The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4.

Targeting of myocyte enhancer binding factor 2 (MEF2) proteins to the nucleus depends on a C-terminal bipartite nuclear localization signal (NLS). By expression of green fluorescent protein (GFP)/MEF2 fusion proteins in transfected myoblasts, we show that MEF2C contains an additional 13 amino acids domain, located immediately upstream of the NLS, which contributes to its nuclear retention. We also show that the NLS present in MEF2 proteins is required for efficient nuclear localization of histone deacetylase 4 (HDAC4). In muscle cells, transfected HDAC4 is largely cytoplasmic or, to a lesser extent, pancellular. Co-transfection of either MEF2A or MEF2C causes HDAC4 to accumulate in the nucleus in association with MEF2. This effect strongly depends on MEF2 NLS; it also requires the specific interaction of HDAC4 with MEF2, since the isolated NLS is not sufficient for targeting HDAC4 to the nucleus and other nuclear proteins, such as NF-Y, cannot substitute MEF2. Therefore, we demonstrate that HDAC4, different from HDAC5, is mainly a cytoplasmic resident protein, requiring a trans-acting NLS for nuclear localization. The physiological implications of MEF2 carrying its own inhibitor to the nucleus are discussed.

Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia.

A cDNA sequence encoding a soluble form of the human low-density lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated. In-frame fusion of the two cDNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-terminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through the pathway of the transferrin receptor. Cells transfected with the LDL-R/transferrin cDNA translate, glycosylate, and secrete the corresponding protein in the culture medium. The secreted protein binds LDL in a ligand-blotting experiment. Finally, the chimeric protein mediates the binding and internalization of LDL in mutant cells lacking the LDL receptor. In fact, Watanabe rabbit fibroblasts, incubated with the chimeric protein show a fourfold increase in LDL binding, a fivefold increase in LDL internalization, and a sixfold increase in LDL degradation, with respect to unincubated fibroblasts.

Inhibition of myogenesis by transforming growth factor beta is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm.

Transforming growth factor beta (TGF-beta) was found to inhibit differentiation of myogenic cells only when they were grown to high density. Inhibition also occurred when myogenic cells were cocultured with other types of mesenchymal cells but not when they were cocultured with epithelial cells. It is therefore possible that some density-dependent signaling mediates the intracellular response to TGF-beta. Within 30 min of treatment, TGF-beta induced translocation of MEF2, but not MyoD, myogenin, or p21, to the cytoplasm of myogenic cells grown to high density. Translocation was reversible on withdrawal of TGF-beta. By using immune electron microscopy and Western blot analysis on subcellular fractions, MEF2 was shown to be tightly associated with cytoskeleton membrane components. To test whether MEF2 export from the nucleus was causally related to the inhibitory action of TGF-beta, we transfected C2C12 myoblasts with MEF2C containing the nuclear localization signal of simian virus 40 large T antigen (nlsSV40). Myogenic cells expressing the chimerical MEF2C/nlsSV40, but not wild-type MEF2C, retained this transcription factor in the nucleus and were resistant to the inhibitory action of TGF-beta. We propose a mechanism in which the inhibition of myogenesis by TGF-beta is mediated through MEF2 localization to the cytoplasm, thus preventing it from participating in an active transcriptional complex.

[Translocation (X; Y) and genetic counseling]. / Translocation (X; Y) et conseil génétique.

A t(X; Y) was discovered in 32 year-old female patient who has had three consecutive miscarriages. Molecular analysis of the loci DXS 31 and DYS 22 was performed which confirmed and precise the breakpoints at Xp22.3 and Yq11. Genetic counselling was based upon the accurate definition of the chromosome rearrangement and the analysis of the outcomes of similar published observations.