Abstract
Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease.
Highlights
The characterization of the genomic elements which govern the spatial and temporal tuning of gene transcription is a key issue in genomic biology, providing with a better understanding of the cell regulatory networks and the functional landscape of the genome [1]
We designed a screening strategy based on transfection in Normal Human Epidermal Keratinocytes (NHEKs) cultured in both conditions of luciferase reporter plasmids containing the promoter region of the gene PADI3, flanked by each of the 19 coding segments (CNS)
We found no sensitivity to nordihydroguaiaretic acid (NDGA) treatment of the PADI1 or PADI2 mRNA levels in calcium-stimulated NHEKs (Fig. 5A)
Summary
The characterization of the genomic elements which govern the spatial and temporal tuning of gene transcription is a key issue in genomic biology, providing with a better understanding of the cell regulatory networks and the functional landscape of the genome [1]. We have reported that the PADI3 minimal promoter region showed a significant, albeit moderate, increase in activity (1.5-fold) in differentiated versus proliferative keratinocytes in vitro, and required the bound transcription factors NF-Y and Sp1/3 [13]. Such co-operation is not specific to differentiated keratinocytes since NF-Y and Sp1 govern the transcription of a variety of genes in epithelial or non epithelial cells [14,15,16,17], as that of the DNp63 isoform of p63, a homolog of the p53 tumour suppressor gene, in proliferative mouse keratinocytes [18]. In the cervical carcinoma HeLa cells, NF-Y and Sp1 enhanced the levels of transcription of the topoisomerase II a and b gene promoters [19,20] whereas the PADI3 gene promoter is not active [13]
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