Differential Regulation of Retinoic Acid Metabolism in Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital abnormalities and increased risk for bone marrow failure and cancer. FA is caused by mutation of any one of 23 genes, the protein products of which function primarily in the maintenance of genome stability. An important role for the FA proteins in the repair of DNA interstrand crosslinks (ICLs) has been established in vitro . While the endogenous sources of ICLs relevant to the pathophysiology of FA have yet to be clearly determined, a role for the FA proteins in a two-tier system for the detoxification of reactive metabolic aldehydes has been established. To discover new metabolic pathways linked to FA, we performed RNA-seq analysis on non-transformed FA-D2 ( FANCD2 -/- ) and FANCD2-complemented patient cells. Multiple genes associated with retinoic acid metabolism and signaling were differentially expressed in FA-D2 ( FANCD2 -/- ) patient cells, including ALDH1A1 and RDH10 , which encode for retinaldehyde and retinol dehydrogenases, respectively. Increased levels of the ALDH1A1 and RDH10 proteins was confirmed by immunoblotting. FA-D2 ( FANCD2 -/- ) patient cells displayed increased aldehyde dehydrogenase activity compared to the FANCD2-complemented cells. Upon exposure to retinaldehyde, FA-D2 ( FANCD2 -/- ) cells exhibited increased DNA double-strand breaks and checkpoint activation indicative of a defect in the repair of retinaldehyde-induced DNA damage. Our findings describe a novel link between retinoic acid metabolism and FA and identify retinaldehyde as an additional reactive metabolic aldehyde relevant to the pathophysiology of FA.

Cyclin-Dependent Kinase-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, which occurs upon exposure to DNA-damaging agents and during the 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 cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the 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.

RI-INBRE: A Statewide NIH Program Grant to Improve Institutional Biomedical Research Capacity in Rhode Island.

The overarching goal of the Rhode Island-IDeA Network of Biomedical Research Excellence (RI-INBRE) is to improve institutional capacity for biomedical research excellence and expand student experiential training opportunities in the State of Rhode Island. RI-INBRE comprises five major core components: The Administrative Core, the Bioinformatics Core, the Centralized Research Core Facility, the Training Core, and the Developmental Research Project Program Core. Since its inception in 2001, RI-INBRE has made significant investments and marked advancements in the biomedical research infrastructure of Rhode Island. RI-INBRE funding has increased the scale and quality of faculty research and engaged undergraduate students, graduate students, and postdoctoral fellows in structured and mentored research training experiences. Over the last 19 years, RI-INBRE has supported 212 faculty researchers and over 533 projects and has provided research-training opportunities for nearly 2,000 students, resulting in 757 publications. Through its student-training program, RI-INBRE has contributed to regional workforce development by engaging students and encouraging them to pursue careers in biomedical fields. Many of these students have been admitted to graduate or medical schools and obtained biomedical industry jobs following graduation. RI-INBRE has been particularly influential in building the research infrastructure at primarily undergraduate institutions, which have seen significant improvements in research quality and output, student training, and research infrastructure.

Application of a Biphasic Mathematical Model of Cancer Cell Drug Response for Formulating Potent and Synergistic Targeted Drug Combinations to Triple Negative Breast Cancer Cells.

Triple negative breast cancer is a collection of heterogeneous breast cancers that are immunohistochemically negative for estrogen receptor, progesterone receptor, and ErbB2 (due to deletion or lack of amplification). No dominant proliferative driver has been identified for this type of cancer, and effective targeted therapy is lacking. In this study, we hypothesized that triple negative breast cancer cells are multi-driver cancer cells, and evaluated a biphasic mathematical model for identifying potent and synergistic drug combinations for multi-driver cancer cells. The responses of two triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, to a panel of targeted therapy drugs were determined over a broad range of concentrations. The analyses of the drug responses by the biphasic mathematical model revealed that both cell lines were indeed dependent on multiple drivers, and inhibitors of individual drivers caused a biphasic response: a target-specific partial inhibition at low nM concentrations, and an off-target toxicity at µM concentrations. We further demonstrated that combinations of drugs, targeting each driver, cause potent, synergistic, and cell-specific cell killing. Immunoblotting analysis of the effects of the individual drugs and drug combinations on the signaling pathways supports the above conclusion. These results support a multi-driver proliferation hypothesis for these triple negative breast cancer cells, and demonstrate the applicability of the biphasic mathematical model for identifying effective and synergistic targeted drug combinations for triple negative breast cancer cells.

A distinct role for recombination repair factors in an early cellular response to transcription-replication conflicts.

Transcription-replication (T-R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T-R conflicts, our understanding of the genetic and temporal nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T-R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-independent function of FANCD2. A hallmark of the TRe response is the rapid co-localization of these three DNA repair factors at sites of T-R collisions. We find that the TRe response relies on basal activity of the ATR kinase, yet it does not lead to hyperactivation of this key checkpoint protein. Furthermore, specific abrogation of the TRe response leads to DNA damage in mitosis, and promotes chromosome instability and cell death. Collectively our findings identify a new role for these well-established tumor suppressor proteins at an early stage of the cellular response to conflicts between DNA transcription and replication.

FANCD2 Binding to H4K20me2 via a Methyl-Binding Domain Is Essential for Efficient DNA Cross-Link Repair.

Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity.

Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2.

Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks.

Understanding the Histone DNA Repair Code: H4K20me2 Makes Its Mark.

Chromatin is a highly compact structure that must be rapidly rearranged in order for DNA repair proteins to access sites of damage and facilitate timely and efficient repair. Chromatin plasticity is achieved through multiple processes, including the posttranslational modification of histone tails. In recent years, the impact of histone posttranslational modification on the DNA damage response has become increasingly well recognized, and chromatin plasticity has been firmly linked to efficient DNA repair. One particularly important histone posttranslational modification process is methylation. Here, we focus on the regulation and function of H4K20 methylation (H4K20me) in the DNA damage response and describe the writers, erasers, and readers of this important chromatin mark as well as the combinatorial histone posttranslational modifications that modulate H4K20me recognition. Finally, we discuss the central role of H4K20me in determining if DNA double-strand breaks (DSB) are repaired by the error-prone, nonhomologous DNA end joining pathway or the error-free, homologous recombination pathway. This review article discusses the regulation and function of H4K20me2 in DNA DSB repair and outlines the components and modifications that modulate this important chromatin mark and its fundamental impact on DSB repair pathway choice. Mol Cancer Res; 16(9); 1335-45. ©2018 AACR.

Modulation of the Fanconi anemia pathway via chemically induced changes in chromatin structure.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway.

Tuning the Multifunctionality of Iron Oxide Nanoparticles Using Self-Assembled Mixed Lipid Layers.

We present an approach to tuning the multifunctionality of iron oxide nanoparticles (IONs) using mixed self-assembled monolayers of cationic lipid and anionic polyethylene glycol (PEG) lipid. By forming stable, monodispersed lipid-coated IONs (L-IONs) through a solvent-exchange technique, we were able to demonstrate the relationship between surface charge, the magnetic transverse relaxivity (r2 from T2-weighted images), and the binding capacity of small interfering ribonucleic acids (siRNAs) as a function of the cationic-to-anionic (PEG) lipid ratio. These properties were controlled by the cationic charge and the PEG conformation; relaxivity and siRNA binding could be varied in the mushroom and brush regimes but not at high brush densities. In vitro results combining cell viability, uptake, and transfection efficiency using HeLa cells suggest that the functional physicochemical and biological properties of L-IONs may be best achieved using catanionic lipid coatings near equimolar ratios of cationic to anionic PEG-lipids.

Proteomic responses to elevated ocean temperature in ovaries of the ascidian Ciona intestinalis.

Ciona intestinalis, a common sea squirt, exhibits lower reproductive success at the upper extreme of the water temperatures it experiences in coastal New England. In order to understand the changes in protein expression associated with elevated temperatures, and possible response to global temperature change, we reared C. intestinalis from embryos to adults at 18°C (a temperature at which they reproduce normally at our collection site in Rhode Island) and 22°C (the upper end of the local temperature range). We then dissected ovaries from animals at each temperature, extracted protein, and measured proteomic levels using shotgun mass spectrometry (LC-MS/MS). 1532 proteins were detected at a 1% false discovery rate present in both temperature groups by our LC-MS/MS method. 62 of those proteins are considered up- or down-regulated according to our statistical criteria. Principal component analysis shows a clear distinction in protein expression pattern between the control (18°C) group and high temperature (22°C) group. Similar to previous studies, cytoskeletal and chaperone proteins are upregulated in the high temperature group. Unexpectedly, we find evidence that proteolysis is downregulated at the higher temperature. We propose a working model for the high temperature response in C. intestinalis ovaries whereby increased temperature induces upregulation of signal transduction pathways involving PTPN11 and CrkL, and activating coordinated changes in the proteome especially in large lipid transport proteins, cellular stress responses, cytoskeleton, and downregulation of energy metabolism.

Recent discoveries in the molecular pathogenesis of the inherited bone marrow failure syndrome Fanconi anemia.

Fanconi anemia (FA) is a rare autosomal and X-linked genetic disease characterized by congenital abnormalities, progressive bone marrow failure (BMF), and increased cancer risk during early adulthood. The median lifespan for FA patients is approximately 33years. The proteins encoded by the FA genes function together in the FA-BRCA pathway to repair DNA damage and to maintain genome stability. Within the past two years, five new FA genes have been identified-RAD51/FANCR, BRCA1/FANCS, UBE2T/FANCT, XRCC2/FANCU, and REV7/FANCV-bringing the total number of disease-causing genes to 21. This review summarizes the discovery of these new FA genes and describes how these proteins integrate into the FA-BRCA pathway to maintain genome stability and critically prevent early-onset BMF and cancer.

The PTEN phosphatase functions cooperatively with the Fanconi anemia proteins in DNA crosslink repair.

Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair.

FANCD2 Facilitates Replication through Common Fragile Sites.

Common fragile sites (CFSs) are genomic regions that are unstable under conditions of replicative stress. Although the characteristics of CFSs that render them vulnerable to stress are associated mainly with replication, the cellular pathways that protect CFSs during replication remain unclear. Here, we identify and describe a role for FANCD2 as a trans-acting facilitator of CFS replication, in the absence of exogenous replicative stress. In the absence of FANCD2, replication forks stall within the AT-rich fragility core of CFS, leading to dormant origin activation. Furthermore, FANCD2 deficiency is associated with DNA:RNA hybrid formation at CFS-FRA16D, and inhibition of DNA:RNA hybrid formation suppresses replication perturbation. In addition, we also found that FANCD2 reduces the number of potential sites of replication initiation. Our data demonstrate that FANCD2 protein is required to ensure efficient CFS replication and provide mechanistic insight into how FANCD2 regulates CFS stability.

The Simple Chordate Ciona intestinalis Has a Reduced Complement of Genes Associated with Fanconi Anemia.

Fanconi anemia (FA) is a human genetic disease characterized by congenital defects, bone marrow failure, and increased cancer risk. FA is associated with mutation in one of 24 genes. The protein products of these genes function cooperatively in the FA pathway to orchestrate the repair of DNA interstrand cross-links. Few model organisms exist for the study of FA. Seeking a model organism with a simpler version of the FA pathway, we searched the genome of the simple chordate Ciona intestinalis for homologs of the human FA-associated proteins. BLAST searches, sequence alignments, hydropathy comparisons, maximum likelihood phylogenetic analysis, and structural modeling were used to infer the likelihood of homology between C. intestinalis and human FA proteins. Our analysis indicates that C. intestinalis indeed has a simpler and potentially functional FA pathway. The C. intestinalis genome was searched for candidates for homology to 24 human FA and FA-associated proteins. Support was found for the existence of homologs for 13 of these 24 human genes in C. intestinalis. Members of each of the three commonly recognized FA gene functional groups were found. In group I, we identified homologs of FANCE, FANCL, FANCM, and UBE2T/FANCT. Both members of group II, FANCD2 and FANCI, have homologs in C. intestinalis. In group III, we found evidence for homologs of FANCJ, FANCO, FANCQ/ERCC4, FANCR/RAD51, and FANCS/BRCA1, as well as the FA-associated proteins ERCC1 and FAN1. Evidence was very weak for the existence of homologs in C. intestinalis for any other recognized FA genes. This work supports the notion that C. intestinalis, as a close relative of vertebrates, but having a much reduced complement of FA genes, offers a means of studying the function of certain FA proteins in a simpler pathway than that of vertebrate cells.

The Fanconi anemia ID2 complex: dueling saxes at the crossroads.

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions.

Cyclic peptide-capped gold nanoparticles for enhanced siRNA delivery.

Previously, we have reported the synthesis of a homochiral l-cyclic peptide [WR]5 and its use for delivery of anti-HIV drugs and biomolecules. A physical mixture of HAuCl4 and the peptide generated peptide-capped gold nanoparticles. Here, [WR]5 and [WR]5-AuNPs were tested for their efficiency to deliver a small interfering RNA molecule (siRNA) in human cervix adenocarcinoma (HeLa) cells. Flow cytometry investigation revealed that the intracellular uptake of a fluorescence-labeled non-targeting siRNA (200 nM) was enhanced in the presence of [WR]5 and [WR]5-AuNPs by 2- and 3.8-fold when compared with that of siRNA alone after 24 h incubation. Comparative toxicity results showed that [WR]5 and [WR]5-AuNPs were less toxic in cells compared to other available carrier systems, such as Lipofectamine.

Coordinate nuclear targeting of the FANCD2 and FANCI proteins via a FANCD2 nuclear localization signal.

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.

Multinucleation regulated by the Akt/PTEN signaling pathway is a survival strategy for HepG2 cells.

Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etoposide. The aim of this study was to examine the survival strategy of the HCC cell line HepG2 after etoposide treatment. Here we analyzed and compared spontaneous and etoposide-induced DNA damage in HepG2 (α-fetoprotein (AFP)-positive) and Chang Liver (AFP-negative) cell lines. Compared to Chang Liver cells, HepG2 cells exhibited a significantly higher degree of micronucleation and a higher nuclear division index, as determined by the cytokinesis-block micronucleus assay, following exposure to etoposide. HepG2 cells were also more resistant to etoposide-induced cytotoxicity compared to Chang Liver cells. We also establish that increased etoposide-induced multinucleation in HepG2 cells is dependent on the catalytic activity of Akt, as phosphatidylinositol-3-kinase inhibitors as well as the overexpression of kinase-defective Akt reversed this phenotype. Moreover, ectopic expression of wild type PTEN reduced the frequency of etoposide-induced multinucleated HepG2 cells, and restored HepG2 etoposide sensitivity. Taken together, these results implicate the Akt/PTEN cellular axis as a major determinant of the etoposide resistance of HCC cells.