Nature Genetics: doi: /ng.3812

Size: px

Start display at page:

Download "Nature Genetics: doi: /ng.3812"

Randall Hubbard
5 years ago
Views:

1 Nature Genetics: doi: /ng.3812

2 Supplementary Figure 1 Smarcd2-knockout mice die perinatally with impaired energy homeostasis. (a) Generation of the Smarcd2 conditional knockout allele. Deletion of exons 3 5 generates a null allele (frameshift and premature stop codon). (b) The Mendelian distribution of animals resulting from Smarcd2-heterozygous parents is shown for the different developmental stages. (c) Histological analysis of a 10-h-old Smarcd2-knockout neonate. Enlarged areas shown in d f are identified as 1, 2 and 3, respectively. Representative images of three independent animals are shown. a, thymus; b, heart; c, lung; d, liver. (d) Hypoplasic white adipose tissue (unfilled arrowhead) and brown adipose tissue (filled arrowhead) in the interscapular area. (e,f) Hypoplasic brown (e) and white (f) adipose tissue in the interscapular region. (g) Periodic acid Schiff (PAS) staining (magenta) showing reduced glycogen storage in the liver. (h) Western blotting showing nearly complete depletion of Smarcd2 protein in the bone marrow, spleen and thymus of pi:pc-treated Smarcd2 fl/fl ; Mx1-Cre + animals on day 28 after treatment. Histone H3 is used as a loading control. (i) The Smarcd2 subunit is biochemically dedicated to Brg1/Brm-containing SWI/SNF complexes in hemopoietic cells. Cofractionation studies on 10 30% glycerol gradients using nuclear extracts from FLA2 leukemic cells. (j) Smarcd2-knockout neonates are smaller than controls. Means ± s.e.m. are shown; P = 0.043, t-test with unequal variance; n = 16 control and n = 8 Smarcd2- deficient mice. (k) Kaplan Meier survival curve of Smarcd2-knockout neonates indicating that they die within 26 h of birth. (l) Giemsa staining of a lung section from a 10-h-old Smarcd2-knockout neonate showing no gross developmental abnormalities. Representative images from three independent animals are shown. Nature Genetics: doi: /ng.3812

3 Nature Genetics: doi: /ng.3812

4 Supplementary Figure 2 Validation of the Smarcd2 conditional allele and Smarcd1, Smarcd2 and Smarcd3 antibodies. (a) Southern blot analysis of the Smarcd2 locus configuration in the bone marrow of Smarcd2 fl/fl and Smarcd2 WT/WT (control) Mx1-Cre + hemopoietic chimeras following pi:pc-induced deletion. Genomic DNA was digested with KpnI enzyme, and an external genomic probe was used. Note the complete genomic recombination of the floxed allele as early as 7 d after pi:pc treatment. The molecular weight of the different Smarcd2 alleles is shown. (b) Southern blot analysis of genomic DNA isolated from the bone marrow of Smarcd2 flox-neo/wt, Smarcd2 flox/wt and Smarcd2 WT/WT (control) animals using the same experimental approach described in a. (c) Validation of the specificity of the affinity-purified Smarcd1, Smarcd2 and Smarcd3 polyclonal rabbit antibodies. Top, experimental design for generating Smarcd1, Smarcd2 and Smarcd3 antibodies. Bottom, nuclear extracts of 293 cells expressing Smarcd1, Smarcd2 or Smarcd3 were used in western blotting with the indicated antibodies. (d) Expression of SWI/SNF subunits is not affected upon Smarcd2 depletion. Antibodies raised against the different SWI/SNF subunits were used in western blotting on nuclear extracts isolated from Smarcd2- knockout E14.5 fetal liver cells. See Supplementary Table 1 for antibody description. WT, wild type; Flox, floxed; KO, knockout; neo, neomycin. Nature Genetics: doi: /ng.3812

5 Nature Genetics: doi: /ng.3812

6 Supplementary Figure 3 Analysis of Smarcd2-deficient bone marrow atypical myeloid colony-forming units. (a) FACS analyses of pooled early-disease (ED) Smarcd2-deficient bone marrow atypical myeloid colony-forming units (CFU-M (aty); n = 20 colonies) showing a high percentage of CD34 + cells but nearly complete absence of Gr1 + and F4/80 + cells (CD34: Ctrl = 8%, KO = 57%; Gr1: Ctrl = 56%, KO = 6%; F4/80: Ctrl = 11%, KO = 0.05%). (b) Wright staining of cytospin preparations of the early-disease (ED) Smarcd2-deficient bone marrow CFU-M (aty) analyzed in a showing a significant reduction in macrophages and an increase in early granulocyte precursors (mainly myeloblasts, promyelocytes and myelocytes). Nature Genetics: doi: /ng.3812

7 Nature Genetics: doi: /ng.3812

8 Supplementary Figure 4 Flow cytometry analyses of early-disease Smarcd2-deficient hemopoietic populations. (a) Smarcd2-deficient bone marrow cells and splenocytes were analyzed 28 d after pi:pc-treatment. Results are expressed as means ± s.e.m Total bone marrow cells: CD34 + progenitors, n = 5 control and Smarcd2-deficient mice; lymphoid cells, n = 5 control and Smarcd2-deficient mice; myeloid lineages, n = 7 control and Smarcd2-deficient mice; erythroid lineages, n = 4 control and Smarcd2- deficient mice; bone marrow progenitors and spleen: n = 5 control and Smarcd2-deficient mice per condition. (b) Smarcd2-deficient bone marrow hemopoietic stem cell (HSC) populations were analyzed 28 d after pi:pc treatment. Results are expressed as means ± s.e.m.; n = 3 control and 3 Smarcd2-deficient mice. (c) Representative FACS profiles of the results shown in b. (d) Smarcd2-deficient bone marrow, spleen and lung monocytic populations were analyzed 28 d after pi:pc treatment. Results are expressed as means ± s.e.m.; P values are from two-tailed t-tests with unequal variance; n = 3 control and 3 Smarcd2-deficient mice, except for lung where n = 2. Ery-I, proerythroblasts (Ter119 low CD71 hi ); Ery-II, basophilic erythroblasts (Ter119 hi CD71 hi ); Ery-III, late erythroblasts (Ter119 hi CD71 med ); Ery-IV, late erythroblasts (Ter119 hi CD71 low ). Nature Genetics: doi: /ng.3812

9 Nature Genetics: doi: /ng.3812

10 Supplementary Figure 5 In vivo administration of G-CSF or FLT3 fails to rescue the granulocyte differentiation defect of Smarcd2-deficient mice. (a) Experimental design for in vivo rescue experiments of the Smarcd2 neutropenic phenotype using human recombinant G-CSF or FLT3 hemopoietic cytokines. (b) Absence of hematological response of Smarcd2-deficient granulocytic progenitors to the major cytokines for neutrophil granulocytes, G-CSF or FLT3, following daily intraperitoneal injections for 10 d. Representative FACS profiles of Smarcd2-deficient CD11b + Ly6G + bone marrow cells analyzed 2 d after the last cytokine injection are shown; n = 3 independent experiments. Nature Genetics: doi: /ng.3812

11 Nature Genetics: doi: /ng.3812

12 Supplementary Figure 6 Smarcd2-deficient end-stage (late-disease) animals display a block at the myelocyte/metamyelocyte stage of maturation with dysplastic features. (a) Differential blood cell counts in Smarcd2-deficient LD and control animals performed by identifying 300 cells per peripheral blood smear based on morphology. Granulocytic precursors include myeloblasts to metamyelocytes. Numbers are given as means ±s.e.m. (b) Dysplastic cells in peripheral blood smears of Smarcd2-deficient LD animals. 1, myeloblast; 2, myelocyte; 3, promyelocyte; 4, myelocyte; 5, myeloblast; 6, promyelocyte; 7 9, myeloblasts; 10 12, WBCs with pseudo-pelger Huët anomaly and abnormal nuclei. (c) Representative images of Wright Giemsa-stained cytospin preparations of LD Smarcd2-deficient total bone marrow cells. Note the relative increase in myelocytes and metamyelocytes and concomitant decrease in band and segmented neutrophils. (d) Differential cell counts performed by identifying 500 cells per bone marrow smear based on cell morphology. Dysplastic granulocytic cells include hyposegmented/atypical neutrophils, hypersegmented neutrophils and pseudo-pelger Huët. Numbers are given as means ± s.e.m.; n = 3 controls and n = 3 Smarcd2-deficient LD mice. (e) Magnification of the indicated area in c. P values are from one-tailed t-tests with unequal variance. PBL, peripheral blood cells; BM, bone marrow; lympho, lymphocytes; granu. prec., granulocytic precursors; seg. neutro, segmented neutrophils; dysplastic gran. cells; dysplastic granulocytic cells; LD, late disease (end stage). Nature Genetics: doi: /ng.3812

13 Nature Genetics: doi: /ng.3812

14 Supplementary Figure 7 Hematological parameters of Smarcd2-deficient primary and secondary transplanted mice. (a) Spleen weight and cellularity of Smarcd2-deficient early- and late-disease animals analyzed either 28 d after pi:pc treatment or when moribund. Results are expressed as means ± s.d.; n = 7 control and Smarcd2-deficient early-disease (ED) mice; n = 5 control and Smarcd2-deficient end-stage (LD) mice, except for the spleen cellularity of Smarcd2-deficient mice, where n = 3 mice. (b) Summary of the hematological parameters of mice transplanted with Smarcd2-deficient primary bone marrow cells (isolated when moribund) showing that the disease is not transplantable. n = 4 primary Smarcd2-deficient and 3 control bone marrows were used for transplantation. P values are from two-tailed t-tests with unequal variance. Nature Genetics: doi: /ng.3812

15 Nature Genetics: doi: /ng.3812

16 Supplementary Figure 8 Severe impairment of lung function in Smarcd2-deficient end-stage (late-disease) animals. (a) Pulmonary abnormalities in Smarcd2-deficient LD animals. Representative images of six independent animals are shown. See also Figure 2b. (b) Increased cellularity of LD Smarcd2-deficient lungs. Results are shown as means ± s.e.m. from five independent mice. (c) Increased frequency of CD34 + CD11b + (dysplastic) and CD11b + F4/80 + populations in LD Smarcd2-deficient lungs. Results are shown as means ± s.e.m. from four independent mice, except for CD11b + F4/80 + cells (n = 3 mice). (d) Anti-CD68 staining of a lung section of a Smarcd2-deficient end-stage animal showing nearly complete tissue consolidation by CD68 + macrophages. (e) Gram staining of a Smarcd2-deficient end-stage lung section showing no reactivity. P values are from two-tailed t-tests with unequal variance. Nature Genetics: doi: /ng.3812

17 Nature Genetics: doi: /ng.3812

18 Supplementary Figure 9 No compensation by homologous family members in Smarcd1- and Smarcd2-deficient cells. (a) The Smarcd1 conditional allele is a null allele (P.P., unpublished data). Western blot analysis of total bone marrow nuclear extracts from pi:pc-treated Smarcd1 fl/fl Mx1-Cre + mice showing complete depletion of Smarcd1 on day 28 after and no compensation by the homologous Smarcd2 protein. An asterisk indicates a non-specific band. (b) mrna levels of Smarcd1, Smarcd2, Smarcd3, CEBPA, CEBPB CEBPE, Smarca2/Brm and Smarca4/Brg1 in Smarcd2-deficient and control granulocyte macrophage progenitors (GMPs). Results are presented as mean TPM values ± s.e.m. from three independent experiments. P values are from two-tailed t-tests with unequal variance. Nature Genetics: doi: /ng.3812

19 Nature Genetics: doi: /ng.3812

20 Supplementary Figure 10 Correlation coefficients of Smarcd2-deficient RNA seq data sets in granulocyte macrophage progenitors. Replicate plots for each of the three independent samples per condition are shown. Pearson s R values, Spearman and slopes (linear fitting model) indicate high correlation. Ctrl, control; KO, knockout; TPM, transcripts per million. Nature Genetics: doi: /ng.3812

21 Nature Genetics: doi: /ng.3812

22 Supplementary Figure 11 Expression patterns of selected genes in purified human hemopoietic cell populations (RNA seq data sets from n = 66 specimens). (a) Expression patterns of human SMARCD2, CEBPE and CEBPA in purified human hemopoietic stem cells, myeloid, erythroid and lymphoid populations. RPKM values are shown. RNA seq gene sets are from the Leucegene project (IRIC). (b,c) Expression patterns of the LTF, LCN2 and CAMP secondary granule genes (b) and the MPO, CTSG and ELANE primary granule genes (c) in the human hemopoietic cell populations shown in a. Cells derived from cord blood, bone marrow and peripheral blood are indicated in gray, red and blue, respectively. Sorting of populations was performed as described (Blood 27, , 2016). Cord blood CD34 +, n = 5; CD34 + CD45RA, n = 12; nbm, n = 3; Ery-I and Ery-II, n = 2; Ery-III, n = 1; Ery-IV, n = 2; pre-b, n = 4; Gran-I, n = 3; Gran-II to Gran-V, n = 2; granulocytes, n = 5; monocytes, n = 5; B and T cells, n = 5; WBCs, n = 3; peripheral blood CD34 +, n = 1 from a pool of 5 donors. Ery-I, most immature proerythroblasts (CD34 + CD71 + GlyA ); Ery-II, early erythroblasts (CD34 CD71 + GlyA ); Ery-III, erythroblasts (CD34 CD71 + GlyA + ); Ery-IV, most mature erythroblasts (CD34 CD71 GlyA + ); nbm, normal bone marrow; pre-b, B cell progenitors and precursors; Gran-I, myeloblasts and myelomonocytic progenitors and precursors; Gran-II, promyelocytes; Gran-III, myelocytes; Gran- IV, metamyelocytes; Gran-V, band and segmented neutrophils; WBCs, white blood cells; RPKM, reads per kilobase per million mapped reads. Nature Genetics: doi: /ng.3812

23 Nature Genetics: doi: /ng.3812

24 Supplementary Figure 12 Genes most correlated with LTF, MPO and CEBPE. (a c) Genes co-regulated with LTF (secondary granule) (a), MPO (primary granule) (b) and (c) in the human hemopoietic stem cells and myeloid, erythroid and lymphoid populations shown in Supplementary Figure 11 (n = 66 specimens). Pearson correlations were performed using log 10 RPKM values from the normal cell populations detailed in Supplementary Figure 11. Correlations are shown for the 5,000 most correlated genes. See Supplementary Table 3 for full lists of genes with a correlation score 0.5. RPKM, reads per kilobase per million mapped reads. Nature Genetics: doi: /ng.3812

25 Nature Genetics: doi: /ng.3812

26 Supplementary Figure 13 Smarcd2 structure function analysis in Smarcd2-deficient Lin differentiation model. bone marrow cells using an in vitro OP9 granulocyte (a) Although all Smarcd2 mutants were expressed at levels similar to (or higher than) that of the exogenously expressed wild-type protein, Smarcd2 mutants lacking either the C1 or SWIB domain failed to rescue the granulocyte differentiation defect of Smarcd2- deficient Lin bone marrow cells. Results are expressed as means ± s.e.m. Biological replicates per condition: Ctrl cells: GFP, n = 9; KO cells: GFP and N(l), n = 8; WT, n = 10; N(s), n = 7; C1 and S, n = 6; INT, n = 3; C2 and C3, n = 2. (b) ChIP on the Onecut (negative control; no predicted CEBP -binding sites) and Ltf promoters in Smarcd2-deficient and control Lin bone marrow cells using anti-brg1/brm (J1) (Onecut; left) and anti-p53 (Ltf; right) antibodies. Chromatin extracts were prepared on day 14 of culture on OP9 stromal cells. Purified rabbit or mouse IgG was used as a control. Primers are within 500 bp of the TSSs. For Ltf, the amplicon was the same as in Figure 6c encompassing the CEBP -binding site. Results are presented as means ± s.e.m. from three independent samples. (c) qpcr analyses of Camp and S100a8 secondary granule gene expression in NIH/3T3 cells stably expressing CEBPE or an empty vector. Results are presented as means ± s.e.m. of three independent samples; P values are from two-tailed t-tests with unequal variance. nd, non-detectable. Nature Genetics: doi: /ng.3812

27 Nature Genetics: doi: /ng.3812

28 Supplementary Figure 14 Bioinformatics analyses of Smarcd1, Smarcd2, Smarcd3 and CEBP family proteins. (a) Bioinformatics studies revealed that the SWIB domain of SMARCD2 (of unknown function) is closely related to hydrophobic regions of MDM2 that allow interaction with the p53 -helix (from Bennett-Lovsey, R., Hart, S.E., Shirai, H. & Mizuguchi, K. The SWIB and the MDM2 domains are homologous and share a common fold. Bioinformatics 18, , 2002, by permission of Oxford University Press). (b) Bioinformatics analysis predicts that the C1 domain of human SMARCD2 (amino acids ) is not conserved in the SMARCD1 and SMARCD3 proteins. Coiled-coil prediction software was from Blue, green and red lines represent domain predictions based on scanning windows of 28, 21 and 14 residues, respectively. (c) Several residues in the human SMARCD2 SWIB domain expected to be involved in -helix interactions (asterisks) are not conserved in the homologous SMARCD1 and SMARCD3 proteins. The position of predicted - helices is indicated (black lines). The SMARCD2 C1 domain (amino acids ) is not conserved in the homologous SMARCD1 and SMARCD3 subunits, potentially because of substitutions of residues involved in protein folding. An arginine serine motif in the basic leucine zipper domain of CEBP (asterisks) expected to be required for its transcriptional activity is not conserved in the homologous CEBP, CEBP, CEPB and CEBP proteins. (d) Coimmunoprecipitation experiments in nuclear extracts from 293 cells expressing the different FLAG-tagged Smarcd2 deletion mutants performed using anti-brg1/brm (J1) antibody. The Smarcd2 C2 and C3 domains are essential for its integration within the SWI/SNF complex. FLAG-tagged BMI1 protein was used as a negative control. Nature Genetics: doi: /ng.3812

Nature Immunology: doi: /ni.3412

Nature Immunology: doi: /ni.3412 Supplementary Figure 1 Gata1 expression in heamatopoietic stem and progenitor populations. (a) Unsupervised clustering according to 100 top variable genes across single pre-gm cells. The two main cell