Regulation of the Human Cathelicidin Antimicrobial Peptide Gene by an Alu SINE by Michael Power Dr. Adrian Gombart Laboratory Linus Pauling Institute/ Department of Biochemistry and Biophysics Oregon State University

 Vitamin D is important for human health.  Bone Health  Immune Function  70% of Americans are Vitamin D insufficient.  Vitamin D increases production of antimicrobial peptides.  Defensins  Cathelicidins Gombart, AF Future Microbiol.

 Part of the innate immune system.  Kills bacteria  Signals other immune cells  Cleans up infection Burchi, et al. Arch. Immunol. Ther. Exp. (2010) 58:15–25 Effect of CAMP on pathogen CAMP geneCAMP mRNACAMP protein Cathelicidin Antimicrobial Peptide (CAMP) CAMP promoter Izadpanah, A. and Gallo, R J Am Acad Dermatol.

Humans Alu SINE u SINE VDRE CAMP Promoter Mice  “Junk DNA”  300 base pairs  1 million copies  10% of genome  Insertion in promoter of hCAMP allows regulatory effect.  Duplication forms a Vitamin D Responsive Element (VDRE).  Mice do not have Alu SINE. Evolution of CAMP promoter Cordaux R. and Batzer M.A Nature Reviews Genetics. CAMP Promoter Alu Short Interspersed Element (Alu SINE) Gombart AF et al BMC Genomics.

Adams, JS et al J Immunol.  Vitamin D induces CAMP expression in humans, but not in mice.  LPS and 19kDa induce CAMP expression in mice, but not in humans.  LPS and 19kDa are components of the bacterial cell membrane. CRAMP is mouse CAMP hCAP is human CAMP A. Human (THP-1)

Hypothesis: The Alu SINE causes the repression of hCAMP in the presence of LPS and 19 kDa. Prediction : Deletion of the Alu SINE will allow induction of hCAMP in the presence of LPS and FSL-1. Question : What causes the repression of hCAMP when human cells are treated with LPS or 19 kDa?

Treatment Gene Expression Messenger RNA Complementary DNA Real-Time PCR No treatment 1,25 D₃ (10⁻⁸ M) LPS (100 ng/ml) FSL-1 (10 ng/ml) THP-1 RAW 264.7

 1,25 D₃ induces hCAMP.  LPS and FSL-1 do not affect hCAMP.  hCAMP is normalized to Beta-Actin.  hCAMP is normalized to 18s.

No mCAMP expression observed.

Promoter Promote  Firefly luciferase gene is inserted under control of hCAMP promoter.  NotI restriction sites are engineered on either side of Alu SINE.  Alu SINE is removed by NotI restriction enzymes. NotI wildtype ∆ Alu luc Alu SINE hCAMP luc

ElectroporationTreatment Gene Expression Harvest Cells Dual-Luciferase Assay U937 and THP-1 No treatment 1,25 D₃ (10⁻⁷ M) LPS (100 ng/ml) FSL-1 (10 ng/ml)

 Vitamin D does not affect expression in cells that contain NotI plasmid.  U937 cells grown and transfected by Chunxiao Guo respond to 1,25 D3

 1,25 D3 induces hCAMP expression in THP-1 cells.  LPS and FSL-1 do not induce hCAMP expression in THP-1 cells.  RAW cells do not express mCAMP.

 Obtain and test mouse cell line J774A.  Expression of mCAMP?  Transfect U937 cells.  Transfect THP-1 cells.  Determine effects of Alu SINE on hCAMP expression.  Does deletion of the Alu SINE allow induction of hCAMP in the presence of LPS and FSL-1?

 Dr. Gombart, Project Mentor  Dr. Ahern, Program Manager  Mary Fantacone, Lab Manager  Members of the Gombart Laboratory  Linus Pauling Institute  Department of Biochemistry and Biophysics  Howard Hughes Medical Institute  Cripps Scholarship Fund  National Institute of Health