Expression Analysis of Activating Transcription Factor 4 (ATF 4) in Zebrafish: Implications for Coffin-Lowry Syndrome

Introduction

Objectives

Methods

Results

Department of Biological Sciences York College of Pennsylvania

Jonathan Coale

Conclusion

- Coffin Lowry Syndrome (CLS) is a genetic disorder that causes skeletal abnormalities due to a mutation in the RSK2 (ribosomal S6 kinase) gene. - Some CLS cases are caused by the lack of phosphorylation of ATF4 (activating transcription factor 4)by RSK2.-ATF 4 is known for a variety of functions but it mostly known for its role in bone development. - ATF 4 has the ability to form homo/heterodimers using its domains.- Activated ATF 4 binds to its target gene, Osteocalcin (Osn), which allows transcription of osteocalcin which is essential for normal mineralization of bone.

-In this study, zebrafish will be used to study the function of ATF4 in development. - The use of morpholino oligos in zebrafish could show similar morphological differences to human cases of CLS.-Advantages of zebrafish ; translucent embryos, quick rate of development, large quantity of offspring, known genome, and ability to perform in situ hybridization and morpholino knockdown techniques.

-Is ATF4 expressed in adult zebrafish? -What tissues show ATF4 expression in zebrafish?-Is ATF4 expressed in embryonic zebrafish?

Amino Acid Sequence

-Expression of ATF4 has been found to occur in various adult tissues and 3 day old embryos.-Key domains of ATF4 are conserved in zebrafish when compared to human amino acid sequence.

Future Research-Finding where ATF4 is expressed in an embryovia in situ hybridization.-Observe morphological changes by introducing morpholino oligos into embryos which examine the effects of knocking down a gene.

Literature Cited-Yang X., Matsuda K., Bialek P., Jacquot S., Masuoka, C., Schinke T., Li L., (…)Karsenty G. 2004. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology: Implication for Coffin-Lowry syndrome. Cell, 117 (3), pp. 387-398.-Ameri, K., Harris, A. 2008. Activating transcription factor 4. Science Direct. 40:14-21.

Acknowledgements: Special thanks to Dr. Wendy Boehmler for her guidance and patience.

Red = βTrCP Recognition SiteBlue = Basic DomainGreen = Leucine Zipper

Whole Adult ZF Selected Tissues 2,3, and 6 day Old ZF Embryos

Extract RNA

Reverse Transcription PCR

cDNA

PCR w/atf4 primers

http://www.unsolvedmysteries.oregonstate.edu/embryo

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=cls&rendertype=figure&id=cls.F1

Forward PrimerReverse Primer

Target Sequence (1019bp)5’ 3’

3’ 5’

ATG (Start Codon) TAG (Stop Codon)

Figure 2. A 1 % agarose gel ran at 240 volts. The numbers at the top of each lane (2,3, and 6) represent the maturity of the embryo (in days) when RNA was reverse transcribed to obtain cDNA samples. The three day old embryo was the only sample with a band at 1000bp meaning ATF 4 shows expression in a 3 day old embryo.

Figure 1. A 0.8% agarose gel of six cDNA tissue samples with ATF4 primers. In well one, 5ul of 500bp ladder was used and 10ul of 500bp ladder was used in well eight. (A=Adult, B=Brain, E=Eye, G=Gut, H=Heart, M=Muscle)