Vitamin D3-Mediated Regulation of Antimicrobial Peptide Expression by Ancient Transposable Elements Conserved Only in Humans and Primates.
Authors: Gombart Adrian F. , Koeffler H. Phillip 
Published: October 2019

Abstract
The seco-steroid hormone 1,25(OH)2D3 (vitamin D3) induces the expression of the cathelicidin antimicrobial peptide (CAMP/CAP18/LL37) and human β defensin 4 (DEFB4/hβ D2) in human bone marrow cells, acute myeloid leukemia cells, monocytes, neutrophils, skin, and epithelial cell lines. It is mediated by a DR3-type vitamin D response element (VDRE; two 6-bp direct repeats [DR] separated by 3-bp) located in the promoter of each gene. We examined a wide range of human cell types and found that induction is most robust in myeloid cell types and does not occur in murine cells. Induction is not conserved in non-primate mammals as indicated by the absence of the VDRE in the murine, rat, and canine CAMP and DEFB4 promoters. The VDRE of the CAMP promoter is located in a short-interspersed nucleotide element (SINE) of the Alu-Sx subfamily of primate transposable elements. The DEFB4 VDRE is not located within such an element. We hypothesized that this unique vitamin D3-regulated expression of antimicrobial peptides is evolutionarily conserved in all primates. To test this, we amplified the promoter regions containing the CAMP and DEFB4 VDREs by PCR from the genomic DNAs of either humans, apes (chimpanzee, bonobo, gorilla & orangutan), Old World Monkeys (OWMs; rhesus macaque, pitailed macaque & African green monkey) or New World Monkeys (NWMs; common marmoset, common woolly monkey, black-handed spider monkey & red-chested tamari). Sequence analysis revealed the CAMP VDRE is highly conserved in all groups. Only African green monkey possessed a G to A change in the second DR6. Interestingly, the size of the PCR product (SINE plus flanking sequence) for rhesus macaque (OWM), tamari (NWM) and marmoset (NWM) was about 880 bp. For all others, the size was about 580 bp. The additional 300 bp of sequence was nearly identical to the first 300 bp of the shorter SINE. In other words, the larger SINE appears to have resulted from a duplication event or insertion of a second SINE into the first. This 880 bp SINE is present in the recently deposited rhesus macaque genome at the UCSC Genome Bioinformatics site. The hBD2/DEFB4 VDRE is highly conserved, too. The bonobo had a C to A change in the second DR6 and the OWM rhesus macaque and African green monkey had a G to A change in the first DR6. All of the NWMs had a T to C change in the second DR6. In addition, the spider monkey had a G to T change in the second DR6. Functional analyses of these sites are underway to assess if these changes affect the binding of VDR to the sites and activation of the promoter. Conservation of vitamin D3 regulation in diurnal primates and not nocturnal mammals (mouse, rat, & dog) suggests that a diurnal lifestyle may have selected for the regulation of the antimicrobial peptides by the vitamin D system. To test this, we are examining the conservation of the VDRE in both diurnal (lemurs) and nocturnal (galagos and lorises) prosimians. We obtained the genomic sequence of the CAMP gene and its promoter for a nocturnal prosimian (Garnett’s galago) from the NCBI trace archive. The SINE and the VDRE were absent from the promoter. Currently, we are cloning the promoter regions from the diurnal ring-tailed lemur and other nocturnal galagos or lorises to determine if conservation correlates with lifestyle. In summary, the data indicate that vitamin D3-mediated regulation of the antimicrobial peptides CAMP and DEFB4 is evolutionarily important for innate immunity in humans and other primates as opposed to non-primate mammals.