Effects Of Acetylation Of HNF1? On Its Biological Function

HNF1? is a protein of the POU family and is an important transcription factor in the liver.HNFla is involved in regulating glucose,amino acid and other metabolic processes.HNF1? interacts with a variety of proteins and regulates transcription,protein synthesis,glucose metabolism,and other biological processes with different protein complexes.The lack of HNF1? secretion or mutations lead to MODY3.MODY3 is a disease caused only by HNF1? mutations.Therefore,exploring the regulation of metabolism of HNF1? has great significance for the diagnosis and treatment of MODY3.We have already tested the post-transcriptional modification of HNF1? by IP-MS.We found HNF1? is phosphorylation at Serine 249,and may be acetylated at Lysine 117.It has been found mutation of HNF1?Lys117 in clinic MODY3.To verify the acetylation of HNF1? at Lys117,we treated cells with the Sirt family inhibitor NAM and HDAC family inhibitor TSA,and we found the acetylation level increased in cells treated.So HNF1? is acetylated.When we mutated Lys117 of HNF1?,the acetylation level was not increased in cells treated,so that HNF1? is acetylated at Lys117.Then we tested the transcriptional activity of HNF1?.HNF1? transcriptional activity is increased when cells was NAM/TSA treated,co-transfected EP300.And when HNF1?Lys117 was mutated,HNF1? transcriptional activity was down-regulated without the effects of inhibitors.EP300(DN)could also turned HNF1? transcriptional activity down.After treated with NAM/TSA,Real-Time PCR shows that the expression of target genes of HNFla was up-regulated.This suggests that HNF1? transcriptional activity may increase when acetylation was enhanced.EMSA and ChIP show that HNFla DNA binding abilities increased after NAM/TSA treated.Co-IP results show that the dimerization was less when HNF1?Lys117 mutated.These results suggest that HNF1? transcriptional activity changes by DNA binding reducing affected by acetylation.We built HNF1?K117E-Knockin mice model to investigate the function of Lys117.We tested organ index and blood chemistry,there was no significance between Knock-in mice and wild type mice.Liver,kidney,intestine were normal.Knock-in mice body weight was lower than wild type mice after 40 weeks.Knock-in mice had lower insulin levels and liver glycogen levels than wild-type mice.Knock-in mice were normal in glucose tolerance and pyruvate tolerance,but knock-in mice blood glucose decreased faster in insulin tolerant than wild type mice.These results suggest that Lys117 may play an important role in liver glycogen synthesis,insulin secretion and sensation.To test the effect of phosphorylation of the HNF1?Ser249,we build an overexpression HNF1?S S249A transgenic mouse model.We tested the levels of plasma glucose,insulin,triglycerides,and non-esterified fatty acids in the model,and found that insulin levels in S249A mice were significantly lower than wild type and triglyceride levels were significantly higher than wild type.At the same time,we also detected the expression of target genes of HNF1? in this model.HNF1?-S249A transgenic mice were significantly down-regulated in G6pc,Gyk,Glut2 and other glucose metabolism and glucose transport genes expression.HNF1?-S249A transgenic mice showed some abnormalities in glucose metabolism and hepatic lipid metabolism.These results show both HNF1? phosphorylation and acetylation have much effect on glucose mentalism by different ways.We need to further explored the interaction between HNF1? and MODY3.