The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression - PubMed (original) (raw)

The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression

Zhen-Biao Xia et al. Proc Natl Acad Sci U S A. 2005.

Abstract

MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27kip1) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias.

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Figures

Fig. 1.

Fig. 1.

Creation and characterization of conditional MLL and MLL-AF4 cell lines. (A) FLAG-tagged MLL and MLL-AF4 fusion proteins expressed in conditional cell lines: full-length MLL (Top), amino portions of MLL (Middle), and MLL-AF4 (Bottom). Numbers refer to amino acid number. MLL-AF4 retains the AT-hook and repression domain of MLL, plus amino acids 347-1221 of AF4. AT, AT hooks; RD1 and RD2, repression domains; PHD, plant homeodomain; AD, activation domain; CS, proteolytic cleavage site; SET, conserved domain with methyltransferase activity. (B) FLAG full-length MLL (a_–_c), FLAG-MLL-AF4 (c, d, and f), or two different amino-terminal FLAG-MLL proteins (e) were expressed in clonally selected stably transfected 293 cell lines or Jurkat cell lines (f) after induction with tetracycline (+), but not without tetracycline (-) for 24 h. After immunoprecipitation with anti-FLAG beads, proteins were detected with anti-FLAG antibodies (a, d, e, and f), anti-MLL activation domain antibodies (b), or anti-AT plus anti-RD antibodies (c). (C) MLL-AF4 inhibits growth of inducible 293 and Jurkat cell lines. Cells were cultured in the absence (-Tet) or presence (+Tet) of 1 μg/ml tetracycline, and cell numbers were counted every 24 h. Results are presented as mean (±SD) of three independent experiments. (D) MLL-AF4 expression does not affect apoptosis and cell cycle. Annexin and PI staining show that the cells were not significantly more apoptotic in 293 cell line with induced MLL-AF4 expression. (E) The relative percent cells in each stage of the cell cycle was not changed after induced MLL-AF4 expression in 293 and Jurkat (data not shown)

Fig. 2.

Fig. 2.

MLL-AF4 decreases p27 expression in 293 epithelial and MEF cells. (A) MLL-AF4 expression decreases CDKN1B RNA in 293 cells. Shown is RT-PCR for CDKN2A (lanes 1 and 2), CDKN1A (lanes 3 and 4), and CDKN1B (lanes 5 and 6), in conditionally expressed MLL-AF4 293 cell line with (+) or without (-) tetracycline. (B) Semiquantitative RT-PCR was used to determine changes of relative expression in CDKN2A, CDKN1A, and CDKN1B, normalized to GAPDH. The relative CDKN1B expression (CDKN1B/GAPDH) was significantly reduced after induction of MLL-AF4 compared with noninduced. Reduced expression was not seen with CDKN2A or with CDKN1A. The data were derived from at least three independent experiments (mean ± SD). (C) CDKN1B was decreased 8-fold by MLL-AF4-expressing 293 cells as compared with uninduced 293 cells by quantitative RT-PCR. CDKN1B expression levels were normalized to GAPDH expression and performed in triplicate. (D) p27kip1 protein levels are reduced after MLL-AF4 expression in 293 cells. p27 protein expression was reduced 24 h after induction of MLL-AF4 (lane 1) in 293 cells compared with the noninduced cells (lane 2). p27 protein levels were not changed with induced expression of MLL(672), MLL(1250) clones 1 and 2, or full-length MLL (lanes 3–10). The upper arrow indicates loading control. (E) p27 protein level was reduced in _Mll_-/- MEFs compared with wild-type Mll+/+ MEFs (upper arrow). p21 protein levels were not changed (lower arrow). (F) MLL rescues CDKN1B expression, and MLL-AF4 further reduces CDKN1B expression in murine _Mll_-/- MEFs. RNA was isolated from _Mll_-/- MEFs (lane 1) or from _Mll_-/- MEFs transfected with MLL-AF4-expressing (lane 2) or MLL-expressing (lane 3) plasmids and grown under hygromycin selection. Data are presented as fold-change in CDKN1B expression levels relative to untransfected _Mll_-/- MEFs as determined by quantitative RT-PCR normalized to GAPDH and performed in triplicate

Fig. 3.

Fig. 3.

MLL-AF4 represses CDKN1B, but not CDKN2A, promoter activity in 293 epithelial cells. Reporter constructs basic pGL3, pGL3-CDKN1B (-3568 to -12) (A), pGL3–2kb_CDKN2A_, or pGL3–0.8kb_CDKN2A_ (B), were cotransfected with pTKRL (Renilla) as internal control, in MLL-AF4-, MLL-, or vector-inducible 293 cell lines. Luciferase activity was detected in the absence (dark bars) or presence (light bars) of tetracycline. The data were normalized to the internal control and the basic vector. The data were from at least three independent experiments (mean ± SD)

Fig. 4.

Fig. 4.

p27 expression is increased in MLL-AF4-expressing hematopoietic cells. (A) p27 protein expression is increased in MLL-AF4-expressing hematopoietic cell lines. (Upper) Western blot analysis shows that induced expression of MLL-AF4 results in increased p27 expression in Jurkat cells (compare lanes 1 and 2) and that p27 is increased in human patient-derived leukemia cell lines expressing the MLL-AF4 translocation (MV4–11 and RS4;11) (lanes 5 and 6) but not in lines expressing the MLL-AF9 translocation (Mono Mac 6 and THP-1) (lanes 3 and 4). (Lower) Blots were stripped and rehybridized with antibody recognizing actin. (B) CDKN1B is up-regulated by MLL-AF4 expression in Jurkat cells. Semiquantitative RT-PCR for CDKN1B in MLL-AF4 Jurkat cell line compared with parental Jurkat cell line (control) induced with tetracycline. Two-fold dilutions of cDNA were used as template for RT-PCR. The relative CDKN1B expression (CDKN1B/GAPDH) was significantly increased after induction of MLL-AF4. (C) CDKN1B is up-regulated by MLL-AF4 expression in primary murine bone marrow progenitor cells. Bone marrow progenitors were infected with control (MSCVneo) or MLL-AF4-expressing (MSCVneo-MLL-AF4) retrovirus. After G418 selection for 1 week, RNA was isolated and analyzed by quantitative RT-PCR for CDKN1B expression. Expression levels were normalized to GAPDH expression and performed in triplicate. The relative CDKN1B expression was increased 2-fold after MLL-AF4 expression

Fig. 5.

Fig. 5.

MLL-AF4 binds to the endogenous CDKN1B promoter. ChIP assays were performed by using antibodies to MLL (AT + RD) or FLAG, or preimmune sera. Immunoprecipitated chromatin was analyzed by PCR with primers specific for either the CDKN1B promoter (A Left; B)orthe CDKN1B coding region (A Right). (A) MLL-AF4 binds to the CDKN1B promoter (Left) but not to the CDKN1B coding region (Right) in induced MLL-AF4 293 cells. (B) Endogenous MLL/MLL-AF4 binds to the CDKN1B promoter in MV4–11 and RS4;11 cells. Endogenous MLL/MLL-AF4 binds to the endogenous CDKN1B promoter in MV4–11 and RS4;11 cells (Upper). Endogenous MLL/MLL-AF9 binds to the endogenous CDKN1B promoter in Mono Mac 6 and THP-1 cells (Lower)

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