A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2

AbstractFanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci −/− mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci−/− Fancd2−/− also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

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RPA resolves conflicting activities of accessory proteins during reconstitution of Dmcl-mediated meiotic recombination

10.1101/356592 ◽

2018 ◽

Author(s):

Yuen-Ling Chan ◽

Annie Zhang ◽

Benjamin P. Weissman ◽

Douglas K. Bishop

Keyword(s):

Meiotic Recombination ◽

Direct Interaction ◽

Multiple Roles ◽

Homology Search ◽

Accessory Proteins ◽

Loop Formation ◽

Ssdna Binding ◽

Ssdna Binding Protein ◽

D Loop

AbstractDmc1 catalyzes homology search and strand exchange during meiotic recombination in budding yeast and many other organisms including humans. Here we reconstitute Dmc1 recombination in vitro using six purified proteins including Dmc1 and its accessory proteins RPA, Rad51, Rdh54/Tid1, Mei5-Sae3, and Hop2-Mnd1 to promote D-loop formation between ssDNA and dsDNA substrates. Each accessory protein contributed to Dmc1’s activity, with the combination of all six proteins yielding optimal activity. The ssDNA binding protein RPA plays multiple roles in stimulating Dmc1’s activity including by overcoming inhibitory effects of ssDNA secondary structure on D-loop reactions, and by stabilizing and elongating D-loops. In addition, we demonstrate that RPA limits inhibitory interactions of Hop2-Mnd1 and Rdh54/Tid1 that otherwise occur during assembly of Dmc1-ssDNA nucleoprotein filaments. Finally, we report interactions between the proteins employed in the biochemical reconstitution including a direct interaction between Rad51 and Dmc1 that is enhanced by Mei5-Sae3.

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Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2162 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2162-2171 ◽

Cited By ~ 66

Author(s):

C S Madsen ◽

S C Ghivizzani ◽

W W Hauswirth

Keyword(s):

Mitochondrial Dna ◽

Protein Binding ◽

Loop Formation ◽

Sequence Element ◽

Conserved Sequence ◽

Loop Region ◽

In Organello ◽

Close Proximity ◽

D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.

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Dosage-Dependent Effects of Akt1/Protein Kinase Bα (PKBα) and Akt3/PKBγ on Thymus, Skin, and Cardiovascular and Nervous System Development in Mice

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10407-10418.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10407-10418 ◽

Cited By ~ 141

Author(s):

Zhong-Zhou Yang ◽

Oliver Tschopp ◽

Nicolas Di-Poï ◽

Elisabeth Bruder ◽

Anne Baudry ◽

...

Keyword(s):

Protein Kinase ◽

Cell Cycle Progression ◽

System Development ◽

Critical Role ◽

Nervous System Development ◽

Mouse Development ◽

Double Knockout ◽

Developmental Defects ◽

Regulation Of Metabolism ◽

Survival And Development

ABSTRACT Akt/protein kinase B (PKB) plays a critical role in the regulation of metabolism, transcription, cell migration, cell cycle progression, and cell survival. The existence of viable knockout mice for each of the three isoforms suggests functional redundancy. We generated mice with combined mutant alleles of Akt1 and Akt3 to study their effects on mouse development. Here we show that Akt1 − / − Akt3 +/ − mice display multiple defects in the thymus, heart, and skin and die within several days after birth, while Akt1 +/ − Akt3 − / − mice survive normally. Double knockout (Akt1 − / − Akt3 − / −) causes embryonic lethality at around embryonic days 11 and 12, with more severe developmental defects in the cardiovascular and nervous systems. Increased apoptosis was found in the developing brain of double mutant embryos. These data indicate that the Akt1 gene is more essential than Akt3 for embryonic development and survival but that both are required for embryo development. Our results indicate isoform-specific and dosage-dependent effects of Akt on animal survival and development.

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Combined knockout of Lrrk2 and Rab29 does not result in behavioral abnormalities in vivo

10.1101/2020.05.13.093708 ◽

2020 ◽

Cited By ~ 2

Author(s):

Melissa Conti Mazza ◽

Victoria Nguyen ◽

Alexandra Beilina ◽

Jinhui Ding ◽

Mark R. Cookson

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Activity ◽

Association Studies ◽

Dopamine Neurons ◽

Substantia Nigra Pars Compacta ◽

Genome Wide Association Studies ◽

Double Knockout ◽

Knockout Mouse Model

AbstractCoding mutations in the LRRK2 gene, encoding for a large protein kinase, have been shown to cause familial Parkinson’s disease (PD). The immediate biological consequence of LRRK2 mutations is to increase kinase activity, leading to the suggestion that inhibition of this enzyme might be useful therapeutically to slow disease progression. Genome-wide association studies have identified the chromosomal loci around LRRK2 and one of its proposed substrates, RAB29, as contributors towards the lifetime risk of sporadic PD. Considering the evidence for interactions between LRRK2 and RAB29 on the genetic and protein levels, here we generated a double knockout mouse model and determined whether there are any consequences on brain function with aging. From a battery of motor and non-motor behavioral tests, we noted only that 18-24 month Rab29-/- and double (Lrrk2-/-/Rab29-/-) knockout mice had diminished locomotor behavior in open field compared to wildtype mice. However, no genotype differences were seen in number of substantia nigra pars compacta (SNc) dopamine neurons or in tyrosine hydroxylase levels in the SNc and striatum, which might reflect a PD-like pathology. These results suggest that depletion of both Lrrk2 and Rab29 is tolerated, at least in mice, and support that this pathway might be able to be safely targeted for therapeutics in humans.Significance statementGenetic variation in LRRK2 that result in elevated kinase activity can cause Parkinson’s disease (PD), suggesting LRRK2 inhibition as a therapeutic strategy. RAB29, a substrate of LRRK2, has also been associated with increased PD risk. Evidence exists for an interactive relationship between LRRK2 and RAB29. Mouse models lacking either LRRK2 or RAB29 do not show brain pathologies. We hypothesized that the loss of both targets would result in additive effects across in vivo and post-mortem assessments in aging mice. We found that loss of both LRRK2 and RAB29 did not result in significant behavioral deficits or dopamine neuron loss. This evidence suggests that chronic inhibition of this pathway should be tolerated clinically.

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Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

10.1101/2020.05.20.106617 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shanaya Shital Shah ◽

Stella Hartono ◽

Frédéric Chédin ◽

Wolf-Dietrich Heyer

Keyword(s):

Real Time ◽

Single Molecule ◽

Detection Methods ◽

Loop Formation ◽

Bisulfite Treatment ◽

Loop Detection ◽

D Loop ◽

Donor Choice ◽

The Relationship

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

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mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

Genetics Research ◽

10.1017/s0016672305007883 ◽

2005 ◽

Vol 86 (3) ◽

pp. 185-191 ◽

Cited By ~ 10

Author(s):

PETTER PORTIN

Keyword(s):

X Chromosome ◽

Meiotic Recombination ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Crossing Over ◽

Dna Double Strand Break ◽

D Loop ◽

Break Repair ◽

Strand Annealing

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.

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Mouse Gonad Development in the Absence of the Pro-Ovary Factor WNT4 and the Pro-Testis Factor SOX9

Cells ◽

10.3390/cells9051103 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1103

Author(s):

Furong Tang ◽

Nainoa Richardson ◽

Audrey Albina ◽

Marie-Christine Chaboissier ◽

Aitana Perea-Gomez

Keyword(s):

Granulosa Cells ◽

Gonad Development ◽

Supporting Cell ◽

Gonadal Development ◽

Ovary Development ◽

Double Knockout ◽

Cell Identity ◽

Knockout Mouse Model ◽

Steroidogenic Cell ◽

Male Specific

The transcription factors SRY and SOX9 and RSPO1/WNT4/β-Catenin signaling act as antagonistic pathways to drive testis and ovary development respectively, from a common gonadal primordium in mouse embryos. In this work, we took advantage of a double knockout mouse model to study gonadal development when Sox9 and Wnt4 are both mutated. We show that the XX gonad mutant for Wnt4 or for both Wnt4 and Sox9 develop as ovotestes, demonstrating that ectopic SOX9 function is not required for the partial female-to-male sex reversal caused by a Wnt4 mutation. Sox9 deletion in XY gonads leads to ovarian development accompanied by ectopic WNT/β-catenin signaling. In XY Sox9 mutant gonads, SRY-positive supporting precursors adopt a female-like identity and develop as pre-granulosa-like cells. This phenotype cannot be fully prevented by the deletion of Wnt4 or Rspo1, indicating that SOX9 is required for the early determination of the male supporting cell identity independently of repressing RSPO1/WNT4/β-Catenin signaling. However, in XY Sox9 Wnt4 double mutant gonads, pre-granulosa cells are not maintained, as they prematurely differentiate as mature granulosa cells and then trans-differentiate into Sertoli-like cells. Together, our results reveal the dynamics of the specific and independent actions of SOX9 and WNT4 during gonadal differentiation: SOX9 is essential in the testis for early specification of male-supporting cells whereas WNT4 functions in the ovary to maintain female-supporting cell identity and inhibit male-specific vascular and steroidogenic cell differentiation.

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iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1505649112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6080-6085 ◽

Cited By ~ 77

Author(s):

Xue Li ◽

Thorsten Maretzky ◽

Gisela Weskamp ◽

Sébastien Monette ◽

Xiaoping Qing ◽

...

Keyword(s):

Heart Valves ◽

Egf Receptor ◽

Transmembrane Domain ◽

Intestinal Barrier ◽

Underlying Mechanism ◽

Egfr Signaling ◽

Mouse Development ◽

Double Knockout ◽

Egfr Ligands ◽

And Function

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17−/− mice die perinatally with open eyes, like Egfr−/− mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2−/− mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2−/− double knockout mice resemble Adam17−/− and Egfr−/− mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2−/− tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.

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Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids

Journal of the American Society of Nephrology ◽

10.1681/asn.2017111205 ◽

2018 ◽

Vol 29 (6) ◽

pp. 1624-1635 ◽

Cited By ~ 13

Author(s):

Clara Vilches ◽

Emilia Boiadjieva-Knöpfel ◽

Susanna Bodoy ◽

Simone Camargo ◽

Miguel López de Heredia ◽

...

Keyword(s):

Amino Acids ◽

Knockout Mouse ◽

Functional Relationship ◽

Tubular Reabsorption ◽

Compensatory Mechanisms ◽

Double Knockout ◽

Knockout Mouse Model ◽

Relationship Of

Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo.Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.

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