Regulation Of Circadian Rhythms

Zatz, Martin

Abstract

Circadian rhythms and environmental lighting regulate a number of endocrine and behavioral functions. Arguably, the best understood endocrine rhythm is that of the pineal gland, which secretes the hormone melatonin almost entirely at night. Unlike cells from rat pineal, dispersed cells from chick pineal remain rhythmic in their synthesis of melatonin, and responsive to light, in culture. We undertook to identify the photopigment that mediates photo-entrainment, the process by which light resets the endogenous clock, in collaboration with Mark Rollag and Ignacio Provencio (USUHS), and Maribeth Eiden (NIMH). We have made retroviral vectors carrying sense and antisense versions of candidate photopigments to test the effects of over- and under-expression of these proteins on the responses of chick pineal cells to light. Melanopsin and pinopsin are each novel photopigments present in chick pineal cells and the best candidates for the photopigments mediating photoentrainment. Iodopsin, a photopigment used for color vision in the retina, but absent from the pineal, provides a control. We previously isolated the cDNAs for melanopsin, pinopsin, and iodopsin, inserted them into plasmids, and prepared retroviral vectors containing these genes. We also determined the endogenous levels of gene expression for each photopigment gene and its tissue distribution using quantitative PCR. Our retroviral particles contained the RNA genome and gag/pol proteins from a Moloney murine leukemia virus, and an envelope from vesicular stomatitis virus. The genome plasmid was modified to contain a gene of interest, driven by the cytomegalovirus (CMV) promoter. We demonstrated transient transduction of each gene by its respective retroviral vector. Unlike the case for HEK293T cells, however, message levels rapidly silenced in primary chick pineal cultures. This year, to overcome silencing, we tried increasing the ?dosage? of particles per well or adding drugs to inhibit histone deacetylation or methylation. These approaches proved only partially effective or otherwise unsuitable. The third and most successful approach was to replace the CMV promoter used to drive gene expression with the phosphoglycerate kinase (pGK) promoter. This promoter, regulating expression of an endogenous housekeeping gene, should not be targeted for silencing by the cell as much as is the viral CMV promoter. In the event, the pGK promoter provided stable expression of the genes of interest though at a level lower than the maxima reached with CMV. New genome plasmids containing pGK driven genes have been synthesized for all of the genes described above and their transduction profile has been characterized. In order to seek the point of entry of photoentraining signals into the clock, we also constructed retroviral vectors to overexpress the clock-component genes cry 1 and 2 and period (per) 2, as well as antisense transcripts targeted against the cryptochromes. We measured cry1, cry2, and per2 message levels using qPCR and demonstrated effective transduction of these genes in both HEK293T cells and chick pineal cells. We also have synthesized polyclonal antibodies targeted against chick CRY1, CRY2, and PER2 and have begun characterizing protein expression in these cells.