Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction

Abstract

Interactions between the cell cycle machinery and transcription factors play a central role in coordinating terminal differentiation and proliferation arrest. We here show that cyclin-dependent kinase 6 (Cdk6) is specifically expressed in proliferating hematopoietic progenitor cells, and that Cdk6 inhibits transcriptional activation by Runx1, but not C/EBPalpha or PU.1. Cdk6 inhibits Runx1 activity by binding to the runt domain of Runx1, interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. Cdk6 expression increased myeloid progenitor proliferation, and inhibited myeloid lineage-specific gene expression and terminal differentiation in vitro and in vivo. These effects of Cdk6 did not require Cdk6 kinase activity. Cdk6-mediated inhibition of granulocytic differentiation could be reversed by excess Runx1, consistent with Runx1 being the major target for Cdk6. We propose that Cdk6 downregulation in myeloid progenitors releases Runx1 from Cdk6 inhibition, thereby allowing terminal differentiation. Since Runx transcription factors play central roles in hematopoietic, neuronal and osteogenic lineages, this novel, noncanonical Cdk6 function may control terminal differentiation in multiple tissues and cell types.

Figure 1

Cdk6 is expressed in the c-Kit⁺ progenitor compartment and inhibits granulocyte differentiation of 32Dcl.3 cells. (A) Fetal liver cells at E14 were fractionated into Lin⁻c-Kit^hi (1), Lin^loc-Kit⁺ (2), and Lin⁺c-Kit⁻ (3) cells by FACS. (B) Expression of cdk4 and cdk6 in fractions from (A) was analyzed by RT–PCR. (C) Control and cdk6-transduced 32Dcl.3 cells were cultured in the presence of G-CSF. Cytospins were prepared from the cultured cells at day 7, and stained with May–Grüenwald–Giemsa solution. (D) Cdk6 expression during G-CSF-induced differentiation of 32Dcl.3 cells. Cells cultured in the presence of IL-3 (lane 1) or G-CSF for 24 h (lane 2); total RNA was analyzed by RT–PCR. (E) Differential counts of cells from (C). In total, 100 cells were counted per sample. The averages from two independent experiments are shown. (MB: myeloblasts; PM: promyelocytes; MM: metamyelocytes; Seg; segmented granulocytes). (F) mRNA expression of myeloid differentiation marker genes in control and Cdk6-transduced 32Dcl3 cells was analyzed by RT–PCR after 2 days of differentiation using two serial fivefold dilutions of cDNA. NE: neutrophil elastase; MPO: myeloperoxidase; HPRT: hypoxanthine–guanine phosphoribosyltransferase.

Figure 2

Cdk6 selectively inhibits Runx1 transactivation function. (A) NIH3T3 cells were transfected with 200 ng of pM-CSF-R-luc reporter and 1 ng of pRL-TK internal control plasmid, 80 ng of pCMV-MTPU.1 and 80 ng of pCMV-Cdk6 as indicated. (B) As in (A), but with 80 ng pcDNA3-C/EBPα, cells were cotransfected as indicated. (C) As in (A), but with 80 ng of pEF-Runx1 and 50 ng pEF-CBFβ, cells were cotransfected as indicated. (D) As in (C), with 80 ng of pCMV-Cdk4 and 80 ng of pCMV-Cdk6, cells were cotransfected as indicated. (E) As in (C), with 80 ng of pCMV-Cdk6 and 80 ng of pCMV-knCdk6, cells were cotransfected as indicated. Equal amount of total cell lysate from cells transfected as in (D) and (E) were electrophoresed on a 12% SDS–PAGE gel and subjected to Western analysis with anti-Runx1 polyclonal antibody or HA-7 monoclonal antibody (detecting the HA-tag on the Cdk4/6 protein; panels D and E). The (−) sign indicates addition of empty expression vector. The averages from two independent transfections are shown.

Figure 3

Cdk6 interacts with Runx1 Runt domain. (A) Lysates of 293T cells transfected with plasmids encoding Flag-Runx1 and cdk6-HA were immunoprecipitated with anti-flag mAb or control mouse IgG. In all, 5% of the input and precipitates were separated by SDS–12% PAGE and analyzed by Western blotting with anti-HA mAb. (B) Lysates of K562 cells were immunoprecipitated with anti-AML1 polyclonal antibody or rabbit IgG and analyzed as in (A) using an anti-Cdk4 or -Cdk6 polyclonal antibodies. (C) FLAG-tagged Runx1 deletion mutants: RHD: Runt homology domain; AD: activation domain; ID: inhibitory domain. (D) Interaction between Cdk6 and FLAG-Runx1 mutants was analyzed as in (A). (E) NIH3T3 cells were transfected with 200 ng of p(CBF)4TK-luc, 1 ng of pRL-TK, 200 ng of pVP16, 200 ng of pAML1-VP16 as indicated. Increasing amounts (100 or 200 ng) of pCMV-cdk6 were cotransfected as indicated. The means of two independent transfections are shown.

Figure 4

Cdk6 inhibits Runx1 DNA binding. (A) 293T cells were transfected with 1 μg pCMV-Flag-Runx1, 1 μg pEF-CBFβ and 1 μg pCMV-cdk6 as indicated. After anti-Flag IP, the input (2.5%) and immunoprecipitates were analyzed by Western blotting with anti-CBFβ antibody (upper panels) or anti-Cdk6 (lower panels). (B) 293T cells were cotransfected with 2 μg pCMV-Flag-Runx1, 1 μg pEF-CBFβ, 0.5μg pRcCMV-Cyclin D3 and 2 μg pCMV-Cdk6HA (lane 3), or 2 μg pCMV-knCdk6HA (lane 4) in a 10 cm plate. Cells were lysed and lysates were precipitated with biotinylated Runx1-binding element oligonucleotides followed by analysis of these complexes using anti-Flag and anti-HA antibodies. (C) As in (B), but with 2 μg pCMV-Cdk6HA, and 2 μg pRcCMV-Cyclin D3HA (lane 2) or 2 μg pRcCMV-Cyclin D3KEHA (lane 3), cells were transfected as indicated, and subjected to the oligonucleotide pulldown (DNAP) assay. Relative levels of DNA-binding Runx1 are indicated on the right. The relative expression level of lane 1 is designed 100%. (D) As in (B), but without pRcCMV-Cyclin D3, cells were transfected as indicated, and subjected to the DNAP assay. (E) Chromatin from mock and Cdk6-transfected LG cells was immunoprecipitated with anti-Runx1 polyclonal antibody and analyzed by PCR with primers that amplify a fragment of Csf1r intronic regulatory element, FIRE region and Mpo promoter.

Figure 5

Cdk6 blocks transcriptional synergy of Runx1 with C/EBPα. (A) NIH3T3 cells were cotransfected with 200 ng of pM-CSFR-luc, 1 ng of pRL-TK, 80 ng of pEF-Runx1, 50 ng pEF-CBFβ (lanes 2, 4–7), 100 ng of pcDNA3-C/EBPα (lanes 4–7) and 50 ng (lanes 5), 100 ng (lanes 6) or 300 ng (lanes 7) of pCMV-cdk6 in a well of 12-well plates. The (−) sign indicates that an empty expression vehicle has been added instead of the corresponding expression plasmid. The means of two independent transfections are shown. (F) 293T cells were cotransfected with 1 μg of pcDNA3-Flag-Runx1, 1 μg of pcDNA3-C/EBPα and 1 μg (lane 2) or 2 μg (lane 3) of pCMV-Cdk6 in a 10 cm plate. Cell lysates of the transfected cells were immunoprecipitated with anti-Flag monoclonal antibody. Immunoprecipitates were analyzed by immunoblotting for the indicated proteins.

Figure 6

Cdk6 inhibits terminal granulopoiesis and Runx1 DNA binding. Control, Cdk4-, Cdk6- and knCdk6-transduced LG cells were cultured with G-CSF. (A) Cytospins were prepared from the culture at day 4, and stained with Giemsa solution. (B) Two hundred cells were counted per sample. An average proportion of segmented granulocytes from three independent studies are shown. (C) Nuclear extracts from the stable LG cells in (A) precipitated with biotinylated Runx-binding element oligonucleotides followed by analysis of these complexes using anti-Runx1 polyclonal antibody. Relative expression levels of DNA-binding Runx1 are indicated on the right. The relative expression level of control LG cells is designed 100%. (D) Cdk6-expressing LG cells in (A) were transduced with Runx1 and selected with Puromycin (1 μg/ml) for 7 days. Subsequently, the selected cells were cultured with G-CSF, and the proportions of segmented granulocytes were monitored by Giemsa staining as shown in (A) and (B). Expression levels of Cdk6 and knCdk6 in stable LG cells were shown in Supplementary Figure S4.

Figure 7

Cdk6 knockdown promotes granulopoiesis, and Cdk6 overexpression inhibits differentiation and enhances expansion of normal myeloid progenitors. (A) Western blot showing knockdown of Cdk6 expression in LG cells expressing a stable vector coding for a siRNA sequence against mouse Cdk6 and corresponding Cdk4 protein expression. (B) Giemsa staining of control and Cdk6-silenced LG cells after 1 day of G-CSF-induced differentiation. (C) Expression of granulocyte-specific (Gr-1) antigen was monitored by flow cytometry after 1 day of G-CSF-induced differentiation. The numbers indicate the average (±s.e.m.) percentage of Gr1⁺ populations (n=3). (D) Lineage distribution of CD45.1⁺EGFP⁺ cells sorted from mice transplanted with control and Cdk6-transduced LSK cells. Number of granulocytic cells (Gran), eosinophils (Eos), Lymphoid (Lym), monocytic (Mono) and nucleated erythroid (Ery) cells as % of total BM cells as indicated; error bars show standard deviation. (E) Distribution of CD45.2⁺EGFP⁺ cells on granylocytic maturation stages as % of total granulocytic cells. MB+PM: myeloblasts/promyelocytes; MC+MM: myelocytes+metamyelocytes; BC+PMN: band cells and polymorphonuclear neutrophils. Error bars show standard deviation. (F) 1 × 10⁴ transduced Lin⁻ cells were plated in M3434 methylcellulose medium. Bulk cultures were harvested after 7–10 days in culture, and 1 × 10⁴ cells were replated for each sample. Error bars show standard deviations.