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Impaired hematopoiesis and leukemia development in mice with a conditional knock-in allele of a mutant splicing factor gene U2af1

Significance Somatic mutations in some splicing factor genes are frequently found in myelodysplastic syndromes (MDS) and MDS-related acute myeloid leukemia (AML), blood cancers with few effective treatment options. However, the pathophysiological effects of these mutations remain poorly characterized. Here, we report the establishment of mouse models to study a common splicing factor mutation, U2AF1(S34F). Production of the mutant protein in the murine hematopoietic compartment disrupts hematopoiesis in ways resembling human MDS. We further identified deletion of the Runx1 gene and other known oncogenic mutations as changes that might collaborate with U2af1(S34F) to give rise to frank AML in mice. However, the U2af1(S34F) mutation was absent in two of the three AML cases, raising the possibility that this mutant protein plays a dispensable role in tumor maintenance.


Generation of mice carrying the MGS34F or IES34F alleles
We have used two strategies to create conditional "knock-in" alleles of U2af1(S34F) in the mouse germ line. The two approaches involve targeting the normal locus with vector DNA that carries the S34F mutation in the context of a MiniGene (MGS34F) or an Inverted Exon (IES34F). Both alleles can be rearranged by the Cre recombinase to allow production of U2af1(S34F) protein from the native U2af1 locus. As shown in Fig.   1A, the targeting vector for MGS34F contains a WT minigene (exons 2 - 8, NM_024187), a transcriptional "STOP" signal (3 repeats of SV40 late poly A sequence), an Frt-Neo-Frt cassette, and a mutant Exon 2 carrying the S34F missense mutation (TCT > TTT). One LoxP sequence precedes the WT minigene, 470 bp upstream;; another LoxP sequence precedes the mutant Exon 2, 475 bp upstream. As shown in  (NHGRI). The mouse ES cell line, named HG3, was a B6/129 F1 hybrid, derived from a cross between a B6 male homozygous for a UBC-GFP transgene (C57BL/6-Tg(UBC-GFP)30Scha/J;; Jackson Laboratory (JAX) stock number: 004353) and a WT 129 female mouse (129S6/SvEvTac. Taconic model number: 129SVE-F). 100 µg of the targeting vector was linearized by digestion with Asc I, the recognition site for which is located immediately upstream of the 5' homology arm on the targeting vectors. Linearized DNA was electroporated into HG3 cells (passage 7). Following G418 selection, the surviving cell clones were expanded for Southern blot analysis to identify those clones that had undergone correct homologous recombination (Figs S1A and S2B) and with normal karyotypes. Positive ES cell clones were then microinjected into C57BL/6J blastocysts.
Resulting chimera mice with a high percentage of GFP positive cells were used to mate with B6 WT mice (C57BL/6J;; JAX stock number: 000664) to test for germline transmission. The genotypes of F1 mice were determined by PCR amplification, using the primers U2af1-A1LoxP-F and U2af1-A1LoxP-R (Table  S2), and the identities of the PCR amplicons were verified by Sanger sequencing. Tail DNA samples carrying the targeted allele were further confirmed by Southern blot analysis (Figs S1A and S2B). F1 mice carrying the targeted allele were crossed with mice expressing Flp (B6.Cg-Tg(ACTFLPe)9205Dym/J. JAX stock number: 005703) to remove the Neo cassette.
Removal of the Neo cassette in the progeny mice was further confirmed by PCR-based genotyping (Table  S2). Flanking sequences surrounding the remaining Frt site in the targeted alleles, as well as the boundaries of the homology arms, were verified by Sanger sequencing. Mice (MGS34F/WT;; Flp or IES34F/WT;; Flp) were crossed with B6 WT mice, and/or B6 mice carrying UBC-CreERT2 (JAX stock number: 008085), Mx1-Cre (JAX stock number: 005673), and/or Runx1 F (1) alleles as specified in the Results section for desired genotypes.

Mouse procedures
Peripheral blood sampling and CBC analysis: Blood was collected by either submandibular or retro-orbital bleeding. The former procedure was done as described (2), except that a 20 G needle instead of a lancet was used to enter the facial vein. The latter procedure involves collecting blood through heparinized microhematocrit capillary tubes (Thermo Fisher Scientific). Freshly collected blood was mixed immediately with EDTA in an EDTA-coated microfuge tube (Sarstedt) before performing complete blood counts (CBC) and other tests. Automatic CBC was conducted at the Department of Laboratory Medicine (NIH Clinical Center) as a paid service. CBC findings were further confirmed by reviewing the corresponding blood smears. four weeks before being returned to normal housing and feeding.
Drug treatment: Poly (IC) (high molecular weight, Invivogen) was reconstituted in saline to 1 mg/ml according to vendor's instruction. Poly (IC) (three doses of 250 µg per mouse) was given every other day via intraperitoneal injection. ENU treatment was as previously described (3). Briefly, ENU (Sigma-Aldrich) was dissolved in pure ethanol at 100 mg/ml, further diluted in phosphate/citrate buffer to a final concentration of 5 mg/ml and given intraperitoneally at 100 mg/kg, one week after the last dose of poly (IC). BrdU (1 mg/mouse at 10 mg/ml, BD Biosciences) was given intraperitoneally 2 hr before euthanasia.
Moribund criteria: The health of the mice were monitored by technicians who were blinded to the genotypes and treatments of the mice. Mice showing signs of pale paws, hunched posture, lethargy, dyspnea, rough hair coat, and severe weight loss were euthanized by an overdose of carbon dioxide, followed by cervical dislocation.
Generation of mouse embryo fibroblasts (MEFs): MEFs were generated from E13.5 embryos, the products of timed mating between MGS34F/WT or IES34F/WT male mice and UBC-CreERT2 hemizygous female mice. Freshly dissected mouse embryos (without the placenta, head, and internal organs) were minced by a sterile single blade and then transferred to a 15 ml conical tube containing 2 ml 0.125% trypsin (0.25% trypsin diluted 1:1 with DPBS). Cells in tissue culture medium were incubated at 37 degrees Celsius for 15 min with gentle shaking. At the end of incubation, 12 ml growth media (DMEM supplemented with 10% FBS and penicillin/streptomycin) were added to the tube to inactivate trypsin. The entire culture was then transferred to a 100 mm dish and incubated at 37 degrees Celsius with 5% CO 2 . The growth medium was changed the next day. The head of the embryo was used for genotyping. MEFs were passaged before they reached 100% confluence, and cells passaged fewer than five times were used for all assays.

Histology
The nuclear features of bone marrow and spleen cells were evaluated using cytospin preparations. 1 - 5 x 10 5 freshly prepared bone marrow or spleen cells (in 100 µl - 200 µl DPBS with 5% FBS) were applied to a slide chamber and spun onto glass microscope slides (pre-wetted with DPBS with 5% FBS) using a Shandon Cytospin 2 centrifuge at 500 g for 5 min with low acceleration. Slides were air dried before Wright-Giemsa staining, which was performed either by the Department of Laboratory Medicine (NIH Clinical Center) as a complimentary service or by the researchers using the PROTOCOL Hema 3 stain set. Stained slides were then sealed with cover glass and Cytoseal 60 (Electron Microscopy Sciences).
Nuclear and cytoplasmic features of peripheral blood, bone marrow, and spleen cells were also evaluated using smear or "touch" preparations. Blood smears were prepared using an Autoprep (Dynamic Diagnostics). Bone marrow smears were prepared by first cutting freshly dissected long bones longitudinally along the bone cavity to expose the bone marrow. A 5-O camel's hair brush was wetted with hypotonic FBS (2:1 (v/v) FBS:distilled water) and used to make a "slurry" of the bone marrow. The suspended bone marrow cells on the brush were then "painted" onto glass microscope slides.
Spleen "touch" preparations were made by touching the freshly cut surface of mouse spleens onto glass slides, which were air dried and stained with Wright-Giemsa as described above.
Evidence of megakaryocyte clustering, excessive extramedullary hematopoiesis, and blast tissue infiltration were examined using tissue sections. Fresh tissues (bone, spleen, liver, lung, heart, etc.) were dissected and fixed in 10% neutral buffered formalin (VWR) for at least 24 hr. Tissue processing (bone decalcification, tissue dehydration, paraffin-embedding and sectioning at 5 µm) and H&E staining were performed as a paid Histological slides were examined by a board-certified anatomic and clinical pathologist and hematopathologist (BD), who was blinded to mouse genotypes and treatment at the time of review. Representative images were acquired using an Axio Observer A1 microscope (Carl Zeiss) at 400x or 600x magnification.

RNA and RT-qPCR: RNA was extracted using Trizol (Molecular Research Center or
Invitrogen) combined with a column "cleanup" step using RNeasy (Qiagen). cDNA was synthesized from RNA (up to 2 μg) using a High-Capacity cDNA Reverse Transcription (RT) kit (Applied Biosystems). Isoform-specific primers (Table  S2) were used to quantify the changes in alternative splicing or polyadenylation. The specificity and efficiency (> 90%) of each primer set were determined as described before (4). Quantitative PCR (qPCR) was performed using either the 7900HT real-time PCR system (Applied Biosystems) with the USB VeriQuest SYBR Green qPCR Master Mix (Affymetrix) or the QX200 Droplet Digital PCR system (ddPCR, Bio-Rad) with the EvaGreen Supermix (Bio-Rad). The RQ Manager software (v1.2, Applied Biosystems) was used for quantifying qPCR results from 7900HT using a relative quantification method (∆∆Ct).
For ddPCR analysis, the concentration of each isoform was calculated using the QuantaSoft Software (Bio-Rad) and results were presented as percent inclusion of the indicated exon or portion of an exon. The allele-sensitive S34F/WT SNP Taqman assay was custom synthesized (Applied Biosystems) and characterized in Fig.  S3C - S3E.
The plasmids for calibrating the assay carry modified versions of the U2af1(WT) or U2af1(S34) cDNA, as described previously (4).
Genotyping PCR: Routine genotyping to detect the MGS34F and IES34F alleles was done by conventional semi-quantitative PCR using the Hotstar Taq master mix (Qiagen), the primers U2af1-A1LoxP-F and U2af1-A1LoxP-R (750 nM final concentration for each primer. Table  S2), and tissue lysate (1 µl) in a 10 µl reaction.

Fluorescent in situ hybridization (FISH): FISH was conducted at the Cytogenetics and
Microscopy Core (NHGRI), as described (6). The GFP probe was made from a fragment released from the pEGFR-C1 vector digested with Nhe I and EcoR I (New England Biolabs).

Bioinformatics
Differential gene expression and splicing patterns were analyzed as described (4,8).
Briefly, reads were first mapped to known mouse or human transcripts using RSEM (9).
Remaining unaligned reads were then mapped to a database of possible junctions between all 5' and 3' splice sites of relevant genes and then subsequently to the GRCm38 mouse genome assembly, using TopHat (10). The number of "reads" were then subjected to analysis by the Wagenmakers's Bayesian alternative to the binomial proportion test (11). Differential splicing was defined as a difference in absolute isoform ratio of at least 10%, based on at least 20 relevant reads in the comparator samples, using MISO v2.0 (12). Differentially expressed genes were defined as those that exhibited at least a two-fold difference in abundance of RNA (mutant vs. WT).
Sequences logos were created using the seqLogo package from Bioconductor (13). The invariant AG at 3' splice sites was not plotted to scale to allow highlighting of the consensus nucleotides at the -3 position. Unsupervised clustering was performed using Ward's method.
Relative levels of expression of WT and mutant alleles were estimated from the mapped mRNA sequencing data in the IGV browser (v2.3.72, Broad Institute). The Sashimi plot for Tet2 mRNA (Fig.  5C) was also generated using the IGV browser.
Analysis of targeted gene panel sequencing (mIMPACT, Table S7) was described previously (7). The DNA-level variants were predicted using the Variant Effect Predictor  Table   S2.
Supplemental Figure  S4. Additional data related to Fig.  2  DNA (see Fig.  S10F and S10G). Table  S1: Crossover of the linked U2af1(S34F) targeted alleles and the GFP transgene. The number of mice in each genotype category is shown. S34F in the column headers represents either the MGS34F or IES34F allele. Table  S2: Primer and probe sequences. Supplemental Table  S3: Splicing alterations in mouse MP cells. Each row of the table corresponds to an isoform of a splicing event that is differentially spliced in MP cells from mice of different genotypes (n = 4 of each genotype) for the indicated comparisons. In each case, the isoform for which information is given is as follows: the inclusion isoform for cassette exons (type "se"), the intron-proximal isoform for competing 5' or 3' splice site events (types "a5ss" or "a3ss"), inclusion of the upstream exon for mutually exclusive exon events (type "mxe"), splicing of retained introns annotated as alternative (type "ri") or constitutive (type "ci"), or canonical splicing of an annotated constitutive junction (type "cj"). The columns specify relevant annotations for each event as follows: "coords" specifies the genomic coordinates of the event;; "gene" specifies the Ensembl gene ID;; "geneName" specifies the gene name;; "geneDescription" is a description of the gene as given by Ensembl;; "type" specifies the type of splicing event;; "nmdTarget" specifies whether the isoform is a predicted substrate for degradation by nonsense mediated decay (NMD) (NA indicates that either all or no isoforms of the event are predicted NMD substrates);; "deltaPsi" give the absolute difference in isoform ratio (Percent Spliced In or "PSI" value) for the indicated comparisons;; "logFoldChange", Log2 fold change values of Psi for the indicated comparisons;; "p-value", p-value computed with the Mann-Whitney U test for group comparisons.

Supplemental
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