Transcriptional regulation of human INSIG2

FERNÁNDEZ ALVAREZ, ANA JULIA; CUCARELLA, CARME; CASADO PINNA, MARTA

Baeza

Workshop; Current trends in Biomedicine Workshop: “Chomatin Domains and Insulators”; 2009

Universidad Internacional de Andalusía

Sterol regulatory-element-binding proteins (SREBPs) are transcription factors with a central role in cholesterol and fatty acid metabolism. Alterations in its functionality have been implicated in the development of several human metabolic diseases such as obesity or type 2 diabetes, which have emerged as global health crises in recent years. SREBPs are synthesized as inactive precursors embedded in ER membranes until they are transported to the Golgi apparatus where they are activated by proteolysis. The ER-to-Golgi migration of SREBPs is crucially dependent on its interaction with two other membrane proteins SCAP (SREBP cleavage–activating protein) and INSIG (insulin-induced gene), both regulated by cell sterol levels. Two INSIG isoforms are known, designated INSIG1 and INSIG2. In humans, both proteins are 59% identical but they differ in their mode of regulation. Whereas transcription of INSIG1 gene in cultured cells requires nuclear SREBPs, INSIG2 activity seems to be constitutive and does not require active SREBP. Transcription of INSIG2 in mice is controlled by two promoters that give rise to alternative mRNA transcripts named INSIG2a and INSIG2b. INSIG2b is ubiquitous but INSIG2a is exclusively expressed in liver and is down-regulated by insulin. The objective was to characterize the human INSIG2 promoter that has not been previously studied. Our results show that INSIG2a transcript is not present in human liver, rat liver or in human derived cell lines. However, this transcript is present in hamster origin CHO cell line and, as previously described, in mouse liver. Luciferase reporter assays allowed us to enclose the minimal human INSIG2 promoter to 300 bp from the transcription start site. Pointed mutations demonstrated the presence of two GGA sites required for optimal expression. Eletrophoretic mobility shift, siRNAs and ChIP assays have shown that Elk1 and Sap1a proteins are able to bind to these sites, although Sap1a is the only one active in vivo. This activation is dependent of its phosphorylation status via Ras/MAPK pathway but is independent of one of its common partners SRF. In conclusion, our data demonstrate for the first time, that members of Ets family of transcription factors are involved in the regulation of human INSIG2 expression and therefore of SREBP processing.