scholarly journals Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination

Cell Reports ◽  
2019 ◽  
Vol 26 (3) ◽  
pp. 564-572.e5 ◽  
Author(s):  
Zhuobin Liang ◽  
Fengshan Liang ◽  
Yaqun Teng ◽  
Xiaoyong Chen ◽  
Jingchun Liu ◽  
...  
Keyword(s):  
Fancd2 Monoubiquitination

scholarly journals Basal level of FANCD2 monoubiquitination is required for the maintenance of a sufficient number of licensed-replication origins to fire at a normal rate

Oncotarget ◽  
2014 ◽  
Vol 5 (5) ◽  
pp. 1326-1337 ◽  
Author(s):  
Jayabal Panneerselvam ◽  
Anna Pickering ◽  
Bing Han ◽  
Liantao Li ◽  
Junnian Zheng ◽  
...  
Keyword(s):  
Normal Rate ◽  
Replication Origins ◽  
Fancd2 Monoubiquitination

scholarly journals FANCL Replaces BRCA1 as the Likely Ubiquitin Ligase Responsible for FANCD2 Monoubiquitination

Cell Cycle ◽  
2004 ◽  
Vol 3 (2) ◽  
pp. 174-176 ◽  
Author(s):  
Amom Ruhikanta Meetei ◽  
Zhijiang Yan ◽  
Weidong Wang
Keyword(s):  
Ubiquitin Ligase ◽  
Fancd2 Monoubiquitination

scholarly journals Clinical severity in Fanconi anemia correlates with residual function of FANCB missense variants

2019 ◽  
Author(s):  
Moonjung Jung ◽  
Ramanagouda Ramanagoudr-Bhojappa ◽  
Sylvie van Twest ◽  
Rasim Ozgur Rosti ◽  
Vincent Murphy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Congenital Abnormalities ◽  
Bone Marrow Failure ◽  
Complementation Group ◽  
Clinical Severity ◽  
List Type ◽  
Missense Variants ◽  
Pathogenic Variants ◽  
Fancd2 Monoubiquitination

ABSTRACTFanconi anemia (FA) is the most common genetic cause of bone marrow failure, and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry (IFAR). Those with FANCB deletion or truncation demonstrate earlier than average onset of bone marrow failure, and more severe congenital abnormalities compared to a large series of FA individuals in the published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization was associated with two missence variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays showed earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is linked to the extent of residual FANCD2 monoubiquitination.KEY POINTSX-linked FANCB pathogenic variants predominantly cause acute, early onset bone marrow failure and severe congenital abnormalitiesBiochemical and cell-based assays with patient variants reveal functional properties of FANCB that associate with clinical severity

The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC

Blood ◽  
2002 ◽  
Vol 100 (7) ◽  
pp. 2457-2462 ◽  
Author(s):  
Toshiyasu Taniguchi ◽  
Alan D. D'Andrea
Keyword(s):  
Mitomycin C ◽  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Autosomal Recessive Disorder ◽  
Nuclear Accumulation ◽  
Retroviral Transduction ◽  
Nuclear Foci ◽  
Fancd2 Monoubiquitination

Fanconi anemia is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. The 6 known Fanconi anemia gene products (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG proteins) interact in a common pathway. The monoubiquitination and nuclear foci formation of FANCD2 are essential for the function of this pathway. FANCA, FANCC, FANCG, and FANCF proteins form a multisubunit nuclear complex (FA complex) required for FANCD2 monoubiquitination. Because FANCE and FANCC interact in vitro and FANCE is required for FANCD2 monoubiquitination, we reasoned that FANCE is a component of the FA complex in vivo. Here we demonstrate that retroviral transduction of Fanconi anemia subtype E (FA-E) cells with the FANCE cDNA restores the nuclear accumulation of FANCC protein, FANCA–FANCC complex formation, monoubiquitination and nuclear foci formation of FANCD2, and mitomycin C resistance. Hemagglutinin (HA)-tagged FANCE protein localizes diffusely in the nucleus. In normal cells, HA-tagged FANCE protein coimmunoprecipitates with FANCA, FANCC, and FANCG but not with FANCD2. Our data indicate that FANCE is a component of the nuclear FA complex in vivo and is required for the monoubiquitination of FANCD2 and the downstream events in the FA pathway.

scholarly journals A homozygous FANCM mutation underlies a familial case of non-syndromic primary ovarian insufficiency

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Baptiste Fouquet ◽  
Patrycja Pawlikowska ◽  
Sandrine Caburet ◽  
Celine Guigon ◽  
Marika Mäkinen ◽  
...  
Keyword(s):  
Dna Damage Response ◽  
Meiotic Recombination ◽  
Chromosomal Abnormalities ◽  
Primary Ovarian Insufficiency ◽  
Homozygous Mutation ◽  
Wild Type ◽  
Ovarian Insufficiency ◽  
Response Gene ◽  
Damage Response ◽  
Fancd2 Monoubiquitination

Primary Ovarian Insufficiency (POI) affects ~1% of women under forty. Exome sequencing of two Finnish sisters with non-syndromic POI revealed a homozygous mutation in FANCM, leading to a truncated protein (p.Gln1701*). FANCM is a DNA-damage response gene whose heterozygous mutations predispose to breast cancer. Compared to the mother's cells, the patients’ lymphocytes displayed higher levels of basal and mitomycin C (MMC)-induced chromosomal abnormalities. Their lymphoblasts were hypersensitive to MMC and MMC-induced monoubiquitination of FANCD2 was impaired. Genetic complementation of patient's cells with wild-type FANCM improved their resistance to MMC re-establishing FANCD2 monoubiquitination. FANCM was more strongly expressed in human fetal germ cells than in somatic cells. FANCM protein was preferentially expressed along the chromosomes in pachytene cells, which undergo meiotic recombination. This mutation may provoke meiotic defects leading to a depleted follicular stock, as in Fancm-/- mice. Our findings document the first Mendelian phenotype due to a biallelic FANCM mutation.

scholarly journals The Genetic and Biochemical Basis of FANCD2 Monoubiquitination

2014 ◽  
Vol 54 (5) ◽  
pp. 858-869 ◽  
Author(s):  
Eeson Rajendra ◽  
Vibe H. Oestergaard ◽  
Frédéric Langevin ◽  
Meng Wang ◽  
Gillian L. Dornan ◽  
...  
Keyword(s):  
Biochemical Basis ◽  
Fancd2 Monoubiquitination

Monoubiquitinated FANCD2 Is Both Necessary and Sufficient for Mitomycin C Resistance in the Absence of a Functional Fanconi Anemia Core Complex.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 722-722
Author(s):  
Akiko Shimamura ◽  
Rebecca Leary ◽  
Maria Stotsky ◽  
Lisa A. Moreau ◽  
Alan D. D’Andrea
Keyword(s):  
Cell Lines ◽  
Fanconi Anemia ◽  
Core Protein ◽  
Diagnostic Feature ◽  
Core Complex ◽  
Chromosomal Breakage ◽  
Pathway Function ◽  
Fanconi Pathway ◽  
Fancd2 Monoubiquitination ◽  
In Cells

Abstract Monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, is necessary for resistance to MMC/DEB-induced chromosomal breakage, which is the diagnostic feature of Fanconi anemia. The upstream Fanconi proteins FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, and FANCL are necessary for FANCD2 monoubiquitination and Fanconi pathway function. We posed the question whether FANCD2 monoubiquitination is sufficient to restore the Fanconi pathway function in the absence of the upstream FA proteins. We constructed a chimeric monoubiquitinated FANCD2 protein by fusing the FANCD2 cDNA sequence contiguously (in-frame) with a ubiquitin coding sequence. This FANCD2-ubiquition fusion protein (D2-ubi) was stably expressed in FANCD2−/−, FANCA−/−, FANCC−/−, and FANCG−/− human patient-derived cell lines. D2-ubi expression corrected the MMC hypersensitivity of these FA cell lines in the absence of a functional upstream FA core complex. By western blot analysis, the D2-ubi protein migrated at a slower rate than the endogenous monoubiquitinated FANCD2 protein, consistent with its higher molecular mass. An even slower migrating D2-ubi form (D2-ubi-L) was detectable in cells harboring an intact Fanconi core protein complex (i.e., FA-D2 cells) but was absent in cells lacking functional Fanconi proteins FANCA, FANCC, or FANCG. These results indicate the D2-ubi can be further monoubiquitinated on lysine 561, but only in the presence of an intact FA complex. Furthermore, D2-ubi-L was upregulated in response to DNA damage in transfected FA-D2 and wild type cells. Like endogenous FANCD2, D2-ubi formed nuclear foci in the presence of the upstream Fanconi core complex. In the absence of the FA core complex, D2-ubi was localized diffusely throughout the nucleus, but unlike endogenous FANCD2, D2-ubi was retained within the nucleus following detergent permeabilization of FA-A, FA-C, and FA-G cells. These results further support the epistatic relationship of the FA genes in a linear pathway. In summary, these data indicate that a constitutively monoubiquitinated FANCD2 polypeptide is able to bypass defects in the upstream FA core complex to restore Fanconi pathway function. These studies identify FANCD2 ubiquitination as a potential therapeutic target for patients lacking the upstream FA core complex.

Correction of Fanconi Anemia Mutation Using the Crispr/Cas9 System

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3622-3622
Author(s):  
Nozomu Kawashima ◽  
Yusuke Okuno ◽  
Yuko Sekiya ◽  
Xinan Wang ◽  
Atsushi Narita ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Point Mutation ◽  
Fanconi Anemia ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Genetically Engineered ◽  
A Cell ◽  
Fancd2 Monoubiquitination

Abstract Introduction Gene therapy has been developed for genetic diseases, either to restore normal function for loss-of-function mutations or to inhibit gain-of-function mutations. Gene addition using genetically engineered viral and plasmid vectors has successfully corrected cell pathophysiology resulting in the production of functional proteins. Therapeutic safety has been reinforced by the use of self-inactivating vectors; however, the potential risk of tumorigenesis raises concerns for insertional mutagenesis combined with acquired somatic mutations. Recent advances in gene editing using an RNA-guided endonuclease (RGEN), known as the CRISPR/Cas9 system, have opened a new frontier for the in situ correction of disease-associated mutations. Genomic DNA of cells harboring mutations can be excised and replaced with a DNA template for the functional gene sequence using homology-directed repair (HDR). The advantages of this repair include fewer off-target effects and a reduced risk of copy number changes compared with gene addition using vectors. Fanconi anemia (FA) is a syndrome of inherited bone marrow failure, characterized by the deficient regulation of DNA double-strand break repair. Clinical trials of gene therapy using viral vectors are still on-going with partial success; therefore, a new gene editing technique deserves attention. However, the feasibility of this approach in diseases with impaired HDR, such as FA, is unknown. Therefore, we used an RGEN to generate a cell line harboring a disease-causing point mutation in an FA-associated gene and elucidated the efficacy of restoring the mutation thereafter. Methods pSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and single guide RNAs (sgRNAs) of interest. The sgRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate sgRNAs were designed to be specific for the FANCC c.67delG:p.D23Ifs*23 mutation type (MT) or wild type (WT): gRNA#4, 5′-ATGGGATCAGGCTTCCACTT-3′ and gRNA#5, 5′-GAAGCTTTCTGTATGGGATC-3′ were specific for the WT sequence; whereas, gRNA M4, 5′-TATGGATCAGGCTTCCACTT-3′ and gRNA M5, 5′-AGAAGCTTTCTGTATGGATC-3′ were specific for the MT sequence. pCAG-EGxxFP, an EGFP-based reporter plasmid for the HDR that harbored the 500-bp target region of the WT or MT FANCC, was constructed for the gRNA selection. An HDR template construct was designed to incorporate a puromycin-resistant gene flanked by two loxP sites and two homologous arms containing the WT or MT sequence. HEK293T cells harboring the WT FANCC sequence were genetically edited by the above-mentioned plasmids. Results To validate an efficient and specific sgRNA for DNA double-strand breaks, we cotransfected pCAG-EGxxFP-FANCC WT or MT and pSpCas9(BB)-FANCC-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR and thus the efficacy and specificity of sequence-specific DNA excision, was observed 48 h later, and we determined that gRNA#4 and gRNA M4 were specific for the WT and MT sequences, respectively. To generate cells harboring the MT FANCC sequence, HEK293T cells were cotransfected with pSpCas9(BB)-FANCC-gRNA#4 and the HDR template plasmid harboring the MT FANCC. A cell harboring biallelic MT FANCC was selected by adding puromycin and single-cell cloning. The transient expression of Cre recombinase in this clone successfully deleted the drug-selection cassette, and 293T-FANCC c.67delG cells were established. This cell showed the loss of FANCD2 monoubiquitination, a hallmark of a deficient FA core complex. Next, the 293T-FANCC c.67delG cells were cotransfected with pSpCas9(BB)-FANCC-gRNA M4 and the HDR template with the WT FANCC. This restoration of the mutated FANCC sequence resulted in a high frequency of at least monoallelic correction and the restoration of FANCD2 monoubiquitination. Conclusions The feasibility of genome editing was demonstrated in cells harboring an FA mutation, which can be a foothold for future therapy using precision gene restoration in patients with impaired HDR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Chk1-Mediated Phosphorylation of FANCE Is Required for the Fanconi Anemia/BRCA Pathway

2007 ◽  
Vol 27 (8) ◽  
pp. 3098-3108 ◽  
Author(s):  
XiaoZhe Wang ◽  
Richard D. Kennedy ◽  
Kallol Ray ◽  
Patricia Stuckert ◽  
Tom Ellenberger ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Mutant Form ◽  
Cross Link ◽  
Core Complex ◽  
Core Enzyme ◽  
Cross Links ◽  
Phase Progression ◽  
Nuclear Foci ◽  
Fancd2 Monoubiquitination

ABSTRACT The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.

Sign in / Sign up

Export Citation Format

Share Document