p21 is a transcriptional target of HOXA10 in differentiating myelomonocytic cells - PubMed (original) (raw)
p21 is a transcriptional target of HOXA10 in differentiating myelomonocytic cells
V C Bromleigh et al. Genes Dev. 2000.
Abstract
The myeolomonocytic cell line U937 differentiates into macrophages in response to a variety of agents. Several genes including the cyclin-dependent kinase inhibitor p21(waf1/cip1) and the homeobox gene transcription factor HOXA10 are induced at the onset of differentiation. Ectopic expression of either gene results in U937 differentiation. In this paper, we describe a mechanism by which p21 and HOXA10 may act in concert, where HOXA10 can bind directly to the p21 promoter and, together with its trimeric partners PBX1 and MEIS1, activate p21 transcription, resulting in cell cycle arrest and differentiation. These experiments for the first time identify p21 as a selective target for a HOX protein and link the differentiative properties of a transcription factor and a cell cycle inhibitor.
Figures
Figure 1
Stable, regulated expression of HOXA10 induces cell cycle arrest and myeloid differentiation. (A) Candidate clones were subjected to 24 h of incubation in the presence or absence of tet. Equal μg amounts of protein from lysates were subjected to SDS-polyacrylamide gel (10%) electrophoresis and immunoblotted for the presence of HOXA10. Shown are two representative clones, one that overexpresses (clone 12) and one that does not overexpress (clone 5) HOXA10 upon tet removal. (B) HOXA10 overexpression in U937 cells results in differentiation. Parental tTA U937 and U937–HOXA10 (clone 12) cells were subjected to four days of incubation in the presence or absence of tet. FACS analysis was carried out to detect the expression of the monocyte/macrophage terminal differentiation cell surface markers CD11b and CD14. The percentage of cells for each maker is indicated in each quadrant. (C) HOXA10 expression in U937 cells results in G1 cell cycle arrest. Cells from the tet removal experiment in B were subjected to cell cycle analysis by staining nuclei with ethidium bromide and detecting fluoresence using flow cytometry. (D) p21 levels increase following HOXA10 expression in U937 cells. Aliquots of cells were taken at the times indicated (in h) from the same tet removal experiment, and equal μg amounts of whole cell extract from each time point were run on parallel gels, subjected to Western blotting and probed with the indicated antibody.
Figure 2
HOXA10 induces p21 transcription. (A) U937 and 10–1 cells were cotransfected with 1.5 μg of either empty CMV5 expression vector or CMV5–HOXA10 and 5 μg of a p21 promoter/luciferase reporter construct (wwp21-luc, containing 2.4 kb of upstream sequence). All transfections included 1.5 μg of CMV–β-galactosidase (β-gal) expression plasmid, and raw luciferase numbers were normalized to β-gal activity. All DNA amounts indicated are per plate of cells transfected. U937 cells were transfected by electroporation and 10–1 cells were transfected by calcium phosphate precipitation. Each transfection was performed in duplicate and repeated a minimum of five times. Shown are the mean and deviations of one representative experiment. (B) p21 induction is specific to HOXA10. U937 cells were cotransfected with 5 μg of wwp21 luc and 1.5 μg of one of three different plasmids, each expressing one of three indicated HOX genes that are known to be involved in myeloid differentiation. (C) Sequence of a potential p21 responsive region containing three (underlined) partially overlapping close matches to the HOXA10 consensus site. HOXA10 core sequences are TTATs, indicated in large lettering. (D) The p21–A10RE confers HOXA10 induction to a minimal promoter. An oligonucleotide containing the sequence shown in C was inserted into a plasmid containing an E1b minimal TATA box/luciferase reporter and tested for activity by transient transfection of both U937 (i) and 10–1 cells (ii).
Figure 3
The p21A10RE contains a cluster of three TTAT core elements. (A) p21 promoter sequence from residues −484 to −450 that contains three TTAT cores. (B) Effect of individual TTAT core mutations on HOXA10 responsiveness. Each core mutation (to GCTG) was tested by transient transfection of U937 cells (B) or 10–1 cells (C) with 5 μg of luciferase construct and 1.5 μg of the expression plasmid CMV5–HOXA10 or CMV-5 vector alone. All transfections included 1.5 μg of CMV–β-gal, and raw luciferase numbers were normalized to β-gal activity.
Figure 4
Interactions between HOXA10 and TALE proteins. (A) Maximal induction from the p21–A10RE occurs with HOXA10, PBX1a, and MEIS1b. U937 cells were cotransfected with 5 μg of p21–A10RE/E1b-luc and all combinations of the homeobox constructs as indicated. (+) 1.5 μg of expression plasmid; (++) 3 μg of expression plasmid (all quantities are per plate). Twice the amount of MEIS1b was transfected because this construct expressed approximately half as well as the PBX1a construct (data not shown). All transfections included 1.5 μg of CMV–β-gal, and raw luciferase numbers were normalized to β-gal activity. (B) MEIS1b interaction with HOXA10 is PBX1a-dependent. GST–HOXA10 or GST alone was used as the bait in combination with in vitro-translated proteins or unprogrammed reticulocyte lysate as indicated. Binding reactions were washed extensively and subjected to SDS–PAGE. Gels were dried and subjected to autoradiography. (C) A putative HOXA10/PBX1a/MEIS1b heterotrimer is formed on DNA. The p21–A10RE double-stranded oligonucleotide was used as a probe for a gel mobility shift assay in the presence of the indicated proteins. Unprogrammed reticulocyte lysate was added as indicated to maintain constant amounts of total lysate. Two bands are indicated that represent the putative heterotrimer, because in vitro translation of MEIS1b yields two products of slightly different sizes (not shown).
Figure 4
Interactions between HOXA10 and TALE proteins. (A) Maximal induction from the p21–A10RE occurs with HOXA10, PBX1a, and MEIS1b. U937 cells were cotransfected with 5 μg of p21–A10RE/E1b-luc and all combinations of the homeobox constructs as indicated. (+) 1.5 μg of expression plasmid; (++) 3 μg of expression plasmid (all quantities are per plate). Twice the amount of MEIS1b was transfected because this construct expressed approximately half as well as the PBX1a construct (data not shown). All transfections included 1.5 μg of CMV–β-gal, and raw luciferase numbers were normalized to β-gal activity. (B) MEIS1b interaction with HOXA10 is PBX1a-dependent. GST–HOXA10 or GST alone was used as the bait in combination with in vitro-translated proteins or unprogrammed reticulocyte lysate as indicated. Binding reactions were washed extensively and subjected to SDS–PAGE. Gels were dried and subjected to autoradiography. (C) A putative HOXA10/PBX1a/MEIS1b heterotrimer is formed on DNA. The p21–A10RE double-stranded oligonucleotide was used as a probe for a gel mobility shift assay in the presence of the indicated proteins. Unprogrammed reticulocyte lysate was added as indicated to maintain constant amounts of total lysate. Two bands are indicated that represent the putative heterotrimer, because in vitro translation of MEIS1b yields two products of slightly different sizes (not shown).
References
- Asada M, Yamada T, Fukumuro K, Mizutani S. p21Cip1/WAF1 is important for differentiation and survival of U937 cells. Leukemia. 1998;12:1944–1950. - PubMed
- Berthelsen J, Viggiano L, Schulz H, Ferretti E, Consalez GG, Rocchi M, Blasi F. PKNOX1, a gene encoding PREP1, a new regulator of Pbx activity, maps on human chromosome 21q22.3 and murine chromosome 17B/C. Genomics. 1998a;47:323–324. - PubMed
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