The 24-hour periodicity of biological rhythms relies on the circadian clock circuitry, made of peripheral oscillators operated by molecular clockworks and synchronized through humoral and neural outputs by central oscillators located in the hypothalamic suprachiasmatic nuclei. Circadian variation of gene expression is driven by the biological clock, operated by a transcriptional-translational negative feed-back loop hardwired by circadian proteins encoded by the core clock genes ARNTL1-2 or BMAL1, CLOCK, CRY1-2, PER1-3, NR1D1-2, RORA. An important paralogue of CLOCK in neural tissues is represented by the Neuronal PAS domain protein 2, NPAS2, a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. NPAS2, whose structure and biochemical facets are presently not well known, appears to reversibly bind heme as a prosthetic group to form a gas-regulated sensor, in which a heme-binding domain controls the DNA-binding activity of an additional domain in the protein triggering its transcriptional function. It is currently not completely clear how heme ligands of the ferrous state (such as the natural ligands CO, NO and O2) or the ferric state (cyanide, azide) may regulate the function of NPAS2, and consequently circadian gene expression. In this project we wish to: i) clone, express and purify recombinant NPAS2, ii) characterize the NPAS2 protein from a biochemical point of view, with particular emphasis on its thermodynamic and kinetic properties by spectroscopic techniques, and its structure features by x-ray crystallography and cryo-EM, and iii) explore its role in neural differentiation of human induced pluripotent stem cells (iPSCs).
