Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia

Complex genetic and biochemical interactions between HOX proteins and members of the TALE (i.e., PBX and MEIS) family have been identified in embryonic development, and some of these interactions also appear to be important for leukemic transformation. We have previously shown that HOXA9 collaborates with MEIS1 in the induction of acute myeloid leukemia (AML). In this report, we demonstrate that HOXB3, which is highly divergent from HOXA9, also genetically interacts with MEIS1, but not with PBX1, in generating AML. In addition, we show that the HOXA9 and HOXB3 genes play key roles in establishing all the main characteristics of the leukemias, while MEIS1 functions only to accelerate the onset of the leukemic transformation. Contrasting the reported functional similarities between PREP1 and MEIS1, such as PBX nuclear retention, we also show that PREP1 overexpression is incapable of accelerating the HOXA9-induced AML, suggesting that MEIS1 function in transformation must entail more than PBX nuclear localization. Collectively, these data demonstrate that MEIS1 is a common leukemic collaborator with two structurally and functionally divergent HOX genes and that, in this collaboration, the HOX gene defines the identity of the leukemia.     

Lock and key binding of the HOX YPWM peptide to the PBX homeodomain

HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.     

Range of HOX/TALE superclass associations and protein domain requirements for HOXA13:MEIS interaction

AbdB-like HOX proteins form DNA-binding complexes with the TALE superclass proteins MEIS1A and MEIS1B, and trimeric complexes have been identified in nuclear extracts that include a second TALE protein, PBX. Thus, soluble DNA-independent protein-protein complexes exist in mammals. The extent of HOX/TALE superclass interactions, protein structural requirements, and sites of in vivo cooperative interaction have not been fully explored. We show that Hoxa13 and Hoxd13 expression does not overlap with that of Meis1-3 in the developing limb; however, coexpression occurs in the developing male and female reproductive tracts (FRTs). We demonstrate that both HOXA13 and HOXD13 associate with MEIS1B in mammalian and yeast cells, and that HOXA13 can interact with all MEIS proteins but not more diverged TALE superclass members. In addition, the C-terminal domains (CTDs) of MEIS1A (18 amino acids) and MEIS1B (93 amino acids) are necessary for HOXA13 interaction; for MEIS1B, this domain was also sufficient. We also show by yeast two-hybrid assay that MEIS proteins can interact with anterior HOX proteins, but for some, additional N-terminal MEIS sequences are required for interaction. Using deletion mutants of HOXA13 and HOXD13, we provide evidence for multiple HOX peptide domains interacting with MEIS proteins. These data suggest that HOX:MEIS interactions may extend to non-AbdB-like HOX proteins in solution and that differences may exist in the MEIS peptide domains utilized by different HOX groups. Finally, the capability of multiple HOX domains to interact with MEIS C-terminal sequences implies greater complexity of the HOX:MEIS protein-protein interactions and a larger role for variation of HOX amino-terminal sequences in specificity of function.     

PBX,MEIS, and IGF-I are potential mediators of retinoic acid-induced proximodistal limb reduction defects

BACKGROUND: Phocomelia, which is primarily due to a disruption in the proximodistal axis, is found in virtually all mouse embryos exposed to high doses of retinoic acid (RA) on 11 days post coitum (dpc). METHODS: To identify genes that potentially mediate the effects of retinoic acid (RA) on limb development, we have examined the expression of 9,000 clones from the IMAGE consortium by microarray analysis of RNA isolated from 11 dpc mouse forelimbs exposed to RA or vehicle for 6 hr. Eight genes that demonstrated altered expression were chosen for further study of their mRNA levels using RT-PCR. Protein levels were determined by Western blot analysis. RESULTS: Of the 9,000 genes examined in the microarray, approximately 111 demonstrated altered expression (33 known genes and 78 ESTs). Of the eight known genes chosen for further study using RT-PCR, four mRNAs (PBX1a, PBX1b, IGF-Ia, and IGF-Ib) demonstrated consistent elevation ( approximately 3-fold) in their levels after RA treatment in both the forelimbs and hindlimbs as early as 3 hr after RA treatment. In addition to the two PBX1 isoforms, the mRNA level of the other two subtypes (PBX2 and PBX3) and the level of PBX1/2/3 protein were also found to be elevated in limb buds after RA treatment. Finally, we examined the expression of MEIS1, MEIS2, and MEIS3 because these proteins are necessary for PBX nuclear localization. The mRNA level of all three subtypes of MEIS were elevated approximately three- to four-fold in both the forelimbs and hindlimbs after RA treatment. CONCLUSIONS: Because both PBX and MEIS (and their orthologs) are believed to be involved in the control of proximodistal axis formation in mouse and fly limbs and IGFs in the development of limbs, we suggest that increases in PBX, MEIS and IGF-1 mRNA levels may contribute to proximodistal limb reduction defects caused by teratogenic doses of RA.     

Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors

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PBX1 intracellular localization is independent of MEIS1 in epithelial cells of the developing female genital tract

While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated.     

J-domain Proteins form Binary Complexes with Hsp90 and Ternary Complexes with Hsp90 and Hsp70

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.     

STAM Adaptor Proteins Interact with COPII Complexes and Function in ER-to-Golgi Trafficking

Signal-transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. In this study, we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells, the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted, the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein–green fluorescent protein trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after Brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with coat protein II (COPII) proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex.     

Variable Requirements for DNA-Binding Proteins at Polycomb-Dependent Repressive Regions in Human HOX Clusters

Polycomb group (PcG)-mediated repression is an evolutionarily conserved process critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We report two novel prospective PcG-dependent regulatory elements within the human HOXB and HOXC clusters and compare their repressive activities to a previously identified element in the HOXD cluster. These regions recruited the PcG proteins BMI1 and SUZ12 to a reporter construct in mesenchymal stem cells and conferred repression that was dependent upon PcG expression. Furthermore, we examined the potential of two DNA-binding proteins, JARID2 and YY1, to regulate PcG activity at these three elements. JARID2 has differential requirements, whereas YY1 appears to be required for repressive activity at all 3 sites. We conclude that distinct elements of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression, similar to what has been seen in Drosophila. These elements, however, have diverse requirements for binding factors, which, combined with previous data on other loci, speaks to the complexity of PcG targeting in mammals.