1. Polycomb repressive complex 2 component Suz12 is required for hematopoietic stem cell function and lymphopoiesis
by Stanley C. W. Lee, Sarah Miller, Craig Hyland, Maria Kauppi, Marion Lebois, Ladina Di Rago, Donald Metcalf, Sarah A. Kinkel, Emma C. Josefsson, Marnie E. Blewitt, Ian J. Majewski, and Warren S. Alexander
Blood
Volume 126(2):
July 9, 2015
©2015 by American Society of Hematology

2. Conditional targeting of the mouse Suz12 gene.
Conditional targeting of the mouse Suz12 gene. (A) The WT (+) Suz12 locus, indicating exons 4-7 is shown, with the fl conditional allele, and recombined knockout allele (Δ) following cre-mediated excision. The WT Suz12 locus was modified after the insertion of a targeting vector containing 2 loxP sites flanking exon 5 of the Suz12 gene, which also contained a neomycin cassette flanked by 2 Flip recombinase target sites located after the 3′-end of exon 5 and just upstream of the second loxP site. Expected fragment sizes in Southern blot analysis and PCR-based genotyping are shown. (B) PCR analysis of genomic DNA extracted from BM and spleen of Suz12/CreERT2 mice 8 days after tamoxifen treatment, using a primer combination that distinguishes WT, fl, and recombined (Δ) alleles. (C) Immunoblot analysis of Suz12 and Ezh2 protein in the thymus, spleen, and BM of Suz12fl/+/CreERT2 and Suz12fl/fl/CreERT2 mice 8 days after tamoxifen treatment. Actin was used as loading control. (D) Immunoblot analysis of Suz12, Ezh2, and H3K27me3 in cultured fetal liver cells from Suz12fl/fl/CreERT2 E14 embryos. Cell lysates were prepared 24 hours after 4-hydroxy tamoxifen (4-OH Tam]) or vehicle treatment. Total histone H3 and actin were used as loading controls.
Stanley C. W. Lee et al. Blood 2015;126:
©2015 by American Society of Hematology

3. Suz12 is required for fetal hematopoiesis.
Suz12 is required for fetal hematopoiesis. (A) Representative plots of flow cytometric analyses of hematopoietic stem and progenitor cells in fetal livers of Suz12fl/+, Suz12fl/Δ, Suz12fl/+/VavCreT, and Suz12fl/Δ/VavCreT E13.5 embryos. Numbers from gated populations represent the proportions of total viable fetal liver cells of the indicated populations: lineage− Sca-1+ c-Kit+ (LSK) cells (top panel), LSK CD150+ CD48− cells (middle panel) and myeloid progenitors (CMP, MEP, and GMP; lower panel) are shown. The absolute numbers of hematopoietic stem and progenitor cells in E13.5 fetal livers are summarized in the adjacent bar charts. (B) Representative flow cytometric plots are shown of erythroid differentiation in fetal livers of Suz12fl/+, Suz12fl/Δ, Suz12fl/+/VavCreT, and Suz12fl/Δ/VavCreT E13.5 embryos. The absolute cell numbers in each stage of erythroid differentiation are summarized in the adjacent bar chart. Data represent mean ± standard deviation from n = 5-14 fetal livers per genotype. One-way analysis of variance followed by Tukey’s post-hoc test was performed to compare differences between genotypes. *P < .05; **P < .01.
Stanley C. W. Lee et al. Blood 2015;126:
©2015 by American Society of Hematology

4. Suz12 is critical for maintaining adult HSC self-renewal and homeostasis.
Suz12 is critical for maintaining adult HSC self-renewal and homeostasis. (A) Experimental design for competitive BM transplantation assays. (B) Analysis of test donor contribution (CD45.2+) to the peripheral blood of mice receiving either Suz12fl/fl/CreERT2 or Suz12+/+/CreERT2 BM cells with tamoxifen (TAM) or vehicle (ethanol [EtOH]) treatment. (C) Representative flow cytometric plots of hematopoietic stem and progenitor cell populations in BM of mice that received Suz12fl/fl/CreERT2 donor BM 16 weeks after tamoxifen (TAM) or vehicle (EtOH) treatment. The numbers represent the proportions of cells gated from the indicated populations. (D) Analysis of test donor contribution (CD45.2+) to the spleen (SPL), mesenteric lymph nodes (MLN), thymus (THY), BM, lineage− Sca-1+ c-Kit+ (LSK) cells and BM lineage− Sca-1− c-Kit+ myeloid progenitors (MP) in mice receiving either Suz12fl/fl/CreERT2 or Suz12+/+/CreERT2 BM cells and tamoxifen (TAM) or vehicle (EtOH) treatment. Data represent mean ± standard deviation from Suz12+/+/CreERT2 (n = 3 donors) and Suz12fl/fl/CreERT2 (n = 4 donors) mice. Each data point represents the average test donor contribution from 3 recipients of BM from an individual donor. A two-tailed Student t test was performed to test statistical significance between vehicle (EtOH) and tamoxifen (TAM)-treated groups. BMT, bone marrow transplant; NS, nonsignificant; PB, peripheral blood. *P < .05; **P < .005; ***P < .001.
Stanley C. W. Lee et al. Blood 2015;126:
©2015 by American Society of Hematology

5. Suz12 is required for B- and T-lymphopoiesis.
Suz12 is required for B- and T-lymphopoiesis. Representative plots of flow cytometric analysis of BM from approximately 8- to 10-weeks old Suz12+/+/Rag1CreKI, Suz12fl/+/Rag1CreKI, and Suz12fl/fl/Rag1CreKI mice. Numbers represent the average proportions of (A) hematopoietic stem and progenitor cells (lineage− Sca-1+ c-Kit+ [LSK]), lymphoid-primed multipotent progenitors ([LMPP], LSK Flt3hi), and common lymphoid progenitors ([CLPs], lineage− Sca-1+ c-Kitint Flt3+ IL7Rα+) populations, and (B) total B cells (B220+ CD19+), pre-B + pro-B (B220+ IgM-), immature B ([Imm. b] B220lo IgM+), recirculating B ([Recirc. B] B220hi IgM+), and pre-proB (NK1.1− CD11c− CD19− B220+ CD43+) populations. Data represent mean ± standard deviation, from approximately 7-12 mice per genotype. (C) Flow cytometric analysis of thymus from approximately 8- to 10-week-old Suz12+/+/Rag1CreKI, Suz12fl/+/Rag1CreKI, and Suz12fl/fl/Rag1CreKI mice. The numbers represent the proportions of cells gated from the indicated populations. Data represent mean ± standard deviation from approximately 7-12 mice per genotype. (D) Competitive BM transplantation with equal numbers of BM cells from 8-week-old Suz12+/+/Rag1CreKI or Suz12fl/fl/Rag1CreKI mice (test; CD45.2) and Suz12+/+ mice (competitor; CD45.1) transplanted into lethally irradiated CD45.1 recipient mice. Test donor-derived (CD45.2+) contribution to specific cell types was assessed in the BM (top panel), thymus (middle panel), and peripheral blood (bottom panel) 8 weeks after transplantation. Gating strategies for BM: LSK, lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitor (CLP), pre-proB, immature, and recirculating B-cell populations are defined as described above, pro-B (B220+ CD19+ c-Kit+ IgM−), pre-B (B220+ CD19+ CD25+ c-Kit− IgM−); thymus: double negative [DN] (CD4− CD8−), DN1 (DN CD44+ CD25−), DN2 (DN CD44+ CD25+), DN3 (DN CD44− CD25+), DN4 (DN CD44− CD25−), DP (CD4+ CD8+); blood: total test donor-derived (CD45.2+), B cells (CD19+), T cells (CD4+ and CD8+), myeloid cells ([M] Mac-1+). Data represent mean ± standard deviation from (n = 3 donor) mice. Each donor marrow is transplanted into 3 recipients per test, and the averaged mean from each test is used to calculate the final mean. A two-tailed Student t test was performed to test statistical significance between genotypes. *P < .05; **P < .005; ***P < .001.
Stanley C. W. Lee et al. Blood 2015;126:
©2015 by American Society of Hematology