Housekeeping promoter 5’pcmah-2 of pig CMP-N-acetylneuraminic acid hydroxylase gene for NeuGc expression

Abstract

In the present study, we isolated pCMAH house-keeping promoter regions (Ph), which are responsible for transcriptional regulation and which are located upstream of the alternative transcript pcmah-2. Luciferase reporter assays using serial construction of each deleted promoter demonstrated that the Ph promoter was highly active in pig-derived kidney PK15. Ph promoter of pcmah lacked a TATA box, but contained three putative Sp1 binding sites. Mutations of these Sp1 binding sites always resulted in the reduction of luciferase activities in Ph-334. In addition, treatment with mithramycin A (25-100 nM) decreased the luciferase activities of the Ph promoters and NeuGc expression in a dose-dependent manner. Electrophoretic mobility shift assay analysis revealed that the probes containing each Sp1 binding site bound to Sp1. Taken together, the results indicate that Sp1 bind to their putative binding sites on the Ph promoter regions of pcmah and positively regulate the promoter activity in pig kidney cells. Interspecies comparison of 5'UTRs and 5'flanking regions shows high homology between pig and cattle, and Sp1 binding sites existing in genomic regions corresponding Ph region are evolutionally conserved.

Gram-Negative Bacterial Endotoxin LPS Induces NeuGc Loss through Ets1-Dependent Downregulation of Intestine-Specific pcmah Transcript in Porcine Intestinal Cells

“…Previously, we demonstrated that the pig cmah gene has two distinct 5′ alternative splicing forms, 5′pcmah-1 and 5′pcmah-2 , which exhibit intestine-specific and housekeeping expression, respectively [ 14 ] ( Figure 2 a). Given our previous finding that 5′pcmah-1 and 5′pcmah-2 encode an identical pCMAH protein [ 15 ] ( Figure 2 a), we questioned which transcript is responsible for the loss of CMAH protein following LPS treatment in IPI-2I cells.…”

“…We next attempted to elucidate the underlying mechanism responsible for transcriptional loss of 5′pcmah-1 following LPS exposure. Since 5′pcmah-1 is mainly controlled by the Pi (intestine-specific) promoter [ 14 , 16 ], we examined the effects of LPS treatment on Pi promoter activity using serially constructed 5′-deletion mutants, Pi-700, Pi-542, Pi-260 and Pi-233. The activity of Pi-542 and Pi-700 was markedly decreased upon LPS treatment in IPI-2I cells, while there was no alteration in the activity of Pi-260 or Pi-230 after LPS treatment ( Figure 2 c).…”

“…Neu5Gc levels were measured with an ELISA assay kit using chicken polyclonal IgY (BioLegend, Sandiego, CA, USA), as described previously [ 14 ].…”

“…The DNA constructs corresponding to the intestine-specific (Pi) promoter of the pig CMAH gene have been described previously [ 14 , 16 ]. Site-directed mutagenesis was performed using a QuickChange XL Site-directed Mutagenesis kit (Stratagene, San Diego, CA, USA) according to the manufacturer’s instructions.…”

“…Previously, we demonstrated that the pig CMAH ( pcmah ) gene has two distinct 5′ alternative splicing forms, 5′pcmah-1 and 5′pcmah-2 , which exhibit intestine-specific and housekeeping expression, respectively [ 14 , 15 ]. In an effort to elucidate the regulatory mechanisms relevant to these pcmah transcripts, we identified two distinct promoters, intestine-specific Pi and housekeeping Ph, which are responsible for the expression of 5′pcmah-1 and 5′pcmah-2 , respectively [ 14 , 16 ]. Furthermore, our previous study established that the transcription factor Ets1 is necessary for intestine-specific activity of the Pi promoter [ 16 ].…”