Myocyte Enhancer Factor 2 Activates Promoter Sequences of the Human AβH-J-J Locus, Encoding Aspartyl-β-Hydroxylase, Junctin, and Junctate

Abstract

Alternative splicing of the locus A␤H-J-J generates three functionally distinct proteins: an enzyme, A␤H (aspartyl-␤-hydroxylase), a structural protein of the sarcoplasmic reticulum membrane (junctin), and an integral membrane calcium binding protein (junctate). Junctin and junctate are two important proteins involved in calcium regulation in eukaryotic cells. To understand the regulation of these two proteins, we identified and functionally characterized one of the two promoter sequences of the A␤H-J-J locus. We demonstrate that the P2 promoter of the A␤H-J-J locus contains (i) a minimal sequence localized within a region ؊159 bp from the transcription initiation site, which is sufficient to activate transcription of both mRNAs; (ii) sequences which bind known transcriptional factors such as those belonging to the myocyte enhancer factor 2 (MEF-2), MEF-3, and NF-B protein families; and (iii) sequences bound by unknown proteins. The functional characterization of the minimal promoter in C2C12 cells and in the rat soleus muscle in vivo model indicates the existence of cis elements having positive and negative effects on transcription. In addition, our data demonstrate that in striated muscle cells the calcium-dependent transcription factor MEF-2 is crucial for the transcription activity directed by the P2 promoter. The transcription directed by the A␤H-J-J P2 promoter is induced by high expression of MEF-2, further stimulated by calcineurin and Ca2 ؉ /calmodulindependent protein kinase I, and inhibited by histone deacetylase 4.

Transcriptional activity and Sp 1/3 transcription factor binding to the P1 promoter sequences of the human AβH‐J‐J locus

“…2B). In contrast, we have previously shown that the expression directed by the P2 promoter is tissue specific, as a high level of transcription is present, in particular, in skeletal muscle, cardiac muscle, and brain [2,13]. The P1 promoter directs transcription also in RD, HepG2, human breast cancer (MCF7), human cervix epithelial carcinoma (HeLa) and human embryonic kidney (Hek293) tumor cell lines (Fig.…”

“…(C) AβH‐J‐J P1 and P2 promoter activity in the RD cell line. Cells were transiently transfected with the reporter constructs that present the maximal transcriptional activity of each promoter [13].…”

“…It should be underlined that the P1 promoter sequences do not share homology regions and signals for transcription factors with the P2 promoter. For instance, no MEF‐2‐binding sites are present within the P1 promoter; this finding is of interest, when related to the different tissue specificities of these two promoters [1,13,16].…”

“…6D, lanes 28–33), but not by J mer or the unrelated oligonucleotide MyD mer (Fig. 6D, lanes 34 and 35) [13] .…”

“…No Sp1‐binding sites are present in the P2 promoter. In contrast, the P1 promoter directs the expression of AAH and humbug in many tissues and contains several functionally active Sp1‐binding sites [1,2,13]. The finding that the sequences present in the upstream P1 promoter are significantly different from those of the P2 promoter is, in our opinion, of great interest.…”