Deciphering the contribution of known cis-elements in the mouse cone arrestin gene to its cone-specific expression.
OBJECTIVE
Cone arrestin (CAR) is highly expressed in all cone photoreceptors of the retina and a subset of pinealocytes in the pineal gland. This study was initiated to examine the cis-elements responsible for the cell-specific expression pattern of CAR.


METHODS
Mutagenesis and specific deletions of known cis-elements in the proximal promoter of the mouse CAR (mCAR) gene were introduced and analyzed in vitro and in vivo. A series of mCAR promoter-luciferase reporter constructs were transiently transfected into COS-7 or Weri-Rb-1 retinoblastoma cells and tested in in vitro promoter assays. Transgenic Xenopus laevis were created with deletional or mutated promoter fragments driving an enhanced green fluorescent protein (EGFP) reporter gene. The resultant EGFP expression pattern in the transgenic animals was analyzed by fluorescence microscopy and immunocytochemistry.


RESULTS
A significant decrease in in vitro transcriptional activities was observed when the minimal 215-bp promoter fragment was mutated in each of the four cone-rod homeobox (CRX)-binding elements (CBEs) or in either of the two TATA-elements. The 215-bp mCAR proximal promoter drove EGFP expression to cone photoreceptors and pinealocytes in transgenic Xenopus laevis; however, the truncated 147-bp fragment drove expression to both cone and rod photoreceptors. Transgenic tadpoles carrying a single mutation in either the TATA-box, the TATA-element or the proximal CBE had undetectable EGFP expression in the retina. However, when one of the other three CBEs was mutated, EGFP expression was observed in muscle and brain tissues, in addition to the eyes. Also, when both TATA elements were mutated, transgenic animals had EGFP expression in all photoreceptors. Because no reporter activity was observed when either a 3.2-kb 5' extended region or the first intron of the mCAR gene was tested in Weri-Rb-1 cells, neither construct was examined in vivo.


CONCLUSIONS
The data demonstrate that the regulatory functions of the known cis-elements in the mCAR promoter are highly dependent on location and nucleotide sequence conservation. The TATA elements and CBEs are crucial for driving both basal transcriptional activity and tissue specificity to cone photoreceptors and pinealocytes.
Pickrell
Shiyi Wei
SW
The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, Los Angeles, CA 90033-9224, USA.
Zhu
Xuemei
X
Wang
Xiaopeng
X
Craft
Cheryl M
CM
D000818:Animals; D030801:Animals, Genetically Modified; D019393:Arrestin / Q000235:genetics*; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D012175:Retinoblastoma / Q000473:pathology; D035181:TATA-Box Binding Protein / Q000235:genetics; D014162:Transfection; D014982:Xenopus laevis
D019393:Arrestin; D035181:TATA-Box Binding Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins