Histone crosstalk between H3S10ph and H4K16ac generates a histone code that mediates transcription elongation.

The phosphorylation of the serine 10 at histone H3 has been shown to be important for transcriptional activation. Here, we report the molecular mechanism through which H3S10ph triggers transcript elongation of the FOSL1 gene. Serum stimulation induces the PIM1 kinase to phosphorylate the preacetylated histone H3 at the FOSL1 enhancer. The adaptor protein 14-3-3 binds the phosphorylated nucleosome and recruits the histone acetyltransferase MOF, which triggers the acetylation of histone H4 at lysine 16 (H4K16ac). This histone crosstalk generates the nucleosomal recognition code composed of H3K9acS10ph/H4K16ac determining a nucleosome platform for the bromodomain protein BRD4 binding. The recruitment of the positive transcription elongation factor b (P-TEFb) via BRD4 induces the release of the promoter-proximal paused RNA polymerase II and the increase of its processivity. Thus, the single phosphorylation H3S10ph at the FOSL1 enhancer triggers a cascade of events which activate transcriptional elongation.

Endothelial cell adhesion to the extracellular matrix induces c-Src-dependent VEGFR-3 phosphorylation without the activation of the receptor intrinsic kinase activity.

RATIONALE: Integrins cooperate with growth factor receptors to promote downstream signaling for cell proliferation and migration. However, the mechanism of receptor activation is still unknown. OBJECTIVE: To analyze the mechanism of phosphorylation of the vascular endothelial growth factor receptor (VEGFR)-3 by cell adhesion. METHODS AND RESULTS: We show that VEGFR-3 phosphorylation, induced by cell attachment to the extracellular matrix, is independent from the intrinsic kinase activity of the receptor, as evidenced from phosphorylation cell adhesion experiments with a mutant kinase dead receptor or in the presence of the specific kinase inhibitor MAZ 51. Cell adhesion experiments in the presence of the c-Src inhibitor PP2 or in fibroblast triple knockout for c-Src, Yes, and Fyn (SYF) demonstrate that VEGFR-3 phosphorylation, induced by extracellular matrix, is mediated by c-Src. Kinase assays in vitro with recombinant c-Src show that VEGFR-3 is a direct c-Src target and mass spectrometry analysis identified the sites phosphorylated by c-Src as tyrosine 830, 833, 853, 1063, 1333, and 1337, demonstrating that integrin-mediated receptor phosphorylation induces a phosphorylation pattern that is distinct from that induced by growth factors. Furthermore, pull-down assays show that integrin-mediated VEGFR-3 phosphorylation activates the recruitment to the receptor of the adaptor proteins CRKI/II and SHC inducing activation of JNK. CONCLUSIONS: These data suggest that cell adhesion to extracellular matrix induces a downstream signaling using the tyrosine kinase receptor VEGFR-3 as scaffold.