Transcriptional Activation of <i>Mina</i> by Sp1/3 Factors

<div><p>Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of <i>Mina</i> gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the <i>Mina</i> 5′ region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080638#pone.0080638-Okamoto1" target="_blank">[1]</a>. In order to explore the mechanisms regulating <i>Mina</i> gene expression, we set out to molecularly characterize the <i>Mina</i> promoter in the region encompassing these SNPs. We used three kinds of assays – reporter, gel shift and chromatin immunoprecipitation – to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of <i>Mina</i> promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of <i>Mina</i> gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.</p></div

Reporter assay analysis of a 2 kb <i>Mina</i> promoter fragment.

<p>The transcriptional start site (TSS), labeled 0, is from NCBI (NM_025910.3). Internal control plasmid pRL-TK encoding renilla luciferase was cotransfected with each firefly luciferase construct. Parental (light gray filled arrow) and nested deletion constructs are diagrammed to the left of the bar graph with numbers representing the 5′ (and in the case of −64/+80, the 3′) termini of each construct. The inferred positions of P1 and P2 are labeled and depicted as dark gray rectangles. <u>Vector</u> is PGL3 basic vector; <u>SV40 pro</u> is PGL3 basic vector containing the SV40 promoter; and <u>SV40 pro+enh</u> is PGL3 basic vector containing the SV40 promoter and enhancer elements. Data are expressed as relative light units (the ratio of firefly to renilla luciferase activity) and presented as the mean and SEM of three independent experiments.</p

Effect on Mina expression of siRNA knockdown of Sp1/3.

<p>EL4 cells were transfected with GAPDH- and two different Sp1/3-specific as well as one non-specific Accell SMART pool siRNA and cultured for 72 hours, prior to real time RT-PCR analysis of relative mRNA levels of GAPDH, Sp1, Sp3 and Mina. Shown is a representative result from 3 independent experiments. Statistical significance of triplicate measurements was determined by the student’s t-test (****p<0.0001).</p

EMSA supershift analysis of Sp1/3 association with the four Sp1/3 binding sites comprising the <i>Mina</i> P1 promoter.

<p>(A) Representative EMSA assay revealing the upper and lower bands of complex 1 to contain Sp1 and Sp3, respectively, and the upper band of complex 2 to contain Sp3. Probes p1–p4 were incubated with EL4 nuclear extracts and antibodies to the indicated targets, prior to analysis by SDS-PAGE. Data are representative of 3 independent experiments. B and C) Chromatin immunoprecipitation (ChIP) analysis of Sp1 (B) and Sp3 (C) binding to the <i>Mina</i> P1 promoter region in EL4 cells. EL4 cell chromatin fragments were immunoprecipitated with antibodies against Sp1 or Sp3 (open bars) or control IgG (filled bars) and analyzed by qPCR. <u>Promoter</u> is the <i>Mina</i> P1-proximal promoter region; <u>Intron 2</u> is located in <i>Mina</i> intron 2, ∼6 kb 3′ of the <i>Mina</i> promoter. Data are from 2 (B) or 3 (C) independent experiments.</p

Electrophoretic mobility shift (EMSA) analysis of four consensus Sp1/3 binding sequences comprising the <i>Mina</i> P1 promoter.

<p>(A) Shown is the sequence of the <i>Mina</i> P1 promoter mapped to region (−64/+19) showing the TSS (bent arrow), the four consensus Sp1/3 binding sequences (boxed and shaded), and probes p1–p4 each containing a single Sp1/3 site (underlined and labeled). (B) Representative EMSA assay showing nucleoprotein complexes 1 and 2 (arrows) formed by combining nuclear extracts from EL4 cells with 5′ biotin labeled probes p1–p4 in the absence or presence of a 100-fold molar excess of unlabeled probes containing autologous (p1–p4) and heterologous (A and B corresponding to Sp1A and Sp1B, respectively, from the OX40 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080638#pone.0080638-Tone1" target="_blank">[16]</a>) Sp1/3 consensus sequences. Data are representative of 2 experiments.</p

Pharmacologic and siRNA analysis of Sp1/3 activation of <i>Mina</i> promoter activity in EL4 cells.

<p>(A–D) Mapping the key nucleotides required for Sp1/3 binding to each Sp1/3 binding site (Sp1.1–Sp1.4) in the <i>Mina</i> P1 promoter. For each site, gel shift assays were performed with wild type (WT) and mutant (M1–6) probes (comprising an overlapping set of mutations, underlined), as shown to the right of each gel shift assay image. Nucleotides critical for Sp1/3 binding are bolded. Data are representative of 2 independent experiments. (E) All four Sp1/3 binding sites act additively to activate <i>Mina</i> promoter activity. Cloned into a firefly luciferase reporter construct, the four Sp1/3 sites of a WT <i>Mina</i> P1 promoter (spanning position −64 to +151) were mutated individually or in combinations, as indicated (Xs) in the schematic to the left of the bar graph depicting reporter activity. Data are presented as the mean and SEM from 3 independent experiments. Statistical significance was determined by the student’s t-test (ns, not significant; *p<0.05; **p<0.01). <u>Vector</u> is the PGL3 basic vector; <u>SV40 Pro</u> is the PGL3 basic vector containing the SV40 promoter. (F) Effect of Sp1 inhibitor Mithramycin A on <i>Mina</i> transcript level. EL4 cells were treated for 24 hr with 0, 10 nM, 100 nM, 1 uM Mithramycin A or 1 µM DMSO carrier prior to RNA harvest and analysis of <i>Mina</i> mRNA level by quantitative RT-PCR.</p

Sp1 and Sp3 binding to the <i>Mina</i> promoter in resting CD4+ T cells.

<p>(A) The <i>Mina</i> promoter was marked as an epigenetically active region in resting CD4+ T cells. Chromatin fragments from CD4⁺CD62L^HiCD44⁻CD25⁻ T cells of C57BL/6 mice were immunoprecipitated with antibody to either H3K27me3 or H3K4me3, and then subjected to high-throughput sequencing (ChIPseq). The ChIPseq signals from both anti-H3K27me3 (upper panel) and anti-H3K4me3 (lower panel) are shown with correlations to the <i>Mina</i> locus. The depth of the peaks in the histograms corresponds to the number of reads mapped to the location. A schematic of the <i>Mina</i> locus is shown above the histograms. Each box represents an exon. The region that enriched for H3K4me3 but was devoid of H3K27me3 modifications is enlarged and shown above the <i>Mina</i> locus schematic. (B–C) ChIP assay confirmed the binding of Sp1 and Sp3 to the <i>Mina</i> promoter in EL4 cells. Chromatin fragments from CD4⁺CD62L^HiCD44⁻CD25⁻ T cells of BALB/c mice were immunoprecipitated with antibodies against Sp1 (B, open bars), Sp3 (C, open bars), or their corresponding IgG controls (B–C, filled bars). Promoter: <i>Mina</i> promoter region; Intron 2: an Intron 2 region located ∼6 kb downstream of the <i>Mina</i> promoter.</p