The T-Cell Oncogenic Protein HOX11 ActivatesAldh1Expression 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 acidsynthesizing 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.The HOX11 protein is a member of the family of proteins carrying a DNA-binding homeodomain. HOX11 was discovered by its activation in a subset of T-cell acute leukemias with t(10;14)(q24;q11) or t(7;10)(q35;q24) (6,10,18,23). This ectopic expression is a contributary factor in the leukemogenesis of those T-cell tumors with translocations affecting chromosome 10, band q24. While activation of HOX11 is a feature of some T-cell tumors, the gene is normally not expressed in T cells. In developing mouse embryos, Hox11 expression is restricted to some parts of the brain, the developing branchial arches, and the developing spleen (3,35,36). The last site of expression seems particularly crucial in mouse development, as creation of null mutations in Hox11 causes mice to be born without spleens (36) as a result of atrophy of the spleen anlage around embryonic days 13 to 14 (E13 to E14) (3).The primary structure of the HOX11 protein suggested that it may be a transcription factor, mainly because of the homeodomain (4). Molecular experiments to dissect functional domains of HOX11 confirmed its ability to bind to specific DNA sequences via the homeodomain (4, 43) and to activate transcription (26, 50). The latter ability was due to the presence of modular transcriptional activation domains (50), of which an NH 2 -terminal module is needed for optimal transcription of a chromosomal target gene (26). In addition, the ability of HOX11 to interact with the catalytic subunits of protein phosphatases 1 and 2A has also been implicated in tumorigenesis (26), but the significance of this interaction for Hox11 function in normal mouse embryogenesis is unclear.Thus, both the expression of Hox11 during embryogenesis (particularly in the develop...

The nuclear oncoprotein TLX1/HOX11 associates with pericentromeric satellite 2 DNA in leukemic T-cells

“…1,[22][23][24]40,41 Our findings strongly suggested that TLX1 directly binds to noncoding satellite 2 DNA sequences in vitro as well as within leukemic T-cells (ALL-SIL) that harbour a chromosomal translocation involving the TLX1 locus at 10q24. Satellite 2 DNA is a poorly conserved, highly repetitive element found near the centromeres of chromosomes 1, 2, 7, 9, 10, 16, 22, Y and possibly 21, that altogether represents about 2% of the human genome.…”

“…To date, several potential downstream targets of TLX1 have been identified by examining correlations in gene transcription following the enforced or blocked expression of this transcription factor in cells or transgenic mice. [22][23][24] This includes the genes encoding aldehyde dehydrogenase 1a1, Fhl1/Slim1, Wt1 and NR4A3. However, although the preferred in vitro binding sequence (CGGTAA T / G TGG) is known, 25,26 as yet no direct in vivo targets of TLX1 have been delineated.…”

G1/S transcriptional networks modulated by the HOX11/TLX1 oncogene of T-cell acute lymphoblastic leukemia

“…The diminished expression of certain Wnt-induced and Wnt-related genes in HOX11 þ T-ALL and Jurkat/ MSCVhyg-HOX11 cells that was observed was in accordance with the documented ability of HOX11 to also act as a negative regulator of gene expression (Greene et al, 1998;Owens et al, 2003). To further investigate this observation, we next performed luciferase reporter assays in 293 T cells using the TOPflash luciferase reporter plasmid.…”

“…The above results notwithstanding, it is necessary to underscore the fact that HOX11 regulation of ISREcontrolled transcription was dependent on an intact homeodomain as is the expression of the HOX11 target gene Aldh1 (Greene et al, 1998;Owens et al, 2003). Using hematopoietic precursor cell immortalization as a surrogate transformation assay (Hawley et al, 1994a(Hawley et al, , 1997, our laboratory previously reported that deletion of the third helix of the homeodomain (HOX11 H3d mutant) or the introduction of point mutations within helix 3 (e.g., HOX11Asn51Ala mutant) abrogated HOX11 immortalizing function .…”

“…In parallel cotransfection experiments with the pAP1(PMA)-TA-Luc plasmid containing the HOX11 target sequence TAAT (Dear et al, 1993), no stimulation of luciferase production was detected (Figure 6a). Since the sequence of the ISRE and the promoter region of pISRE-Luc does not contain this or any other known HOX11-binding motif (Dear et al, 1993;Allen et al, 2000;Owens et al, 2003), stimulation of transcription by HOX11 through the ISRE may be mediated in a manner analogous to Aldh1 (Greene et al, 1998), either via protein-protein interactions or indirectly by an as yet unidentified intermediate HOX11 target gene. Interestingly, cotransfection of pcDNA3-HOX11 Lys55Gln , a mutant of HOX11 in which lysine 55 in the third helix of the homeodomain was changed to glutamine, stimulated ISRE-driven reporter gene expression at levels 2.3-fold higher than wild-type HOX11 (3.5-fold greater than empty vector) (Figure 6a).…”