Rbm15-Mkl1 enhances the proliferation of murine 6133 megakaryoblastic leukemic cells in a SPOC domain dependent manner.

<p>A. Diagram of pCL6IEGwo lentiviral expression constructs. GFP is expressed from an internal ribosome entry site (IRES). B. Murine 6133 megakaryoblastic leukemic cells were transduced with lentivirus encoding Rbm15, Rbm15 (K795A), Rbm15-Mkl1, Rbm15-Mkl1 (K795A), or with empty vector. After four days of transduction, GFP-positive cells were sorted and cultured in medium containing 10 ng/ml SCF. Whole cell extracts were prepared and analyzed by Western blotting using the indicated antisera. Arrows indicate the position of Rbm15, Mkl1, and Rbm15-Mkl1. C. GFP-positive 6133 megakaryoblastic leukemic cells were cultured in medium containing SCF and cells were enumerated every two or three days after staining with trypan blue. The error bars indicate the mean standard deviation from three experiments, and asterisks indicate <i>p</i> values compared to vector control (*, <i>p</i> = 0.008; **, <i>p</i> = 0.001, ***, <i>p</i> = 0.0001). D. Analysis of apoptosis in transduced 6133 megakaryoblastic leukemic cells. GFP-positive 6133 leukemic cells were analyzed for apoptosis using APC-annexin V and 7-ADD staining and flow cytometry. UR, upper right panel corresponding to dead cells; LL, lower left panel corresponding to healthy cells; LR, lower right panel corresponding to apoptotic cells. Numeric values represent a summary of data from three experiments. Asterisks indicate <i>p</i> values compared to vector control (<i>p</i><0.05).</p

Rbm15-Mkl1 fusion protein decreases the steady-state level of Rbm15.

<p>A. Inducible T-REx HEK293 cell lines that express FLAG-tagged Rbm15-Mkl1 (RM), Rbm15-Mkl1 (K795A), or carry the empty expression vector were induced with 0.5 ug/ml doxycycline for three days. The expression level of each protein before and after induction was analyzed by Western blotting using the indicated antisera. Arrows indicate the position of Rbm15, Mkl1, and Rbm15-Mkl1. B. Inducible T-REx HEK293 cell lines that express FLAG-tagged Rbm15-Mkl1, Rbm15 (aa 1–507), Rbm15 (aa 671–977), or carry the empty expression vector were induced with 0.5 ug/ml doxycycline for three days. Whole cell extracts were prepared and analyzed by Western blotting using the indicated antisera. Arrows indicate endogenous Rbm15 and Mkl1.</p

The leukemogenic Rbm15-Mkl1 fusion protein associates with histone H3-Lys4 methyltransferase activity.

<p>A. Nuclear extracts isolated from inducible T-REx HEK293 stable cell lines that express FLAG-Rbm15, FLAG-Rbm15 (K795A), FLAG-Rbm15-Mkl1, FLAG-Rbm15-Mkl1 (K795A), FLAG-Mkl1, or carry empty vector were subjected to FLAG immunoprecipitation and bound proteins were eluted by FLAG peptide after extensive washing. HMT activity was assayed by incubating immunoprecipitates with core histones or recombinant histone H3 in the presence of [³H]methyl-S-adenosyl methionine. Reaction products were resolved by SDS-PAGE and examined by Coomassie blue staining or fluorography. B. Recombinant human histone H3 purified from <i>E.coli</i> was incubated with immunoprecipitates in the presence of methyl-S-adenosyl methionine. Reaction products were analyzed by Western blotting using the indicated modification-specific antibodies. Core histones purified from HEK293 cells were used as a positive control.</p

Over-expression of Rbm15-Mkl1 transforms murine 6133 megakaryoblastic leukemic cells in a SPOC domain dependent manner.

<p>Following sorting, GFP-positive cells were cultured in the presence of SCF for several days. Cells were then plated in medium lacking SCF, and cells were enumerated every two or three days after staining with trypan blue. The error bars indicate the mean standard deviation from three experiments.</p

The SPOC domain of Rbm15 interacts with the Setd1b complex.

<p>A. Schematic diagram of various Rbm15 expression constructs are shown. Numbers indicate amino acid residues. HEK293 cells were transiently transfected with various expression constructs of Rbm15 fragments carrying an N-terminal FLAG epitope. Whole cell extracts were prepared and subjected to FLAG immunoprecipitation, and immunoprecipitates were analyzed by Western blotting using the indicated antisera. B. SPOC domains are highly conserved in <i>Spen</i> family proteins. Primary sequences of SPOC domains from human Rbm15, mouse Rbm15, xenopus Rbm15, human SHARP, and drosophila <i>Spen</i> proteins were aligned. Secondary structures of SPOC domains were adapted from the previously published SHARP SPOC domain (37). Amino acids that form the pocket of basic patches in SPOC domains are shaded with gray boxes, and arrows indicate amino acid residues that were mutated in the experiment. C. Expression vectors encoding various point mutants of Rbm15 were transiently transfected into HEK293 and analyzed as described as above.</p

The leukemogenic Rbm15-Mkl1 fusion protein associates with the Setd1b HMT complex.

<p>A. Schematic diagram of Rbm15, Rbm15-Mkl1 fusion protein, and Mkl1. Numbers indicate aa residues. B. Nuclear extracts from T-REx HEK293 stable cell lines that express FLAG-tagged Rbm15, Rbm15-Mkl1 (RM), or Mkl1 (or carrying empty vector) were subjected to FLAG immunoprecipitation, and immunoprecipitates were analyzed by Western blotting using the indicated antisera. C. Constructs that express empty vector, Myc-Rbm15, Myc-Rbm15-Mkl1, or Myc-Mkl1 were transiently transfected into HEK293 cells. Nuclear extracts were prepared and subjected to immunoprecipitation using anti-Setd1b antiserum. Immunoprecipitates (Setd1b-IP) were analyzed by Western blotting using the indicated antisera. D. Whole cell extracts from murine 6133 megakaryoblastic leukemic cells were subjected to immunoprecipitation using antisera directed against Setd1b or Rbm15. Immunoprecipitates were analyzed by Western blotting using the indicated antisera.</p

The LSD motif of Setd1b is responsible for the interaction with Rbm15.

<p>A. A schematic diagram of various Setd1b expression constructs is shown. Numbers indicate amino acid residues of the human Setd1b protein. Inducible T-REx HEK293 cell lines that express various FLAG-tagged Setd1b fragments were induced for 3 days with doxycycline. Nuclear extracts were prepared and immunoprecipitated by FLAG-IgG agarose beads, and immunoprecipitates were analyzed by Western blotting using the indicated antisera. B. HEK293 cells were transiently transfected with expression vectors encoding various FLAG-tagged deletion constructs of Setd1b. Whole cell extracts were prepared and subjected to FLAG immunoprecipitation and analyzed by Western blotting using the indicated antisera. C. Conservation of the LSD motif in SPOC domain-interacting proteins. The LSD motifs from drosophila SMARTER, human SMRT, human N-CoR, Xenopus Setd1b, and human Setd1b are aligned. Conserved amino acid residues are shaded with gray boxes. Arrows indicate the mutated amino acids in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042965#pone-0042965-g003" target="_blank">Figure 3D</a>. D. Mutation of the LSD motif of Setd1b abolishes the interaction with Rbm15. Conserved leucine and aspartic acid residues within the Setd1B LSD motif were mutated. HEK293 cells were transiently transfected with expression vectors encoding FLAG-Setd1b fragment (1–867 aa) or FLAG-Setd1b (1–867 aa)(LPD:APA), or empty vector, and protein interactions were analyzed as described above.</p

Rbm15 interacts with the Setd1b HMT complex.

<p>A. The homology between Setd1a and Setd1b is shown. Areas of identity and similarity between human Setd1a (NP_055527) and Setd1b (JF813787) were determined by ClustalW (1.82) (43). RRM, RNA recognition motif; HBM, Hcf1 binding motif; N-SET, N-terminal region of the SET domain; SET, catalytic SET histone methyltransferase domain. Numbers indicate amino acid residues of human Setd1a and Setd1b proteins. B. Nuclear extracts were isolated from inducible T-REx HEK293 cell lines that express FLAG-tagged versions of the central divergent domains of Setd1a or Setd1b (or carrying the empty expression vector). Extracts were subjected to FLAG immunoprecipitation and bound proteins were eluted by FLAG peptide after extensive washing. Proteins were analyzed by SDS-PAGE and stained with Coomassie Blue. Arrows indicate proteins identified by mass spectrometry, and “a” or “b” in front of each identified protein indicates Setd1a and Setd1b interactions, respectively. C. Constructs that express FLAG-tagged full length Setd1a, Setd1a middle fragment, full length Setd1b, or Setd1b middle fragment (or empty vector) were transiently transfected into HEK293 cells. Nuclear extracts were isolated and subjected to FLAG immunoprecipitation, and immunoprecipitates were analyzed by Western blotting using the indicated antisera. D. HEK293 cells were transiently transfected with a FLAG-Rbm15 vector or empty expression vector. Nuclear extracts were isolated and subjected to FLAG immunoprecipitation, and immunoprecipitates were analyzed by Western blotting using the indicated antisera. E. Nuclear extracts isolated from HEK293 cells were subjected to immunoprecipitation (IP) using antisera directed against Setd1b or Rbm15. Immunoprecipitates were analyzed by Western blotting using the indicated antisera.</p

Hematopoietic progenitor cell populations are sensitive to Cfp1 depletion.

<p>Mutant and control mice were induced with poly(I:C) injections at days 0, 2, and 4. Tissues were collected on day 8 following initiation of Cre induction for all analyses. (A) Bone marrow and spleen cells were each collected and analyzed by colony forming assay for myeloid hematopoietic progenitors. For both tissues, N = 3 for Cre- controls and N = 8 for Cre+ mutants. (B–D) Bone marrow cells were collected, stained for the indicated cell surface markers, and analyzed by flow cytometry. (B) Scatter plot showing a representative experiment. (C) Summary for frequency per total LDBMCs (percent) of CMP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III lo CD34+), GMP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III hi CD34+), MEP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III lo CD34-), and CLP (Lin- Sca1 lo Kit lo IL7Ra+). (D) Summary for number of stained cells per femur. (For C–D, N = 11 for Cre- controls and N = 16 for Cre+ mutants for three independent experiments.) Asterisks denote statistically significant differences in cell number or frequency (P≤0.03).</p

Additional file 10: of Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

Size distribution of sequencing reads from H3K4me3 ChIP-seq data. (PDF 203 kb