Analysis of intronless genes involved in oscillation and differentiation

The genomes of higher eukaryotes are replete with intron-containing genes. Transcription of these genes produces precursor mRNAs containing intervening sequences, which are subsequently removed and the exons spliced together to form the mature mRNA. However, a small proportion of eukaryotic protein-coding genes are intronless and therefore bypass post-transcriptional splicing events. Although a large proportion of intronless genes are known to code for certain types of proteins, their specific role in the genome of higher organism is perplexing. This research set out to elucidate the functions of intronless genes in humans by studying their involvement in the expression pattern of oscillatory gene that occurs in the pre-somitic mesoderm of developing embryo. Twenty-seven (27) human homologs of mouse oscillatory genes were analysed to determine the number of exons present in them using various bioinformatics databases. The result obtained identified two intronless genes –NRARP and ID1 – which are associated with the Notch signalling pathway of the segmentation clock. This represented 7.4% of the total oscillatory genes analysed. No intronless gene was found in the Wnt and FGF signalling pathways – two other pathways famous for oscillatory gene expression. The proteins encoded by the intronless genes are involved in several important biological processes including angiogenesis, cell cycle control and in the regulation of cellular senescence. Although oscillatory genes had fewer numbers of introns compared to the non-oscillatory genes, the intronless genes were not implicated in the regulation of the precise timing events of the segmentation clock. This result may also point to the fact that the rapid expression rate of the oscillatory genes in the PSM may favour the reduced intron length of the oscillatory genes.

Auto-Regulation of Transcription and Translation: Oscillations, Excitability and Intermittency

Several members of the Hes/Her family, conserved targets of the Notch signalling pathway, encode transcriptional repressors that dimerise, bind DNA and self-repress. Such autoinhibition of transcription can yield homeostasis and, in the presence of delays that account for processes such as transcription, splicing and transport, oscillations. Whilst previous models of autoinhibition of transcription have tended to treat processes such as translation as being unregulated (and hence linear), here we develop and explore a mathematical model that considers autoinhibition of transcription together with nonlinear regulation of translation. It is demonstrated that such a model can yield, in the absence of delays, nonlinear dynamical behaviours such as excitability, homeostasis, oscillations and intermittency. These results indicate that regulation of translation as well as transcription allows for a much richer range of behaviours than is possible with autoregulation of transcription alone. A number of experiments are suggested that would that allow for the signature of autoregulation of translation as well as transcription to be experimentally detected in a Notch signalling system.

Optogenetic control of NOTCH1 signalling

The Notch signalling pathway is a crucial regulator of cell differentiation as well as tissue organisation. Dysregulation of Notch signalling has been linked to the pathogenesis of different diseases. Notch plays a key role in breast cancer progression by controlling the interaction between the tumour cells and the microenvironment as well as by increasing cell motility and invasion. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). Here, we circumvent this step by regulating Notch activity by light. To achieve this, we have engineered a membrane-bound optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures. OptoNotch allows fine-tuning ligand-independent regulation of N1ICD to understand the spatiotemporal complexity of Notch signalling.

The role of delta like non-canonical Notch ligand 1 (DLK1) in cancer

Delta like non-canonical Notch ligand 1 (DLK1) is a cleavable single-pass transmembrane protein and a member of the Notch/Delta/Serrate family. It is paternally expressed and belongs to a group of imprinted genes located on chromosome band 14q32 in humans and 12qF1 in mice. DLK1 is expressed in many human tissues during embryonic development but in adults expression is low and is mostly restricted to (neuro)endocrine tissues and other immature stem/progenitor cells (notably hepatoblasts). However, DLK1 is expressed at a high frequency in many common malignancies (liver, breast, brain, pancreas, colon and lung). More recently, high levels of expression have been identified in endocrine related cancers such as ovarian and adrenocortical carcinoma. There is growing evidence that DLK1 expression in cancer is associated with worse prognosis and that DLK1 may be a marker of cancer stem cells. Although the exact mechanism through which DLK1 functions is not fully understood, it is known to maintain cells in an undifferentiated phenotype and has oncogenic properties. These effects are partly exacted through interaction with the Notch signalling pathway. In this review, we have detailed the functional role of DLK1 within physiology and malignancy and posit a mechanism for how it exacts its oncogenic effects. In describing the expression of DLK1 in cancer and in healthy tissue, we have highlighted the potential for its use both as a biomarker and as a potential therapeutic target.

Notch Signalling in Breast Development and Cancer

The Notch signalling pathway is a highly conserved developmental signalling pathway, with vital roles in determining cell fate during embryonic development and tissue homeostasis. Aberrant Notch signalling has been implicated in many disease pathologies, including cancer. In this review, we will outline the mechanism and regulation of the Notch signalling pathway. We will also outline the role Notch signalling plays in normal mammary gland development and how Notch signalling is implicated in breast cancer tumorigenesis and progression. We will cover how Notch signalling controls several different hallmarks of cancer within epithelial cells with sections focussed on its roles in proliferation, apoptosis, invasion, and metastasis. We will provide evidence for Notch signalling in the breast cancer stem cell phenotype, which also has implications for therapy resistance and disease relapse in breast cancer patients. Finally, we will summarise the developments in therapeutic targeting of Notch signalling, and the pros and cons of this approach for the treatment of breast cancer.

Effects of serum starvation and vascular endothelial growth factor stimulation on the expression of Notch signalling pathway components

Brain arteriovenous malformation (BAVM) is an abnormality in the cerebral vascular system. Although the upregulation of the Notch signalling pathway is a deterministic factor in BAVM, the mechanism by which this pathway is upregulated in patients with BAVM is uncertain. The effects of serum starvation and vascular endothelial growth factor (VEGF) stimulation on the Notch signalling pathway in brain microvascular endothelial cells (MECs) and mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial cells were investigated in this study. The duration of serum starvation and VEGF concentration were changed, cell viability was measured, and reasonable time and concentration gradients were selected for subsequent studies. Protein and mRNA expression levels of Notch signalling pathway components in both MECs and mES/EB-derived endothelial cells were detected using western blotting and real-time PCR, respectively. Expression levels of the Notch1, Notch4, Jagged1, delta-like ligand 4 (Dll4) and Hes1 proteins and mRNAs were upregulated by lower VEGF concentrations and shorter-term serum starvation but inhibited by higher VEGF concentrations and longer-term serum starvation. This study revealed effects of changes in the duration of serum starvation and VEGF concentration on the expression of Notch signalling pathway components in both MECs and mES/EB-derived endothelial cells, potentially contributing to BAVM formation.

Comparative and evolutionary analyses reveal conservation and divergence of the notch pathway in lophotrochozoa

Lophotrochozoan species exhibit wide morphological diversity; however, the molecular basis underlying this diversity remains unclear. Here, we explored the evolution of Notch pathway genes across 37 metazoan species via phylogenetic and molecular evolutionary studies with emphasis on the lophotrochozoans. We displayed the components of Notch pathway in metazoans and found that Delta and Hes/Hey-related genes, as well as their functional domains, are duplicated in lophotrochozoans. Comparative transcriptomics analyses allow us to pinpoint sequence divergence of multigene families in the Notch signalling pathway. We identified the duplication mechanism of a mollusc-specific gene, Delta2, and found it displayed complementary expression throughout development. Furthermore, we found the functional diversification not only in expanded genes in the Notch pathway (Delta and Hes/Hey-related genes), but also in evolutionary conservative genes (Notch, Presenilin, and Su(H)). Together, this comprehensive study demonstrates conservation and divergence within the Notch pathway, reveals evolutionary relationships among metazoans, and provides evidence for the occurrence of developmental diversity in lophotrochozoans, as well as a basis for future gene function studies.