Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.DOI: http://dx.doi.org/10.7554/eLife.09406.001

SummaryPhosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.

The transforming growth factor–β (TGF-β) and bone morphogenetic protein (BMP) family of cytokines critically regulates vascular morphogenesis and homeostasis. Impairment of TGF-β or BMP signaling leads to heritable vascular disorders, including hereditary hemorrhagic telangiectasia (HHT). Drosha, a key enzyme for microRNA (miRNA) biogenesis, also regulates the TGF-β and BMP pathway through interaction with Smads and their joint control of gene expression through miRNAs. We report that mice lacking Drosha in the vascular endothelium developed a vascular phenotype resembling HHT that included dilated and disorganized vasculature, arteriovenous fistulae, and hemorrhages. Exome sequencing of HHT patients who lacked known pathogenic mutations revealed an over-representation of rare nonsynonymous variants of DROSHA. Two of these DROSHA variants (P100L and R279L) did not interact with Smads and were partially catalytically active. In zebrafish, expression of these mutants or morpholino- directed knockdown of Drosha resulted in angiogenesis defects and abnormal vascular permeability. Together, our studies point to an essential role of Drosha in vascular development and the maintenance of vascular integrity, and reveal a previously unappreciated link between Drosha dysfunction and HHT.

Phosphoinositides are a family of seven lipids that are present in various subcellular compartments at low levels, compared to other phospholipids. Their levels are regulated by an array of kinases and phosphatases that respond to extrinsic and intrinsic signals in order to change the phosphoinositide profile in different compartments. For example in the nucleus the phosphoinositide PtdIns5P is phosphorylated by the lipid kinase PIP4K2B to generate PtdIns(4,5)P2. In response to activation of stress pathways, PIP4K2B becomes phosphorylated and less active and this in part leads to an increase in nuclear PtdIns5P. Changes in nuclear PtdIns5P are sensed by nuclear proteins that contain lipid interaction motifs which act to transduce the changes in the level of nuclear PI into changes in functional output. PIP4K2B is present in muscle tissue and in myoblasts and we have investigated how its expression regulates myogenic differentiation. Using C2C12 cells as a model of myogenesis we have identified a pathway that illustrates how signalling through PIP4K2B/PtdIns5P can directly regulate gene transcription. Further characterisation of nuclear proteins that can interact with phosphoinositides suggest that changes in nuclear PI are likely to impact on many aspect of chromatin regulation and eventually on transcriptional output.