The speci®c loss of pRB or p107 together with p130 disrupts the normal development of only a very limited spectrum of tissues. These developmental defects have been attributed primarily to deregulation of E2F activity and consequent uncontrolled proliferation. We hypothesized, however, that the tissue-speci®c nature of these defects may also re¯ect deregulation of pRB-family associated factors that are speci®cally involved in determining cell fate. We report here that the pRBfamily members interact with transcription factors which contain paired-like homeodomains such as MHox, Chx10 and Pax-3. The interaction between the pRBfamily and the paired-like homeodomain proteins was initially identi®ed in a yeast two-hybrid screen where the N-terminal portion of p130 was used to isolate interacting factors from an embryonic mouse library. This interaction was con®rmed by in vitro binding and co-immunoprecipitation assays. We show further that coexpression of Pax-3 dependent pRB, p107 or p130 with Pax-3 causes repression of activated transcription from the c-met promoter. These data demonstrate that the pRB-family proteins can modulate the activity of factors which speci®cally control cell fate and/or dierentiation as well as controlling cell cycle regulators.

No abstract

We have recently demonstrated that the retinoblastoma family of negative cell cycle regulators can form complexes with a class of developmental factors which contain paired‐like (PL) homeodomains (Wiggan et al. [1998] Oncogene 16:227–236). Our screens led to the isolation of a novel PL‐homeodomain protein which had been isolated independently by another group and called Alx‐4 (Qu et al. [1997] Development 124:3999–4008). Mice homozygous for a targeted null mutation of Alx‐4 have several abnormalities, including preaxial polydactyly, suggesting that Alx‐4 plays a role in pattern formation in limb buds. In data that we present here, we show that Alx‐4 is expressed in mesenchymal condensations of a diverse group of tissues whose development is dependent on epithelial–mesenchymal interactions, many of which are additionally dependent on expression of the HMG‐box–containing protein, LEF‐1. Alx‐4–expressing tissues include osteoblast precursors of most bones, the dermal papilla of hair and whisker follicles, the dental papilla of teeth, and a subset of mesenchymal cells in pubescent mammary glands. We show further that Alx‐4 strongly activates transcription from a promoter containing the homeodomain binding site, P2. Optimal activation requires specific sequences in the N‐terminal portion of Alx‐4, as well as a proline‐rich region downstream of the PL‐homeodomain, but not the paired‐tail at the C terminus. Taken together, our results demonstrate that Alx‐4 is a potent transcriptional activator that is expressed at sites of epithelial–mesenchymal interactions during murine embryonic development. Dev. Dyn. 1998;213:159–169. © 1998 Wiley‐Liss, Inc.

The thioester bond in complement components C3 and C4 and the protease inhibitor alpha2-macroglobulin have traditionally been thought of as fulfilling the dual roles of mediating covalent attachment and maintaining the native conformational states of these molecules. We previously reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond formation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a native-like conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H2O) species is not. Subsequently, a species referred to as C3(NH3)* was described which potentially could account for the observations with the above mutants. C3(NH3)* is a transient species formed on aminolysis of native C3 that can spontaneously re-form the thioester bond. Importantly, it has a mobility on cation-exchange HPLC that is distinct from both native C3 and C3(H2O), but like the native molecule, it is cleavable by an alternative-pathway C3 convertase. In this study we showed by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3)*. Instead they displayed a chromatographic behaviour that was indistinguishable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by C1s as the conformational probe. A possible interpretation of our data is that the adoption of the native conformational state during biosynthesis of C3 and C4 is an energetically permissible process, even if it is not locked in via thioester-bond formation. Whereas this conformational state is stable in mature C3, it is unstable in mature C4, perhaps reflecting the additional post-translational cleavage of C4 before its secretion.

We have recently demonstrated that the retinoblastoma family of negative cell cycle regulators can form complexes with a class of developmental factors which contain paired-like (PL) homeodomains (Wiggan et al. [1998] Oncogene 16:227-236). Our screens led to the isolation of a novel PL-homeodomain protein which had been isolated independently by another group and called Alx-4 (Qu et al. [1997] Development 124:3999-4008). Mice homozygous for a targeted null mutation of Alx-4 have several abnormalities, including preaxial polydactyly, suggesting that Alx-4 plays a role in pattern formation in limb buds. In data that we present here, we show that Alx-4 is expressed in mesenchymal condensations of a diverse group of tissues whose development is dependent on epithelial-mesenchymal interactions, many of which are additionally dependent on expression of the HMG-box-containing protein, LEF-1. Alx-4-expressing tissues include osteoblast precursors of most bones, the dermal papilla of hair and whisker follicles, the dental papilla of teeth, and a subset of mesenchymal cells in pubescent mammary glands. We show further that Alx-4 strongly activates transcription from a promoter containing the homeodomain binding site, P2. Optimal activation requires specific sequences in the N-terminal portion of Alx-4 as well as a proline-rich region downstream of the PL-homeodomain, but not the paired-tail at the C terminus. Taken together, our results demonstrate that Alx-4 is a potent transcriptional activator that is expressed at sites of epithelial-mesenchymal interactions during murine embryonic development.