Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression

Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1

Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

<p>Abstract</p> <p>Background</p> <p>Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp <it>Chelonus inanitus </it>(Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences.</p> <p>Results</p> <p>About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein.</p> <p>An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the <it>Chelonus </it>lineage. Venom components specific to <it>C. inanitus </it>included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins.</p> <p>Conclusions</p> <p>The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of <it>C. inanitus </it>appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.</p

Begegnung / sich begegnen

Wiese C. Begegnung / sich begegnen. In: Blohm M, Brenne A, Hornäk S, eds. Irgendwie anders: Inklusionsaspekte in den künstlerischen Fächern und der ästhetischen Bildung. 1st ed. Hannover: fabrico; 2017: 169-173

Computational aqueous solubility prediction for drug-like compounds in congeneric series

It was the aim of the present work to develop a quantitative structure-property relationship (QSPR) model for predicting the aqueous solubility of drug-like compounds in congeneric series. Lipophilicity combined with structural fragment information, fragmental based correction factors and congeneric series indices were used as descriptors for a principal component analysis (PCA) followed by multivariate partial least squares regression statistics (PLS). The derived PLS regression model for the prediction of solubility parameters was based on an in-house data set of 2473 drug-like compounds. The generated PLS model had a coefficient of determination (R(2))=0.844 and a root-mean-square (rms) error of 0.51 log units. It predicted the solubility of the test data set with a high degree of accuracy (R(2)=0.81). In addition, the PLS model was successful in predicting the solubility of new congeneric test sets when solubility values of corresponding scaffolds were accessible

Involvement of a Leishmania thymidine kinase in flagellum formation, promastigote shape and growth as well as virulence

Leishmania promastigote cells transmitted by their insect vector get phagocytosed by macrophages and convert into the amastigote form. In a recently performed proteomic study, a thymidine kinase (TK) was found to be preferentially expressed in amastigotes. Western blot analysis showing a marked increase in TK protein synthesis during stage differentiation from promastigotes to amastigotes confirmed this result. After comparison of the amino acid sequence of Leishmania donovani and Leishmania major thymidine kinases with thymidine kinases of other organisms the Leishmania protein has to be classified as a type II TK. Therefore, in accordance with the nomenclature of other thymidine kinases we named the Leishmania enzymes LdTK1 and LmTK1, respectively. The LdTK1 is localised within the cytoplasm of promastigotes. In amastigotes, increased expression and a clustered distribution of the protein can be observed. Lmtk1 single allele gene replacement mutants have significantly elongated flagellum. In contrast, lmtk1 double allele gene replacement mutants show a remarkably reduced flagellar length, diminished overall size and a deformed body shape. In addition, they have a 12-fold reduced growth rate. For both mutant strains, macrophage infectivity is clearly reduced compared to a L. major wildtype infection