The T-cell oncogenic protein HOX11 activates Aldh1 expression in NIH 3T3 cells but represses its expression in mouse spleen development

Abstract

Hox11 is a homeobox gene essential for spleen formation in mice, since atrophy of the anlage of a developing spleen occurs in early embryonic development in Hox11 null mice. HOX11 is also expressed in a subset of T-cell acute leukemias after specific chromosomal translocations. Since the protein has a homeodomain and can activate transcription, it probably exerts at least some of its effects in vivo by regulation of target genes. Representational difference analysis has been used to isolate cDNA clones corresponding to mRNA species activated following stable expression of HOX11 in NIH 3T3 cells. The gene encoding the retinoic acid-synthesizing enzyme aldehyde dehydrogenase 1 (Aldh1), initially called Hdg-1, was found to be ectopically activated by HOX11 in this system. Study of Aldh1 gene expression during spleen development showed that the presence of Aldh1 mRNA inversely correlated with Hox11. Hox11 null mouse embryos have elevated Aldh1 mRNA in spleen primordia prior to atrophy, while Aldh1 seems to be repressed by Hox11 during organogenesis of the spleens of wild-type mice. This result suggests that expression of Aldh1 protein is negatively regulated by Hox11 and that abnormal expression of Aldh1 in Hox11 null mice may cause loss of splenic precursor cells by aberrant retinoic acid metabolism.

FIG. 1

Enrichment of 3T3-HOX11 DPs by cDNA RDA. (A) Ethidium-stained agarose gel electrophoresis of starting cDNA DpnII fragment representations and various RDA enriched populations obtained by mutual subtraction of NIH 3T3 cells (3T3) with 3T3-HOX11 (left lanes show products obtained with NIH 3T3 cells as the tester and right lanes show products obtained with 3T3-HOX11 cells as the tester). Tester representation (R; unselected cDNA) and DP1, DP2, DP3, and DP4 are shown. The arrow indicates an enriched HOX11 DpnII fragment identified by Southern filter hybridization with a HOX11-specific probe. (B) Southern filter hybridizations (with Slim1, Aldh1, HOX11, and β-actin probes as indicated) of the agarose gel-fractionated RDA DPs shown in panel A. Fragment sizes are indicated. (C) Northern filter hybridization of 10 μg of total RNA prepared from untransfected NIH 3T3 cells and three independent 3T3-HOX11 clones and hybridized with HOX11, Slim1, Aldh1, and ATP synthase probes (as indicated). 3T3-HOX11 clones 5 and 18 were found to express HOX11 protein, while clone 11 did not (data not shown). Hybridization of the filter with ATP synthase was used to assess the quality of the RNA transferred.

FIG. 2

Expression of ALDH1 and SLIM1 mRNAs in T- and B-lymphoid cell lines. Results of Northern filter hybridization of total RNAs from the indicated cell lines with Slim1, Aldh1, HOX11, TCRβ, and ATP synthase probes are shown. RNAs were extracted from the T-cell lines (T) KOPT-K1, RPMI 8402, ALL-SIL, PER-255 (the last two carrying 10q24 translocations involving HOX11), SUP-T1, SUP-T13, CCRF-CEM (CEM), NALM, HBP-ALL, PER-117, and Jurkat or from the B-cell lines. (B) 38B9, WEHI-231, RAJI, and ACVA-1. N417 is a neuroblastoma line; LUDLU is a small cell lung carcinoma line; HEL, K562, and 707 are erythroid lines, and HepG2 is a hepatoma line.

FIG. 3

Specificity of Aldh1 induction by ectopic expression of HOX11 RNA, extracted from NIH 3T3 cell clones stably transfected with various HOX11 or Hox2.9 expression constructs, was analyzed by Northern filter hybridization with Aldh1, HOX11, Hox2.9, and ATP synthase probe as indicated. (A) Induction of Aldh1 mRNA by ectopic expression of HOX11 protein. RNAs were isolated from three independent NIH 3T3 clones stably transfected with either a HOX11 expression construct (HOX11 controlled by the pEF-BOS expression cassette [HOX11]), a HOX11 expression construct encoding a mutant HOX11 protein lacking the third helix of the homeodomain (ΔH3-HOX11), or the expression vector only (BOS). Aliquots were fractionated and transferred to nylon membranes prior to hybridization with an Aldh1, a HOX11, or an ATP synthase probe. Protein extracts were also prepared from these NIH 3T3 clones and analyzed by Western blotting. The presence of HOX11 protein was assayed (bottom section) with anti-HOX11 antiserum (26). (B) Stimulation of Aldh1 mRNA expression by HOX11 via the MT promoter. NIH 3T3 clones expressing HOX11 were incubated in the presence or absence of 50 μM zinc for 36 h. RNAs were prepared, fractionated, and transferred to nylon membranes. These were hybridized to Aldh1 and ATP synthase probes. Clone 1 is a control not expressing HOX11 protein (as shown by the Western blot developed with anti-HOX11 antiserum [bottom section]), and clone 2 is a HOX11-expressing line (see bottom section). (C) Aldh1 gene expression in response to HOX11 protein expression. We obtained NIH 3T3 clones which were stably transfected with pEF-BOS-HOX11 or pEF-BOS-Hox2.9. Two clones were selected for their expression of HOX11 or Hox2.9 mRNA. RNA was prepared, electrophoresed, and transferred to nylon filters. These filters were hybridized with Aldh1, HOX11, Hox2.9, and ATP synthase probes as indicated.

FIG. 4

Aldh1 expression in embryonic mouse spleen is inversely correlated with Hox11. Wild-type (+/+) or Hox11 homozygous mutant (−/−) mouse embryos were taken at E12.5 or E13.5, and parasagittal cryosections were hybridized in situ with oligonucleotide probes specific for Hox11, Aldh1, Aldh2, or Tal1/Scl mRNA. The upper section shows E12.5 embryos. Consecutive sections of +/+ or −/− embryos were hybridized with Hox11, Aldh1, and Aldh2 probes. The lower section shows E13.5 embryos. Consecutive sections of +/+ or −/− embryos were hybridized with Hox11 and Aldh1 probes in the order shown. For comparison, we hybridized Tal1/Scl, which showed its specific location within the hindbrain and fetal liver. Nissl-stained embryo sections are shown for comparison. SP, spleen; HB, hind brain; L, fetal liver.