ABSTRACT
Objective:Cardiogenesis is a precise process controlled by sequential gene regulatory steps.Heart-specific transcription factors (TFs)GATA4,Nkx2.5,MEF2C and Tbx5 play critical roles in controlling heart development.But the genetic basis for the temporal expression of genes during heart development still remains unclear.Substantial studies displayed gene regulation partly attributes to histone acetylation.However,the functions of individual histone acetyltransferases (HATs)in specific developmental transcription programs are not well elucidated.p300,a histone acetyltransferase and coactivator,plays key role in many physiological processes,moreover our previous study suggests that expression of p300 is developmentally regulated during mouse cardiogenesis,but the underlying molecular mechanism remains elusive.Methods:The whole hearts from embryonic mice on embryonic days (E)10.5 and 16.5 were collected accordingly,and then total RNA was extracted.Heart-specific TFs GATA4,Nkx2.5,MEF2C and Tbx5 mRNA from mouse myocardium at differential embryonic days during cardiac development were analyzed by quantitative (Q)-PCR.The levels of histone H3 acetylation on these heart-specific TFs promoter and transcription factor binding sites of p300 to these genes were identified by chromatin immunoprecipitation assays (ChIP).Results:The present study furnishes a comparative temporal expression map of cardiac transcriptional factors,during murine heart development (E10.5 and E16.5).The mRNA levels of these genes present dynamic change.On E10.5,the expressions of GATA4 and MEF2C were remarkable lower than those on E16.5 (P
ABSTRACT
Eukaryotic DNA element called Matrix Attachment Regions (MARs) can function on regulating the structure and activity of chromosome. Traditional quantitation in vitro and indirect functional analysis can not always reflect MAR-involved physiological state. In order to study transcription regulation and make a try in methodism,? 1-antitrypsin MAR (?1-AT MAR) is cloned and incorporated into pEGFP-C1 vector. Non-MAR-containing and MAR-containing plasmids were then transfected into HEK-293 cells with LipofectamineTM 2000 respectively. Positive cell clones were assayed after 20 days of selection by G418. Semiquantitative RT-PCR and fluorescence microscope analysis show that this MAR has a positive effect on modulating nearby gene expression. Further, co-localization with newly CMV promoter and RNA polymeraseⅡ(RNAPⅡ) was detected by chromatin immunoprecipitation (ChIP), The PCR result demonstrates that more RNAPⅡwas recruited to the CMV promoter to initiate transcription in presence of MAR. ChIP can be used to confirm the MAR-mediated transcriptional activation and provide more reliable information than RT-PCR in real time. The technology is also providing a platform for our research in gene expression regulation.