FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2904293039> ?p ?o ?g. }

• W2904293039 endingPage "84.e6" @default.
• W2904293039 startingPage "70" @default.
• W2904293039 abstract "SMC3 encodes a subunit of the cohesin complex that has canonical roles in regulating sister chromatids segregation during mitosis and meiosis. Recurrent heterozygous mutations in SMC3 have been reported in acute myeloid leukemia (AML) and other myeloid malignancies. In this study, we investigated whether the missense mutations in SMC3 might have dominant-negative effects or phenocopy loss-of-function effects by comparing the consequences of Smc3-deficient and -haploinsufficient mouse models. We found that homozygous deletion of Smc3 during embryogenesis or in adult mice led to hematopoietic failure, suggesting that SMC3 missense mutations are unlikely to be associated with simple dominant-negative phenotypes. In contrast, haploinsufficiency was tolerated during embryonic and adult hematopoiesis. Under steady-state conditions, Smc3 haploinsufficiency did not alter colony forming in methylcellulose, only modestly decreased mature myeloid cell populations, and led to limited expression changes and chromatin alteration in Lin–cKit+ bone marrow cells. However, following transplantation, engraftment, and subsequent deletion, we observed a hematopoietic competitive disadvantage across myeloid and lymphoid lineages and within the stem/progenitor compartments. This disadvantage was not affected by hematopoietic stresses, but was partially abrogated by concurrent Dnmt3a haploinsufficiency, suggesting that antecedent mutations may be required to optimize the leukemogenic potential of Smc3 mutations. SMC3 encodes a subunit of the cohesin complex that has canonical roles in regulating sister chromatids segregation during mitosis and meiosis. Recurrent heterozygous mutations in SMC3 have been reported in acute myeloid leukemia (AML) and other myeloid malignancies. In this study, we investigated whether the missense mutations in SMC3 might have dominant-negative effects or phenocopy loss-of-function effects by comparing the consequences of Smc3-deficient and -haploinsufficient mouse models. We found that homozygous deletion of Smc3 during embryogenesis or in adult mice led to hematopoietic failure, suggesting that SMC3 missense mutations are unlikely to be associated with simple dominant-negative phenotypes. In contrast, haploinsufficiency was tolerated during embryonic and adult hematopoiesis. Under steady-state conditions, Smc3 haploinsufficiency did not alter colony forming in methylcellulose, only modestly decreased mature myeloid cell populations, and led to limited expression changes and chromatin alteration in Lin–cKit+ bone marrow cells. However, following transplantation, engraftment, and subsequent deletion, we observed a hematopoietic competitive disadvantage across myeloid and lymphoid lineages and within the stem/progenitor compartments. This disadvantage was not affected by hematopoietic stresses, but was partially abrogated by concurrent Dnmt3a haploinsufficiency, suggesting that antecedent mutations may be required to optimize the leukemogenic potential of Smc3 mutations. Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy characterized by the accumulation of myeloblasts in the blood or bone marrow (BM) with maturation arrest and retained self-renewal [1Saultz JN Garzon R. Acute myeloid leukemia: a concise review.J Clin Med. 2016; 5: E33Crossref PubMed Scopus (191) Google Scholar]. Tremendous progress has been made in identifying recurrent gene mutations in AML, yet we are still in the early stages of understanding the mechanisms through which these genetic alterations contribute to the onset of the disease [2Lagunas-Rangel FA Chávez-Valencia V Gómez-Guijosa MÁ Cortes-Penagos C Acute myeloid leukemia: genetic alterations and their clinical prognosis.Int J Hematol Oncol Stem Cell Res. 2017; 11: 328-339PubMed Google Scholar]. Recurring mutations in the cohesin complex occur in four core components: SMC3, SMC1A, RAD21, and STAG2, and have been identified in AML and other myeloid malignancies [3Ley TJ Miller C Ding L et al.Cancer Genome Atlas Research Network,Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.N Engl J Med. 2013; 368: 2059-2074Crossref PubMed Scopus (3095) Google Scholar, 4Kon A Shih LY Minamino M et al.Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms.Nat Genet. 2013; 45: 1232-1237Crossref PubMed Scopus (260) Google Scholar, 5Thota S Viny AD Makishima H et al.Genetic alterations of the cohesin complex genes in myeloid malignancies.Blood. 2014; 124: 1790-1798Crossref PubMed Scopus (153) Google Scholar]. More than 50% of patients with Down syndrome-associated acute megakaryocytic leukemia have cohesin mutations, specifically in STAG2 [6Yoshida K Toki T Okuno Y et al.The landscape of somatic mutations in Down syndrome-related myeloid disorders.Nat Genet. 2013; 45: 1293-1299Crossref PubMed Scopus (238) Google Scholar]. Somatic cohesin mutations have also been observed in a variety of solid cancers, including colorectal carcinoma, ovarian carcinoma, glioblastoma, bladder carcinoma, and Ewing's sarcoma [7Barber TD McManus K Yuen KWY et al.Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers.Proc Natl Acad Sci. 2008; 105: 3443-3448Crossref PubMed Scopus (299) Google Scholar, 8Gorringe KL Ramakrishna M Williams LH et al.Are there any more ovarian tumor suppressor genes? A new perspective using ultra high-resolution copy number and loss of heterozygosity analysis.Genes Chromosomes Cancer. 2009; 48: 931-942Crossref PubMed Scopus (50) Google Scholar, 9Bailey ML O'Neil NJ van Pel DM Solomon DA Waldman T Hieter P Glioblastoma cells containing mutations in the cohesin component STAG2 are sensitive to PARP inhibition.Mol Cancer Ther. 2014; 13: 724-732Crossref PubMed Scopus (46) Google Scholar, 10Solomon DA Kim J-S Bondaruk J et al.Frequent truncating mutations of STAG2 in bladder cancer.Nat Genet. 2013; 45: 1428-1430Crossref PubMed Scopus (142) Google Scholar, 11Balbás-Martínez C Sagrera A Carrillo-de-Santa-Pau E et al.Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy.Nat Genet. 2013; 45: 1464-1469Crossref PubMed Scopus (188) Google Scholar, 12Solomon DA Kim T Diaz-Martinez LA et al.Mutational inactivation of STAG2 causes aneuploidy in human cancer.Science. 2011; 333: 1039-1043Crossref PubMed Scopus (319) Google Scholar]. Additionally, germline mutations of the cohesin complex are causally related to developmental disorders, particularly cohesinopathies such as Cornelia de Lange syndrome [13Remeseiro S Cuadrado A Gómez-López G Pisano DG Losada A A unique role of cohesin-SA1 in gene regulation and development.EMBO J. 2012; 31: 2090-2102Crossref PubMed Scopus (95) Google Scholar, 14Mannini L Cucco F Quarantotti V Krantz ID Musio A Mutation spectrum and genotype-phenotype correlation in Cornelia de Lange syndrome.Hum Mutat. 2013; 34: 1589-1596Crossref PubMed Scopus (122) Google Scholar]. SMC3 and RAD21 mutations are nearly universally heterozygous, whereas mutations in SMC1A and STAG2 may be hemizygous because they are X-linked. Cohesin mutations also tend to be mutually exclusive, implying that alteration in one component may be sufficient to disrupt the entire complex or alternatively, they may not be tolerated by a cell when co-occurring [15Welch JS Ley TJ Link DC et al.The origin and evolution of mutations in acute myeloid leukemia.Cell. 2012; 150: 264-278Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar, 16Thol F Bollin R Gehlhaar M et al.Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications.Blood. 2014; 123: 914-920Crossref PubMed Scopus (136) Google Scholar]. Cohesin mutations are often observed as early subclonal events in AML, conceivably facilitating disease initiation, although they are not observed in cases of clonal hematopoiesis of indeterminate potential (CHIP), suggesting they are unlikely to be the initiating event [15Welch JS Ley TJ Link DC et al.The origin and evolution of mutations in acute myeloid leukemia.Cell. 2012; 150: 264-278Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar, 17Conese M Liso A. Cohesin complex is a major player on the stage of leukemogenesis.Stem Cell Investig. 2016; 3: 18Crossref PubMed Scopus (1) Google Scholar, 18Xie M Lu C Wang J et al.Age-related mutations associated with clonal hematopoietic expansion and malignancies.Nat Med. 2014; 20: 1472-1478Crossref PubMed Scopus (1094) Google Scholar, 19Jaiswal S Natarajan P Silver AJ et al.Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease.N Engl J Med. 2017; 377: 111-121Crossref PubMed Scopus (956) Google Scholar]. The majority of SMC3 mutations are missense mutations; only one-third of SMC3 mutations are nonsense or splice-site variants. The missense mutations are scattered across all domains, although a few recurrently mutated nucleotides have been observed (e.g., R381Q, R661P). This pattern suggests that many of these mutations may result in simple loss-of-function consequences, although novel dominant-negative activities cannot be dismissed within the hot-spot variants. Intriguingly, DNMT3A mutations, one of the most commonly mutated genes in AML, frequently coincides with SMC3 mutations, suggesting there may be leukemogenic interactions between these mutations [5Thota S Viny AD Makishima H et al.Genetic alterations of the cohesin complex genes in myeloid malignancies.Blood. 2014; 124: 1790-1798Crossref PubMed Scopus (153) Google Scholar, 15Welch JS Ley TJ Link DC et al.The origin and evolution of mutations in acute myeloid leukemia.Cell. 2012; 150: 264-278Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar, 16Thol F Bollin R Gehlhaar M et al.Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications.Blood. 2014; 123: 914-920Crossref PubMed Scopus (136) Google Scholar, 20Cole CB Russler-Germain DA Ketkar S et al.Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies.J Clin Invest. 2017; 127: 3657-3674Crossref PubMed Scopus (51) Google Scholar]. In yeast- and cell-line-based studies, cohesin has been shown to play essential roles in sister chromatid segregation during the cell cycle, DNA damage repair, transcriptional regulation via chromatin looping, and maintenance of chromatin architecture [21Ball AR Chen YY Yokomori K Mechanisms of cohesin-mediated gene regulation and lessons learned from cohesinopathies.Biochim Biophys Acta. 2014; 1839: 191-202Crossref PubMed Scopus (40) Google Scholar, 22Wendt KS Yoshida K Itoh T et al.Cohesin mediates transcriptional insulation by CCCTC-binding factor.Nature. 2008; 451: 796-801Crossref PubMed Scopus (852) Google Scholar, 23Merkenschlager M Odom DT CTCF and cohesin: linking gene regulatory elements with their targets.Cell. 2013; 152: 1285-1297Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 24Schmidt D Schwalie PC Ross-Innes CS et al.A CTCF-independent role for cohesin in tissue-specific transcription.Genome Res. 2010; 20: 578-588Crossref PubMed Scopus (284) Google Scholar]. AML patients who harbor cohesin mutations typically have normal karyotype, indicating that hematopoietic cohesin mutations do not lead directly to chromosomal instability [3Ley TJ Miller C Ding L et al.Cancer Genome Atlas Research Network,Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.N Engl J Med. 2013; 368: 2059-2074Crossref PubMed Scopus (3095) Google Scholar, 16Thol F Bollin R Gehlhaar M et al.Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications.Blood. 2014; 123: 914-920Crossref PubMed Scopus (136) Google Scholar]. To define the hematopoietic consequences of SMC3 mutations and to determine whether these could reflect dominant-negative or loss-of-function phenotypes, we characterized the in vivo effects of Smc3 deficiency and Smc3 haploinsufficiency on murine hematopoiesis using conditionally deleted strategies. In contrast to our expectations that these leukemia-associated mutations would lead to expansions of hematopoietic stem cell populations or augmented self-renewal, we observed a competitive disadvantage in Smc3-deficient and -haploinsufficient BM cells in vivo without an associated increase in maturation-arrested stem cells. Smc3trap mice were obtained from the European Conditional Mouse Mutagenesis Program (EUCOMM) (Smc3<tm1a(EUCOMM)Wtsi>, MGI:4434007). To generate Smc3fl mice, the gene-trap was removed by crossing Smc3trap mice with Flp deleter mice (B6.129S4-Gt(ROSA)26Sortm2(FLP*)Sor/J) and subsequently outbreeding the Flp allele with C57BL/6J intercrosses. We generated Smc3 conditional-deficient mice by breeding the Smc3fl/fl mice with Vav1-Cre (B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J), ERT2-Cre (B6.Cg-Tg(cre/Esr1)5Amc/J), and CMV-Cre (B6.C-Tg(CMV-cre)1Cgn/J) mice obtained from The Jackson Laboratory. We characterized Smc3 conditional-deficient mice at 6–8 weeks of age and both genders were used. Whenever possible, littermate controls were used for all experiments. Complete blood counts were measured using Hemavet 950 (Drew Scientific Group). All mice were on the C57BL/6 background and were cared for in the Experimental Animal Center of Washington University School of Medicine. The Washington University Animal Studies Committee approved all animal experiments. Intracellular Smc3 was detected with the Pharmingen Transcription Factor Buffer Set (BD Biosciences, #562574) according to the manufacturer's instructions. BM cells were isolated from femurs and tibias and lysed with ACK lysis buffer (150 mmol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L Na2EDTA [Na2-ethylenediaminetetraacetic acid], pH 7.2–7.4). Cells were stained with cell-surface markers to identify cell type by flow cytometry and then fixed for 40 minutes at 4°C. Cells were washed with permeabilization wash buffer and incubated with primary antibody against Smc3 (1:100 dilution, Abcam, #ab9263) for 30 minutes at 4°C. Cells were washed in permeabilization wash buffer and incubated in secondary antibody (1:500 dilution, chicken anti-rabbit Alexa Fluor 647, Molecular Probes) for 30 minutes at 4°C. Cells were rinsed in permeabilization wash buffer and analyzed by flow cytometry. The mean fluorescence intensity was calculated for the AF647 signal. After lysis of red blood cells by ACK lysis buffer, peripheral blood, BM, spleen cells, or thymocytes were treated with anti-mouse CD16/32 (eBioscience; clone 93) and stained with the indicated combinations of the following antibodies (all antibodies are from eBioscience unless noted otherwise): CD34 FITC (clone RAM34), CD11b PE (clone M1/70), c-Kit PECy7 (clone 2B8) or BV421 (BioLegend, clone 2B8), Sca1 PE-Dazzle 594 (BioLegend, clone D7) or APC (clone D7), Gr-1 FITC, PECy7, APC (clone RB6-8C5), or BV421 (BioLegend, clone RB6-8C5), B220 PE, PECy7, APC (clone RA3-6B2), or APC-Cy7 (BioLegend, clone RA3-6B2), CD3 PECy7 (clone 145-2C11), CD71 PE(clone R17217), Ter-119 PECy7 or APC (clone TER-119), CD16/32 BV510 (clone 93), CD150 PE (BioLegend 115903, clone TC15-12F12.2), CD48 APC-Cy7 (BioLegend, clone HM48-1), Ly5.1 APC (clone A20) or AF700 (BioLegend, clone A20), Ly 5.2 PE or e450 (clone 104). The following flow phenotypes were used for stem and progenitor cell flow: Lin- (lineage negative): B220–, CD3e–, Gr-1–, Ter-119–, CD4–, CD8–, CD19–, CD127–; KL: Lin–, cKit+, Sca1–; KLS: Lin–, cKit+, Sca-1+; KLS-SLAM: Lin–, cKit+, Sca-1+, CD150+, CD48–; GMP: Lin–, cKit+, Sca-1–, CD34+, CD16/32+; CMP: Lin–, cKit+, Sca-1–, CD34+, CD16/32–; and MEP: Lin–, cKit+, Sca-1–, CD34–, CD16/32–-. Analysis was performed using a FACScan (Beckman Coulter) or Gallios flow cytometer (Beckman Coulter). Cell sorting was performed using a I-Cyt Synergy II sorter (I-Cyt Technologies). Flow cytometry data were analyzed with FlowJo Version 10 (TreeStar), Excel (Microsoft), and Prism 7.02 (GraphPad Software) software. Competitive transplantation was performed using 0.5 × 106 whole BM cells from indicated donor mice (CD45.2) mixed with 0.5 × 106 competitor whole BM cells wild-type CD45.1 (Ly5.1) × CD45.2 mice. Mixture cells were injected intravenously into 6- to 8-week-old CD45.1 recipient mice that received 1100 cGy of total body irradiation (Mark 1 Cesum irradiator, J.L. Shepard) 24 hours prior to transplantation. For Smc3fl/fl/ERT2-Cre+/– or Smc3fl/+/ERT2-Cre+/– transplantation, recipient mice were treated with tamoxifen (TAM) (dissolved in sterile corn oil, Sigma-Aldrich) 6 weeks after transplantation via oral gavage for nine doses (3 mg/day/mouse, 3 days/week). Peripheral blood was examined for donor cell chimerism at the indicated time points after transplantation. Recipient mice BM were analyzed at the end of the experiment. BM cells were harvested and plated in duplicate (10,000 BM cells/plate) in complete mouse methylcellulose medium with stem cell factor, interleukin-3 (IL-3), IL-6, and erythropoietin (Epo) (R&D Systems). Colonies were counted on day 7 and cells were collected from methylcellulose in warm Dulbecco-modified Eagle medium (DMEM) containing 2% fetal bovine serum, washed, and replated as before. An aliquot of cells was taken for analysis of myeloid (Gr1, CD11b) and mast cell markers (cKit, FcER1) by flow cytometry. This process was repeated for 4 weeks or until colony formation failed. Multipotent progenitors (KLs) were sorted from three wild-type or Smc3fl/+/Vav1-Cre+/– mice into DMEM. Flow cytometry of samples after sorting validated >93% sort accuracy. RNA was extracted from cell pellets using a miRNeasy kit (QIAGEN) and genomic DNA was removed by the RNase-Free DNase Set (QIAGEN). RNA was analyzed for degradation using the RNA Nano Chip (Agilent Technologies, #5067-1521). An input of 300 ng was taken forward for each sample using the TruSeq Stranded Total RNA with Ribo-Zero Globin Kit (Illumina, #20020612). Final libraries were analyzed using a high-sensitivity DNA chip (Agilent Technologies, #5067-4626). All libraries were pooled and run across three lanes of HiSeq4000. RNAseq data were aligned to the human reference with Tophat version 2.0.8 (denovo mode, params: –library-type fr-firststrand –bowtie-version = 2.1.0). Expression levels were calculated with Cufflinks version 2.1.1 (params: –max-bundle-length 10000000 –max-bundle-frags 10000000) [25Trapnell C Roberts A Goff L et al.Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.Nat Protoc. 2012; 7: 562-578Crossref PubMed Scopus (153) Google Scholar]. Chromatin accessibility assays using the bacterial Tn5 transposase were performed using multipotent progenitors (KLs) sorted from Smc3fl/+ or Smc3fl/+/Vav1-Cre+/– mice in triplicate. DNA was prepared from 75,000 sorted cells and >93% sorting accuracy verified with post-sort analysis. Assay for transposase-accessible chromatin (ATAC) libraries were generated exactly as described previously [26Buenrostro JD Giresi PG Zaba LC Chang HY Greenleaf WJ Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.Nat Methods. 2013; 10: 1213-1218Crossref PubMed Scopus (2891) Google Scholar] and pooled and sequenced on a HiSeqX instrument (Illumina) to obtain between 133 and 152 million 2 × 150 bp paired-end reads. Raw sequencing reads were adapter trimmed with trim galore using cutadapt version 1.8.1 (Martin EMBnet 2011) and then aligned to the mouse reference genome (mm10) using bwa mem (Li H. arXiv:1303.3997v1 (2013)). Peaks in each sample were identified with macs2 [27Zhang Y Liu T Meyer CA et al.Model-based analysis of ChIP-Seq (MACS).Genome Biol. 2008; 9: R137Crossref PubMed Scopus (7878) Google Scholar] using the -f BAMPE parameter and then filtered to retain peaks with q < 0.01. Peak summits from all samples were merged together with BEDtools merge [28Quinlan AR Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features.Bioinforma Oxf Engl. 2010; 26: 841-842Crossref PubMed Scopus (10658) Google Scholar] using parameters to combine summits within 50 bp of each other. Read counts at the merged peak summits were obtained for all samples using the deepTools multiBamSummary command [29Ramírez F Dündar F Diehl S Grüning BA Manke T deepTools: a flexible platform for exploring deep-sequencing data.Nucleic Acids Res. 2014; 42: W187-W191Crossref PubMed Scopus (1093) Google Scholar] with the minimum mapping quality set to 1 and then processed using DESeq2 [30Love MI Huber W Anders S Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (27180) Google Scholar] with default parameters to obtain normalized counts for each peak summit and to perform differential analysis across all peaks between wild-type and mutant mice. Statistical analysis was performed using Prism version 7.02 (GraphPad Software) and Excel (Microsoft) software. Unpaired two-tailed t test and one-way and two-way ANOVA with Tukey's multiple-comparisons tests were performed as appropriate. p values < 0.05 were considered statistically significant. Error bars represent standard deviation (SD). Data points without error bars have SD below Prism 7.02’s limit to display. To investigate the effects of Smc3 loss on hematopoiesis, we generated Smc3 conditionally deficient and haploinsufficient mice using Smc3<tm1a(EUCOMM)Wtsi> mice obtained from EUCOMM (Smc3trap). The Smc3trap allele has a lacZ-neomycin-gene-trap cassette inserted in intron 4 with two Frt sites on each side of the cassette and two loxP sites flanking exon 4. The gene trap is predicted to lead to an early transcription stop after splicing into lacZ-neomycin. The conditional knockout Smc3fl allele was created by excising the gene-trap cassette with Flp recombinase and was used for further characterizations because homozygous deletion could be achieved using the Smc3fl allele (Figure 1A). We validated the integration of the loxP sites surrounding exon 4 in the Smc3fl allele using whole-genome sequencing (Figure 1B). We examined the transcriptional consequences of the Smc3fl allele using RNA sequencing (RNA-Seq) and intracellular flow cytometry. In BM cells from three Smc3fl/+/Vav1-Cre+/– mice, nearly 50% (48.4%) of transcripts spliced from exons 3 to 5, consistent with deletion of exon 4, whereas all of the wild-type transcripts spliced from exons 3 to 4 and exons 4 to 5 (Figure 1C and Supplementary Figure E1A-F). Analysis of reads spanning exons 3 to 5 suggests that this results in a frameshift mutation and a stop codon after 59 amino acids, although this truncated protein could not be detected using N-terminal antibodies. Using C-terminal antibodies, the intracellular Smc3 protein level was reduced to approximately half of littermate control, as would be expected with a heterozygous allele and confirming Smc3 haploinsufficiency (Figure 1D). In addition, the Smc3 protein level was regulated during normal hematopoiesis, with higher expression in KLS stem/progenitor cells versus SLAM stem cells (Figure 1E). Representative primary intracellular flow data are shown in Supplementary Figure E2 (online only, available at www.exphem.org). To determine whether SMC3 mutations might have dominant-negative effects or phenocopy loss-of-function effects, we compared the consequences of Smc3-deficient and -haploinsufficient mouse models. We found that hematopoietic homozygous deletion of Smc3 led to embryonic lethality. In heterozygous Smc3fl/+/Vav1-Cre+/– intercrosses, we observed 0 out of 75 pups with homozygous Smc3 alleles (Figure 2A). To determine whether the cause of death in Smc3fl/fl/Vav1-Cre+/– embryos was from hematopoietic failure, we examined embryonic day 13.5 (E13.5) embryos. Grossly, the Smc3fl/fl/Vav1-Cre+/– embryos were indistinguishable in size and appearance from other genotypes except for the lack of obvious fetal livers (Figures 2B and 2C). A severe decrease in fetal liver hematopoietic cells was verified by cell count and flow cytometry with near-complete absence of CD45+ Gr1+ CD11b+ cells demonstrating myeloid-biased hematopoietic failure (Figures 2D–2F). We investigated somatic homozygous Smc3 deletion in adult mice using the Smc3fl/fl/ERT2-Cre+/– mice. Smc3 deletion was achieved by treating mice with oral TAM at 6 weeks of age and reduction in Smc3 protein confirmed with Western blot (Supplementary Figure EE3A, online only, available at www.exphem.org). After four doses of TAM, mice were moribund and therefore were sacrificed for analysis. Complete blood count (CBC) data showed the Smc3fl/fl/ERT2-Cre+/– mice had lower white blood cell counts; percentages of lymphocytes and monocytes; and fewer platelets than TAM-treated littermates (Figure 3A). The Smc3fl/fl/ERT2-Cre+/– mice had decreased spleen weights (Figure 3B) and their spleens were smaller in size (Supplementary Figure E3B, online only, available at www.exphem.org). Total numbers of cells in the BM, spleen, and thymus of the Smc3fl/fl/ERT2-Cre+/– mice were significantly reduced compared with Smc3fl/fl mice after TAM treatment (Figure 3C). The reduction of cells occurred across all lineages in the BM (Figure 3D), spleen, and thymus (Supplementary Figures E3C and E3D, online only, available at www.exphem.org) of the Smc3fl/fl/ERT2-Cre+/– mice, suggesting complete hematopoietic collapse. Because activation of ERT2-Cre leads to Smc3 deletion in a wide range of cells and tissues, we repeated these studies, isolating hematopoietic cells via a competitive transplantation. Equivalent engraftment of transgenic CD45.2+ and competitor CD45.1+ CD45.2+ cells was verified 6 weeks after transplantation. Following TAM-induced Smc3 deletion, the Smc3fl/fl/ERT2-Cre+/– donor cells were quickly outcompeted, indicating complete loss of hematopoietic stem and progenitor cell (HSPC) functions in the Smc3fl/fl/ERT2-Cre+/– BM. Once again, the effect was most pronounced within the myeloid compartment (Figures 3E and 3F), suggesting that myeloid hematopoiesis is sensitive to Smc3 deletion, so the AML-associated SMC3 mutations are unlikely to have simple dominant-negative effects. In the ExAC database (exac.broadinstitute.org), no SMC3 loss-of-function mutations are observed in available human data (0 observed vs. 58.5 expected mutations), suggesting potential embryonic lethality or reduced fitness associated with Smc3 haploinsufficiency. We therefore determined whether Smc3 haploinsufficiency might be tolerated in mice. Because CMV-Cre is X-linked and expressed during early embryogenesis, we examined the ratio of male: female pups and compared difference between genders to determine whether embryonic Smc3 haploinsufficiency altered hematopoiesis. We found that Smc3 haploinsufficiency led to a normal number of female pups in CMV-Cre intercrosses (Supplementary Figure E4A, online only, available at www.exphem.org) and the female pups had no obvious defects in CBCs, total numbers of BM cells, and percentages of HSPCs and cells in different lineages (Supplementary Figures E4B–E4E, online only, available at www.exphem.org). Therefore, embryonic Smc3 haploinsufficiency could be tolerated and did not grossly perturb steady-state hematopoiesis in mice. We next assessed the effects of somatic Smc3 haploinsufficiency on hematopoiesis using the inducible Smc3fl/+/ERT2-Cre+/– mice. Smc3 haploinsufficiency did not alter the proportions of immunophenotypic HSPCs and cells of different lineages (Figures 4A and 4B). Furthermore, Smc3 haploinsufficiency did not increase the number of colonies formed in methylcellulose or the average number of cells per colony and the Smc3 haploinsufficient BM cells did not replate beyond 2 weeks (Figures 4C–4E). At the end of each week, the colonies on each plate were collected, washed, and characterized by immunophenotype. At the end of week 1, the cells were predominantly Gr1+ CD11b+ for both the Smc3fl/+/ERT2-Cre+/– and Smc3fl/+ genotypes. However, starting week 2, the colonies shifted to cKit+ FcER1+ mast cells. In weeks 3 and 4, the few colonies left were exclusively mast cells (Supplementary Figures E5A and E5B, online only, available at www.exphem.org). Similar results were observed using BM cells from Smc3fl/+/Vav1-Cre+/– mice. We performed RNA-Seq to measure global gene expression in Smc3-haploinsufficient hematopoietic progenitors (Lin-cKit+Sca1–) using the constitutive Smc3fl/+/Vav1-Cre+/– model. This model was chosen because it required minimal manipulation of the mice, provided hematopoietic-restricted deletion, and would evaluate steady-state hematopoietic conditions. Multipotent progenitors (KLs) were sorted from age-matched individual wild-type and Smc3fl/+/Vav1-Cre+/ mice for RNA-Seq. KLs were selected because Smc3 haploinsufficiency resulted in severe multilineage competitive disadvantage in vivo, suggesting potential defect in the functions of Smc3-haploinsufficient KLs. However, minimal global transcriptional changes were detected. Using t tests and significance analysis of microarrays [31Tusher VG Tibshirani R Chu G Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci U S A. 2001; 98: 5116-5121Crossref PubMed Scopus (9640) Google Scholar], 149 genes were identified with differential expression in Smc3-haploinsufficient KLs compared with wild-type controls (most with less than twofold changes) (Figure 4F). KEGG pathway analysis showed significance (p < 0.002 and p < 0.005) for progesterone-mediated oocyte maturation and toxoplasmosis, respectively, but these are not related to hematopoiesis [32Kanehisa M Sato Y Kawashima M Furumichi M Tanabe M KEGG as a reference resource for gene and protein annotation.Nucleic Acids Res. 2016; 44: D45-D462Crossref PubMed Scopus (3280) Google Scholar]. Smc3 expression was not observed to be different when" @default.
• W2904293039 created "2018-12-22" @default.
• W2904293039 creator A5002400585 @default.
• W2904293039 creator A5006673204 @default.
• W2904293039 creator A5035002013 @default.
• W2904293039 creator A5052203075 @default.
• W2904293039 creator A5069032154 @default.
• W2904293039 creator A5081851296 @default.
• W2904293039 date "2019-02-01" @default.
• W2904293039 modified "2023-10-12" @default.
• W2904293039 title "Smc3 is required for mouse embryonic and adult hematopoiesis" @default.
• W2904293039 cites W1255676896 @default.
• W2904293039 cites W1506037097 @default.
• W2904293039 cites W1536775300 @default.
• W2904293039 cites W1554405213 @default.
• W2904293039 cites W1794578906 @default.
• W2904293039 cites W1882792479 @default.
• W2904293039 cites W1952907727 @default.
• W2904293039 cites W1965239343 @default.
• W2904293039 cites W1973384207 @default.
• W2904293039 cites W1973736002 @default.
• W2904293039 cites W1975004069 @default.
• W2904293039 cites W1992826811 @default.
• W2904293039 cites W1994856499 @default.
• W2904293039 cites W2007530277 @default.
• W2904293039 cites W2012753533 @default.
• W2904293039 cites W2014658249 @default.
• W2904293039 cites W2024748833 @default.
• W2904293039 cites W2026954946 @default.
• W2904293039 cites W2047005748 @default.
• W2904293039 cites W2047547960 @default.
• W2904293039 cites W2054143975 @default.
• W2904293039 cites W2056227778 @default.
• W2904293039 cites W2078336790 @default.
• W2904293039 cites W2100305481 @default.
• W2904293039 cites W2102619694 @default.
• W2904293039 cites W2107910135 @default.
• W2904293039 cites W2110256992 @default.
• W2904293039 cites W2115551519 @default.
• W2904293039 cites W2117757143 @default.
• W2904293039 cites W2119799188 @default.
• W2904293039 cites W2126816979 @default.
• W2904293039 cites W2141208987 @default.
• W2904293039 cites W2143192498 @default.
• W2904293039 cites W2154457034 @default.
• W2904293039 cites W2154968323 @default.
• W2904293039 cites W2157783208 @default.
• W2904293039 cites W2158343538 @default.
• W2904293039 cites W2158789637 @default.
• W2904293039 cites W2171808845 @default.
• W2904293039 cites W2179299570 @default.
• W2904293039 cites W2179438025 @default.
• W2904293039 cites W2180981534 @default.
• W2904293039 cites W2205870734 @default.
• W2904293039 cites W2246096828 @default.
• W2904293039 cites W2288260164 @default.
• W2904293039 cites W2338039072 @default.
• W2904293039 cites W2461360940 @default.
• W2904293039 cites W2511911618 @default.
• W2904293039 cites W2520178460 @default.
• W2904293039 cites W2628145035 @default.
• W2904293039 cites W2751498819 @default.
• W2904293039 cites W2797367925 @default.
• W2904293039 cites W2897292730 @default.
• W2904293039 cites W4294107304 @default.
• W2904293039 doi "https://doi.org/10.1016/j.exphem.2018.11.008" @default.
• W2904293039 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6639053" @default.
• W2904293039 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30553776" @default.
• W2904293039 hasPublicationYear "2019" @default.
• W2904293039 type Work @default.
• W2904293039 sameAs 2904293039 @default.
• W2904293039 citedByCount "11" @default.
• W2904293039 countsByYear W29042930392021 @default.
• W2904293039 countsByYear W29042930392022 @default.
• W2904293039 countsByYear W29042930392023 @default.
• W2904293039 crossrefType "journal-article" @default.
• W2904293039 hasAuthorship W2904293039A5002400585 @default.
• W2904293039 hasAuthorship W2904293039A5006673204 @default.
• W2904293039 hasAuthorship W2904293039A5035002013 @default.
• W2904293039 hasAuthorship W2904293039A5052203075 @default.
• W2904293039 hasAuthorship W2904293039A5069032154 @default.
• W2904293039 hasAuthorship W2904293039A5081851296 @default.
• W2904293039 hasBestOaLocation W29042930391 @default.
• W2904293039 hasConcept C104317684 @default.
• W2904293039 hasConcept C109159458 @default.
• W2904293039 hasConcept C145103041 @default.
• W2904293039 hasConcept C28328180 @default.
• W2904293039 hasConcept C54355233 @default.
• W2904293039 hasConcept C86803240 @default.
• W2904293039 hasConcept C95444343 @default.
• W2904293039 hasConceptScore W2904293039C104317684 @default.
• W2904293039 hasConceptScore W2904293039C109159458 @default.
• W2904293039 hasConceptScore W2904293039C145103041 @default.
• W2904293039 hasConceptScore W2904293039C28328180 @default.
• W2904293039 hasConceptScore W2904293039C54355233 @default.
• W2904293039 hasConceptScore W2904293039C86803240 @default.
• W2904293039 hasConceptScore W2904293039C95444343 @default.
• W2904293039 hasFunder F4320337351 @default.

• next