Ectopic expression of Id1 or Id3 inhibits transcription of the <i>GATA-4</i> gene in P19CL6 cells under differentiation condition

Abstract

Inhibitor of DNA binding, GATA transcription factor, cardiomyocyte differentiation, transcription Inhibitor of DNA binding (Id) is a dominant negative form of the E-box binding basic-helixloop-helix (bHLH) transcription factor since it is devoid of the basic region required for DNA binding and forms an inactive hetero dimer with bHLH proteins. The E-box sequence located in the promoter region of the GATA-binding protein 4 (GATA-4) gene is essential for transcriptional activation in P19CL6 cells. These cells differentiate into cardiomyocytes and start to express GATA-4, which further triggers cardiac-specific gene expression. In this study, expression plasmids for Ids tagged with human influenza hemagglutinin (HA)-FLAG were constructed and introduced into P19CL6 cells. The stable clones expressing the recombinant Id proteins (Id1 or Id3) were isolated. The GATA-4 gene expression in these clones under differentiation condition in the presence of 1% dimethyl sulfoxide (DMSO) was repressed, with concomitant abolishment of the transcription of α-myosin heavy chain (α-MHC), which is a component of cardiac myofibrils. Thus, the increased expression of Id protein could affect GATA-4 gene expression and negatively regulate the differentiation of P19CL6 cells.

Cyclic AMP (cAMP)-dependent proteolysis of GATA6 by proteasome: Zinc-finger domain of GATA6 has signals for nuclear export and proteolysis, both of which are responsive to cAMP

“…The sequence of the cloned DNA was determined by the dideoxy chain-termination method with sequence primers for the pME18S vector (16) and a BigDye TM terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA), using an ABI PRISM TM 310 Genetic Analyzer (Applied Biosystems). The molecular biological methods for DNA manipulations were based on standard procedures as described in our previous study (17). The primers for PCR and sequencing are listed in Table S1 (http:// www.ddtjournal.com/action/getSupplementalData.…”

“…Each plasmid construct was introduced into Cos-1 cells (ATCC, Manassas, VA, USA) by means of the diethylaminoethyl (DEAE)-dextran method (17) to verify expression of the recombinant protein. Cells were grown for two days in Dulbecco's modified Eagle medium (DMEM) (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 7% fetal bovine serum (FBS) (GIBCO BRL) and antibiotics [100 units/mL benzylpenicillin (Wako, Osaka-city, Osaka, Japan), 100 μg/mL streptomycin sulfate (Wako), and 2.5 μg/mL fungison (GIBCO BRL)], and the transiently expressed proteins were detected immunologically as described in 2.3.…”

“…Chinese hamster ovary (CHO)-K1 cells (11) were grown in Ham's F12 medium (GIBCO BRL) supplemented with FBS and antibiotics as above. Each expression plasmid for GATA6 derivatives was introduced into CHO-K1 cells by means of the calciumphosphate method (17) together with phyg (17) in the ratio of 15:1 (w/w). Resistant colonies were selected in the presence of 200 μg/mL hygromycin (Wako).…”

“…A protein sample (10 μg) was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis and Western blotting (17); the proteins were electro-blotted onto a Hybond-P Polyvinylidene Difluoride (PVDF) membrane (GE Healthcare, Chicago, IL, USA). The concentration of the separation gel was 7.5% (w/v) for Myc-tagged proteins, 15% (w/v) for hZf, and 10% (w/v) for others.…”

“…The concentration of the separation gel was 7.5% (w/v) for Myc-tagged proteins, 15% (w/v) for hZf, and 10% (w/v) for others. Procedures for membrane blocking and washing were essentially the same as described previously (13,17).…”