Meis1a suppresses Oct4 expression.

<p>(<b>A</b>) Reduction of Oct4 by ectopic expression of <i>Meis1a</i> RNA. P19 cells were transfected with the vacant vector or pcDNA3-EF1-α-<i>Oct4</i> and after 24 h, these aggregated cells were treated with RA for 0 and 6 h. Thereafter, expression levels of Oct4 and Meis1a proteins were analyzed by WB with anti-Oct4 and anti-Meis1 antibodies. (<b>B</b>) The suppressive effect of Meis1a on <i>Oct4</i> promoter activity. P19 cells were co-transfected with <i>Oct4</i>(−1059)-Luc and various amounts of pcDNA3-EF1-α-<i>Meis1a</i>. After 24 h, luciferase activities and Meis1a expression levels were analyzed. *<i>p</i><0.001 significantly different from vacant vector-introduced cells. <i>n = </i>3. (<b>C</b>) Schematic presentation of <i>Oct4</i> promoter-introduced luciferase reporters. <i>Oct4</i>(−1059)-Luc has four putative Meis1-BEs. (<b>D</b>) Functional analysis of Meis1-BEs. P19 cells were transfected with <i>Oct4</i>(−1059)-Luc, <i>Oct4</i>(−698)-Luc, <i>Oct4</i>(−506)-Luc, or <i>Oct4</i>(−254)-Luc together with 60 ng pcDNA3-EF1-α-<i>Meis1a</i>. After 24 h, luciferase activities were analyzed. *<i>p</i><0.001 significantly different from <i>Oct4</i>(−698)-Luc, <i>Oct4</i>(−506)-Luc, and <i>Oct4</i>(−254) Luc. <i>n = </i>3. (<b>E</b>) Occupation of the <i>Oct4</i> promoter by Meis1a and HDAC1. Aggregated P19 cells were treated with RA for 0 and 6 h. Genomic chromatin fragments were immunoprecipitated with anti-Oct4 and anti-HDAC1 antibodies and DNAs were extracted. PCR was carried out using the primer set covering the −1062 to −778 region of the <i>Oct4</i> promoter. Aliquots of 10% antibody-untreated DNA samples were used for input DNA. (<b>F</b>) Quantification of the <i>Oct4</i> promoter-bound Meis1a and HDAC1 indicated in <b>E</b>. (<b>G</b>) Schematic presentation of the adjacent Meis1-BEs3/4 region. In this region, putative Pbx- and Hox-BEs also existed.