Oct4 activates Meis1a expression.

<p>Monolayer-cultured P19 cells were transfected with the pcDNA3-EF1-α-<i>Oct4</i> expression vector and after 24 h <i>Meis1a</i> and <i>Oct4</i> mRNAs and proteins were analyzed by RT-PCR (<b>A</b>) and WB (<b>B</b>), respectively. (<b>C</b>) Stimulatory effect of Oct4 on <i>Meis1</i> promoter activity. P19 cells were transfected with <i>Meis1</i>(−926)-Luc and various amounts of pcDNA3-EF1-α-<i>Oct4</i>. After 24 h, luciferase activities and expression levels of Oct4 were analyzed. *<i>p</i><0.001 significantly different from vacant vector introduced control cells. (<b>D</b>) Schematic presentation of <i>Meis1</i> promoter-inserted luciferase reporter vectors. <i>Meis1</i>(−926)-Luc possesses three putative Oct4-BEs. (<b>E</b>) Functional analysis of Oct4-BEs. P19 cells were transfected with indicated <i>Meis1</i>-Luc vectors and pcDNA3-EF1-α-<i>Oct4.</i> After 24 h, luciferase activities were assayed. *<i>p</i><0.001 significantly different from <i>Meis1</i>(−926)-Luc and <i>Meis1</i>(−335)-Luc. <i>n = </i>3. (<b>F</b>) Association of Oct4, HDAC1, AcH3, 5mC and 5hmC with the <i>Meis1</i> promoter. Genomic chromatin fragments from RA-treated aggregation form of P19 cells for 0 and 6 h were immunoprecipitated with the indicated antibodies and then DNAs were extracted. PCR was carried out using the primer set covering the −360 to −67 region of Meis1 promoter, in which 12 CpG sites exist. Aliquots of 10% antibody-untreated DNA samples were used for input DNA. (<b>G</b>) Quantification of the <i>Meis1</i> promoter-bound Oct4, HDAC1, AcH3, 5mC and 5hmC indicated in <b>F</b>.