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Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair.

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.

NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology, allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of preleukemic cells to full-blown disease.

Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes. Previously, conditional alleles were introduced in the mouse germline by classic gene targeting in embryonic stem cells, a challenging and time-consuming procedure. Now, conditional alleles can be generated directly in fertilized mouse eggs (zygotes) using the CRISPR/Cas9 technology. This one-step generation of mice is easier in design and faster. Here, we describe our achieved success rate, the considerations in design of a conditional allele, a detailed protocol to prepare the zygote injection mix, and the screening procedure to identify the new conditional knockout mouse strain.

Pax3, a member of the paired-class homeodomain family of transcription factors, plays an important role in embryonic development of neurepithelium and mesenchyme-derived tissues in the mouse and is an early marker for myogenic differentiation. In the present work we identify an alternative splicing event for endogenous Pax3 in primary mouse myoblasts. The resulting splice variant arises through the utilization of a previously unreported splice donor consensus sequence present at the junction between exons 7 and 8 in the Pax3 sequence. The use of this splice donor site in conjunction with the splice acceptor site present between intron 8 and exon 9 results in the deletion of exon 8 and removes a majority of the Pax3 transcriptional activation domain. Consistent with this fact, we demonstrate that the alternatively spliced form of Pax3 is transcriptionally inactive and that the presence of this isoform can effectively inhibit the activity of the full-length protein.

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p–Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non–WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98–RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

An expanding subgroup of chromosomal translocation-generated oncoproteins in human acute myeloid leukemias (AML) involve the FG repeat-containing nuclear pore complex (NPC) proteins NUP98 (39) and CAN/NUP214 (13, 22). The NUP98 gene is found at the breakpoints of two distinct chromosomal rearrangements: t(7;11)(p15;p15) (7, 18, 33), and inv(11)(p15;q22) (2), which link NUP98 to the class I homeotic transcription factor HOXA9 and the putative RNA helicase DDX10, respectively. In each rearrangement, the chromosomal breakpoints are located within two flanking introns of the NUP98 gene that separate the FG repeat-rich N terminus of NUP98 from its C terminus, which contains a ribonucleoprotein (RNP)-binding motif (39). Although each translocation generates two reciprocal chimeric products, only those driven by the NUP98 promoter and containing the FG repeat region are predicted to mediate leukemogenesis (2, 7, 33). Another nucleoporin gene, CAN/NUP214, is found at the breakpoint of two independent chromosomal rearrangements: t(6;9)(p23;q34), which fuses CAN/NUP214 to DEK (49), and inv(9;9)(q34;q34), which links it to SET (50). The leukemia-specific transcripts, DEK-CAN/NUP214 and SET-CAN/NUP214, both encode nuclear fusion proteins. The proteins contain identical C-terminal portions of CAN/NUP214, including its FG repeat-rich region, and a coiled-coil domain (13, 22). DEK and SET are both nuclear proteins that have no sequence similarity other than the presence of acidic motifs that may participate in DNA binding (13, 14, 31). The involvement of two FG repeat-containing nucleoporins in multiple translocations associated with human leukemia raises intriguing questions about their role in leukemogenesis. In particular, the consistent presence of FG repeat regions suggests that such domains could serve a common function in the transformation of hematopoietic cells. Many of the known components of the NPC have regions rich in FXFG, GLFG and/or FG repeats (amino acids are given in single-letter code, with X indicating any amino acid). Such repeats (called FG for simplicity) are presumed contact sites for soluble nucleocytoplasmic transport factors carrying different kinds of cargo; however, their precise functions in vivo remain to be determined (35, 36). HOXA9, expressed in both the primitive pluripotent precursors and the myeloid progenitors of human bone marrow (42), is the only nucleoporin fusion partner with an established physiological role in hematopoietic development. HOXA9 knockout mice have multiple hematopoietic defects, including reduced numbers of peripheral blood granulocytes and lymphocytes, as well as myeloid and pre-B-cell progenitors, and their spleens and thymuses are smaller than normal (25). Besides its involvement in t(7;11)-mediated myeloid leukemogenesis, HOXA9 has been implicated in the formation of myeloid leukemias in the BXH-2 strain of mice (29). BXH-2 mice carry an endogenous murine leukemia virus that acts as a viral mutagen predisposing the animals to myeloid malignancies (4, 5). In this experimental tumor model, about 3% of all leukemias in BXH-2 mice display proviral activation of HOXA9 (34). Constitutive expression of HOXA9 alone is not sufficient for efficient transformation of murine hematopoietic cells; it requires coexpression of MEIS1 (23, 34), a PBX1-related divergent homeodomain-containing protein that cooperatively binds DNA with HOXA9 in vitro (44). In this study, we show that the t(7;11)-derived fusion gene generates two chimeric proteins via alternative splicing within NUP98. Investigation of the structural and functional regions of the chimeric NUP98-HOXA9 proteins demonstrated that HOXA9-mediated DNA binding and interaction with PBX are essential for transformation of NIH 3T3 fibroblasts. In both chimeras, the NUP98 portions contained very potent transcription activation domains, which replace a strong transcriptional repressor domain within the amino-terminal half HOXA9. Interestingly, the transcriptional coactivators CREB binding protein (CBP) and potentially p300 interacted and functionally cooperated with the NUP98 FG-repeat-rich portions. Abbrogation of NUP98-HOXA9-mediated transformation corresponded to the loss of NUP98-mediated transcriptional activity and CBP binding. Thus, NUP98-HOXA9 seems to recruit CBP/p300 as part of its oncogenic mechanism. Because CBP and p300 are coactivators for a number of gene-specific transcription factors, they could also be critical accessory factors for other fusion proteins that deregulate transcription.