification is defective in >90% of FA patients. However, the function, and regulation of FANCD2, particularly under unperturbed cellular conditions, remains remarkably poorly characterized. In this study, we describe a novel mechanism of regulation of the FANCD2 protein during S-phase of the cell cycle. CDK-mediated phosphorylation of FANCD2 on S592 promotes the ubiquitination of FANCD2 during S-phase. Disruption of this phospho-regulatory mechanism results in compromised mitotic fidelity and an increase in mitotic chromosome instability.

biochemistry↗

bioRxiv2020

Endemic and epidemic human alphavirus infections in Eastern Panama, An Analysis of Population-Based Cross-Sectional Surveys

BackgroundMadariaga virus (MADV), has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June, 2017, a fatal MADV infection was confirmed in a community of Darien province. MethodsWe conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. Additionally, by applying a catalytic, force-of-infection statistical model to two serosurveys from Darien province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. ResultsIn 2017, MADV and VEEV IgM seroprevalence was 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%; VEEV: 16.8%; Una virus (UNAV): 16.0%; and Mayaro virus (MAYV): 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households -- raising questions about its vectors and enzootic transmission cycles. Insomnia was associated with MADV and VEEV infection, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in some regions. ConclusionsThe lack of additional neurological cases suggest that severe MADV and VEEV infections occur only rarely. Our results indicate that, over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased -- potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially. Author summaryPrior to 2010, it was believed that the Madariaga virus (MADV) was primarily associated with equine disease. However, an outbreak reported in Panama, in an endemic area where Venezuelan equine encephalitis virus (VEEV) also circulates, suggested a change in its epidemiological profile. We aimed to reconstruct the epidemiological dynamics of MADV and VEEV, as well as additional alphaviruses known to circulate in the region in order to understand MADV emergence. For this, cross-sectional serosurveys were used to demonstrate that the Alphaviruses MADV, VEEV and Una virus have repeatedly infected humans in eastern Panama over the past five decades. Whilst their historical transmission has been low, we confirm that the transmission has recently increased for both MADV and VEEV.

microbiology↗

bioRxiv2019

The RIF1-Long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

molecular biology↗