Regulatory Analysis of miR-484 Promoter

The sequence of 1,442 bp promoter–enhancer region of miR-484 (Hu et al, 2019 (link)) was analyzed using ALGGEN-PROMO (version 3.0.2) (https://alggen.lsi.upc.es/) to identify putative cis-regulatory elements. 500 bp fragment of the proximal promoter region, from −495 to +5, containing the two putative RAREs was amplified from mouse genomic DNA using primer pairs (forward primer: 5′- ATAAAATCTAGAGGCGCCAACAGTGACAGTAGT-3′; reverse primer, 5′- ATAAAAGGATCCAGGCTGCAGGGCCGCGA-3′) and was subsequently cloned into the Xba I and BamHI sites of the vector pEZX-FR01 (GeneCopoeia Inc.). The fragment of the same promoter sequence containing mutation at the second RARE was synthesized by Sangon Biotech and was cloned into the same sites of pEZX-FR01. The promoter activities were evaluated in 293T cells induced with or without 1 μM atRA for 48 h using the dual-luciferase reporter assay system (Promega), according to the manufacturer’s instructions.