Identification Of LPL Gene Enhancer And Its Function In Fat Deposition

Adipose tissue is the main source of energy storage and has a significant impact on overall metabolic homeostasis.It mainly maintains energy balance in animals through the synthesis and decomposition of triglycerides.It can maintain energy balance in animals through the synthesis and decomposition of triglycerides.As a complex enzyme for fat breakdown,lipoprotein lipase(LPL)plays an important role in the metabolism and transportation of triglycerides.In agricultural animals,LPL is significantly associated with important economic traits such as backfat thickness and meat quality in livestock such as pigs and sheep.LPL can regulate the levels of nutrients required during the development of adipose and muscle tissues,thereby affecting carcass traits such as eye muscle area,lean meat percentage,and fat ratio.Therefore,identifying the important regulatory elements of LPL will provide new targets for pig molecular breeding.In this experiment,the cyclic chromatin conformation capture sequencing(4C-Seq)technique was used to draw the interaction map of LPL gene before and after adipogenic differentiation of preadipocytes.Combined with chromatin immune coprecipitation(CHIP seq)technique,it was found that the 200 kb intergenic region interacting with the promoter of lipoprotein lipase LPL gene was related to the expression of lipoprotein lipase LPL gene.The two active enhancer LPL-E1 and LPL-E2 of LPL gene were identified,revealing that PPARG and RXRA have a significant impact on the activity of LPL-E1 and LPL-E2.The main results are as follows:(1)The three-dimensional chromatin interaction map of LPL gene before and after3T3-L1 lipogenic differentiation was constructed using 4C seq technology.Pearson correlation coefficient between technical repetitions of 4C seq library was greater than 0.4.A total of 51 interaction sites were identified in the pre differentiation library,while 17 interaction sites were identified in the post differentiation library.(2)Compared to the pre differentiation library,the post differentiation library exhibited a higher proportion of trans interactions(pre differentiation: 58.4%;post differentiation: 62.55%).The differences in LPL gene interaction sites before and after adipogenic differentiation were compared using DESeq2 software.Compared to the pre differentiation samples,there were 34 significantly upregulated interaction sites and 2280 significantly downregulated interaction sites in the post differentiation samples,indicating significant changes in LPL gene interaction before and after differentiation.(3)Combining the Chi IP seq data of histones H3K4me1 and H3K27me3 after 3T3-L1 differentiation,eight potential active enhancer(LPL-E1-E8)of LPL gene were identified in the 200 kb intergenic region(chr8: 68840513 – 69040028),which showed significant enrichment of H3K27 Ac histones.The activity of eight potential enhancer was evaluated using the dual Luciferase reporting system.Compared with the control vector,the two enhancer LPL-E1 and LPL-E2 significantly increased Luciferase activity.Among them,LPL-E1 increased by 1.74 times(P < 0.01),and LPL-E2 increased by 1.78 times(P <0.01).(4)In order to clarify the effect of LPL gene enhancer activity change on adipogenic differentiation ability of adipose precursor somatic cell cells,three groups(CRISPRi-E1,CRISPRi-E2 and CRISPRi Control)were set up in this study.The results at 7 days after adipogenic differentiation showed that compared with the control group,the expression of LPL gene and triglyceride content in 3T3-L1 cells were significantly reduced(P < 0.01).The RNA Seq results indicate that,the genes downregulated by CRISPRi-E1 and CRISPRi-E2 were mainly enriched in functional items such as monocarboxylic acid metabolism,fatty acid metabolism,adipocyte differentiation,neutral lipid metabolism,and triglyceride metabolism compared to CRISPRi-Control.(5)The transcription factor(TF)motif enrichment analysis found that LPL-E1 and LPL-E2 enhancer showed significant enrichment of PPARG and RXRA motif.Combined with the public Chi IP seq(PPARG,RXRA)dataset,the results showed that PPARG and RXRA were significantly enriched in the LPL-E1 and LPL-E2 enhancer regions.The constructed LPL-E1 and LPL-E2 double Luciferase report vector,PPARG and RXRA overexpression vector and p RL-TK plasmid were co transfected into H293 T cells to measure Luciferase activity.The experimental results on Luciferase report showed that PPARG and RXRA overexpression could significantly increase the post Luciferase activity of LPL-E1 and LPL-E2 double fluorescent vector.This study drew a high-resolution whole genome interaction map of the LPL gene before and after adipogenic differentiation;The transcriptional activity of LPL gene enhancer was identified and evaluated;Clarified the functions of LPL-E1 and LPL-E2 in adipogenic differentiation;Analyzed the regulatory effects of two important lipogenic transcription factors(PPARG and RXRA)on the activity of LPL-E1 and LPL-E2.In conclusion,this study provides a theoretical reference for clarifying the transcriptional regulation of chromatin interaction on adipogenic differentiation genes.