Involvement of NF‐Y in transcriptional regulation of the phospholamban gene

Abstract

To understand the transcriptional regulation of the phospholamban gene, we analyzed a 5′-upstream region of the gene. Using a series of deletion constructs, we demonstrated that the region from Ϫ96 bp to Ϫ78 bp, containing the CCAAT sequence, is essential for transcription of this gene. This region specifically bound to nuclear proteins extracted from rat hearts, and gel-shift assays using competitive oligonucleotides, antibodies and recombinant proteins showed that this region binds to the NF-YA and NF-YB, members of the CCAAT-binding nuclear protein family. This region-dependent transcription in cardiac myocytes transfected with antisense cDNAs encoding NF-YA and NF-YB was decreased to approximately 50% of that seen in cells transfected with the same sense cDNAs. We, therefore, conclude that the region from Ϫ96 bp to Ϫ78 bp plays a critical role in expression of the phospholamban gene, which is regulated by binding of the nuclear protein NF-Y.Keywords : phospholamban; transcriptional regulation ; CCAAT-binding proteins.Phospholamban (PLN), a pentameric protein composed of 52 amino acid subunits, is a major substrate for the cAMP-dependent protein kinase in sarcoplasmic reticulum (SR) of cardiac myocytes [1,2]. PLN has been found to regulate activity of cardiac SR Ca-ATPase (SERCA2) by the proteinϪprotein interaction ; the de-phosphorylated PLN functions as an inhibitory co-factor of SERCA2 and the phosphorylation of PLN relieves its inhibition for SERCA2 [3,4]. The subsequent activation of SERCA2 leads to enhanced muscle relaxation rates, thereby contributing to the inotropic response to β-adrenergic stimulation.The expression of PLN, detectable in the embryonic heart at an early stage [5,6], is restricted to cardiac, slow-twitch skeletal [7,8] and smooth muscles [9] at adult stage, and its amino acid sequences show a high degree of similarity among many species [8,10,11]. When cardiac muscles are subjected to neurohumoral and hemodynamic stresses, the amounts of PLN mRNA expressed in cardiac SR are greatly altered [12Ϫ14].In previous studies, we have demonstrated that the PLN gene, which is localized in human chromosome 6, and consists of two exons and a 5′ upstream regulatory region [10,11]. In this study, we characterized the transcriptional regulation of the PLN gene. By measuring the transcriptional activity of deleted PLN gene, we found that the region containing a CCAAT sequence is important for transcriptional activity of the PLN gene. Using gel-shift assays, we showed that this region associates with the transcriptional factors, NF-YA and NF-YB. Suppression of these two proteins in cardiac myocytes by over-expression of antisense cDNAs encoding them decreased the transcriptional activity of the PLN gene. We therefore concluded that transcription of the PLN gene is regulated by the association of this region with NF-Y. EXPERIMENTAL PROCEDURESCell culture. Neonatal rat cardiac myocytes were isolated and cultured as described previously [15]. Hearts were removed from 20 2-day-old neonatal Wistar-Kyoto ra...

Mutational screening of phospholamban gene in hypertrophic and idiopathic dilated cardiomyopathy and functional study of the PLN –42 C>G mutation

“…Two additional regions also show a marked conservation: an AT‐rich region (−217/−170) is approximately 70% conserved and the region between −169 and −134 shows 85% conservation. PLN promoter is a strong promoter, although all the transcriptional proteic factors still remain unknown [18]. Our experiments showed that the PLN −42 C<G mutation affects PLN promoter strength: PLN −42 C<G results in a 43% and 47% decrease of promoter activity compared to wild‐type PLN promoter in C6 and C2C12 cell lines, respectively.…”

“…These cell lines had been previously used in the study of the PLN promoter. C6 allows a high basal luciferase expression under the control of PLN promoter and C2C12 had been chosen for the study of PLN promoter activity in a muscular tissue without endogenous phospholamban expression [18,19]. Both C6 and C2C12 cell lines were cultured in Dulbecco's‐modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotic/antimicotic mix (Gibco.…”

ZBTB20 Regulates SERCA2a Activity and Myocardial Contractility Through Phospholamban

“…The study identifies ZBTB20 as a novel regulator of PLN expression, adding to the existing knowledge of transcriptional regulation of PLN. Previous research has demonstrated the involvement of various transcriptional factors, including glucocorticoid nuclear receptor, 17 TRα1 (thyroid hormone receptor α1), 16 NF-YA (subunit A of nuclear factor Y), and NF-YB (subunit B of nuclear factor Y), 41 in the regulation of PLN at the transcriptional level. In cardiac myocytes, the expression of PLN is significantly decreased by T 3 , a process that is facilitated by TRα1 and involves the recruitment of histone-modifying enzymes associated with transcriptional silencing.…”

Organization and Evolution of the Human Growth Hormone Receptor Gene 5′-Flanking Region*

“…Within the proximal promoter sequence for V9, we have identified a binding site for the nuclear transcription factor NF-Y. NF-Y can act on tissuerestricted promoters such as phospholamban (37) and the Na,K-adenosine triphosphatase ␣ 3 -subunit (38), even though binding sites for NF-Y (CCAAT-boxes) exist in many mammalian promoters, and NF-Y itself may be ubiquitously expressed (39). Clearly, promoter context is important in determining the specificity of gene expression (40).…”