At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GIRE) of the L-PK gene is built around two noncanonical E boxes (element IA) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)-and COUPITF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GIRE function. Mutations transforming one or two noncanonical E boxes of element LA into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GIRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GIRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway. It is coordinately regulated at the transcriptional and posttranscriptional levels, positively by carbohydrates in the presence of insulin and negatively by glucagon via cyclic AMP (8,32). The regulatory region of the L-PK gene, responsible for its transcriptional response to carbohydrates and hormones, has been ascribed to a -120/ -183-bp proximal promoter fragment (2, 30), ex vivo by transient expression assays in hepatocytes in primary culture, and in vivo in transgenic mice (7). We had previously characterized the important cis-acting DNA elements in the L-PK gene promoter: in the 3'-5' direction upstream from the TATA box, we found, respectively, box Li, a binding site for hepatocyte nuclear factor 1 (HNF1); box L2, a binding site for nuclear factor 1; box L3, a binding site for HNF4; and box LA, a weak in vitro binding site for major late transcription factor (MLTF)/USF ( Fig. 1) (33). Box IA contains the L-PK gene glucose/insulin response element (GlRE) and is also indispensable for the action of glucagon and cyclic AMP. When L4 is oligomerized, it is able to confer glucose/insulin and cyclic AMP responsiveness on a

Melanocytic nevi frequently harbor oncogenic BRAF mutations, but only a minority progress to melanoma. In human melanocytes, persistent BRAF(V600E) expression triggers oncogene-induced senescence, which implies that bypass of oncogene-induced senescence is necessary for malignant transformation of melanocytes. We show that a subpopulation of primary human melanocytes with persistent expression of BRAF(V600E) do not enter oncogene-induced senescence, but instead survive despite heightened MAPK activity. Disruption of the p53 pathway using short-hairpin RNA initiated rapid growth of these V600E(+) melanocytes in vitro. The resultant V600E(+)/p53(sh) melanocytes grew anchorage-independently in soft agar, formed pigmented lesions reminiscent of in situ melanoma in artificial skin reconstructs, and were weakly tumorigenic in vivo. Array comparative genomic hybridization analysis demonstrated that the transformed melanocytes acquired a substantial deletion in chromosome 13, which encodes the Rb1 tumor suppressor gene. Gene expression profiling study of nevi and melanomas showed that p53 target genes were differentially expressed in melanomas compared with nevi, suggesting a dysfunctional p53 pathway in melanoma in vivo. In summary, these data demonstrate that a subpopulation of melanocytes possesses the ability to survive BRAF(V600E)-induced senescence, and suggest that p53 inactivation may promote malignant transformation of these cells.

Mouse and human somatic cells can either be reprogrammed to a pluripotent state or converted to another lineage with a combination of transcription factors suggesting that lineage commitment is a reversible process. Here we show that only one factor, the active intracellular form of Notch1, is sufficient to convert mature pigmented epidermal-derived melanocytes into functional multipotent neural crest stem-like cells. These induced neural crest stem cells (iNCSCs) proliferate as spheres under stem cell media conditions, re-express neural crest-related genes and differentiate into multiple neural crest derived mesenchymal and neuronal lineages. Moreover, iNCSCs are highly migratory and functional in ovo. These results demonstrate that mature melanocytes can be reprogrammed toward their primitive neural crest cell precursors through the activation of a single stem cell-related pathway. Reprogramming of melanocytes to iNCSCs may provide an alternate source of NCSCs for neuroregenerative applications.