Non-Coding-Regulatory Regions Of Human Brain Genes Delineated By Bacterial Artificial Chromosome Knock-In Mice

Background The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX (\u27high-throughput human genes on the X chromosome’) strategy to expand our understanding of human gene regulation in vivo. Results In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. Conclusions We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression

Ionomycin-induced activation of LFA-1 is independent of calpain-mediated cleavage of talin

Integrins are heterodimeric surface receptors that play a critical role in many cellular processes due to their ability to mediate the interactions between cells as well as between cells and the extracellular matrix. Integrin function can be regulated by conformational changes in their extracellular domains that increase their affinity for their ligand. Inside-out signalling is thought to initiate conformational changes through the binding of cytoplasmic proteins such as talin to integrin cytoplasmic domains. Calpains, calcium-dependent proteases, cleave talin upon activation, generating a free talin head domain that exhibits higher affinity for integrins than the intact talin protein. Therefore, the activation of calpains by receptor-induced calcium fluxes may lead to talin cleavage and could be an important step in the activation of integrins via inside-out signalling. In this study, the effects of the calcium ionophore ionomycin on the activation of LFA-1 in murine T cells were tested. Ionomycin treatment induced LFA-1-mediated adhesion in the helper T cell hybridoma line T28, in in vitro generated cytotoxic T cells, and in primary resting splenic T cells. The calpain inhibitors calpeptin and PD 150606 abrogated LFA-1 activation by ionomycin, implicating calpains in this process. However, calpeptin also inhibited LFA-1 activation by PMA, which did not induce calcium influx, suggesting that calpeptin is not entirely specific to calpains. Additionally, both calpain inhibitors induced the rapid apoptosis of cells treated with ionomycin, indicating that their effects on adhesion are non-specific. In contrast, two other calpain inhibitors, namely ALLN and calpain inhibitor III, did not impair LFA-1 activation by ionomycin. Western blotting did not detect cleavage of talin following ionomycin treatment of T cells, or cleavage of a-spectrin, another well-established substrate of calpains. These findings suggest that ionomycin-induced activation of LFA-1 is independent of the calpain-mediated cleavage of talin.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Targeted Cell-to-Cell Delivery of Protein Payloads via the Granzyme-Perforin Pathway

There is great potential for engineering cellular therapeutics by repurposing biological systems. Here, we report utilization of the granzyme-perforin pathway of cytotoxic lymphocytes as a cell-to-cell protein delivery module. We designed and constructed granzyme B-derived chaperone molecules fused to a fluorescent protein payload and expressed these constructs in natural killer (NK) cells. Using confocal microscopy and flow cytometry, we investigated the co-localization of the chaperones with lytic granules and the chaperone-mediated transfer of the fluorescent protein payload from NK to target cells in co-culture experiments. A synthetic chaperone consisting of the granzyme B ER signal peptide and a domain encompassing putative N-linked glycosylation sites in granzyme B is insufficient for payload transfer to target cells, whereas full-length granzyme B is sufficient for payload delivery. Combining our functional data with an analysis of the crystal structure of granzyme B suggests that the necessary motifs for granzyme B loading into lytic granules are dispersed throughout the primary amino acid sequence and are only functional when contiguous in the tertiary structure. These results illustrate that by using granzyme B as a molecular chaperone the granzyme-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies