Identification and functional characterisation of novel conserved Hes1 cis-regulatory modules

• Main Author: Jeziorska, Danuta M.
• Published: University of Warwick 2011
• Subjects: 570; QH426 Genetics
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.560162

The bHLH transcriptional repressor HES1 plays a pivotal role in progenitor cell maintenance and cell fate decisions. Hes1 is cyclically expressed during somitogenesis and in a variety of cell types. In order to better understand the mechanism of Hes1 expression, we used comparative genomics to identify additional potential Hes1 cis-regulatory modules (CRMs). This revealed seven phylogenetically conserved sequence blocks within 57 kb upstream of the Hes1 transcription start site in mouse. In vitro reporter assays revealed that these regions have a transcriptional regulatory function as they modulate the activity of both a heterologous and the endogenous Hes1 promoter. Notch signalling is believed to play a role in Hes1 regulation. Consistent with this, sequence analysis revealed that all of the newly identified CRMs contain consensus motifs for the RBP-Jĸ Notch effector. ChIP assays confirmed that RBP-Jĸ binds to all the identified regions in proliferating C2C12 cells, with the exception of CRM7. Further CRM7 sequence analysis identified a conserved M-CAT motif which we showed is specifically bound by TEAD2 and its co-activator YAP using ChIP. The loss of RBP-Jκ or TEAD2 binding correlated with impaired CRM function. Furthermore, mapping of the chromatin architecture of the endogenous Hes1 locus showed that several of the identified CRMs loop onto the Hes1 promoter, consistent with their regulatory function. This CRM-promoter communication is preserved throughout oscillatory expression of the gene. These data suggest a molecular mechanism for Notch and Hippo signalling in Hes1 regulation in proliferating C2C12 cells. Additionally, we have engineered destabilised Venus fluorescent reporters for live cell imaging of CRM function. Venus was modified using protein degradation motifs, including the PEST motif from c-myc and the CL1 yeast sequence, resulting in proteins with half-lives of approximately 76.5 and 28.7 minutes, respectively. These reporters were subsequently multimerised using a 2A peptide to enhance their brightness. The destabilised Venus reporters represent an excellent tool for real-time live cell imaging.