Identification Of Porcine Alveolar Macrophages Enhancer And Its Mechanism

In the mechanism of porcine disease treatment,porcine alveolar macrophages3D4/21,as a kind of innate immune cells,play a very important role by secreting cytokines.Recent studies have shown that,as a cis-regulatory element,enhancers can significantly increase gene expression levels and regulate the spatiotemporal selective expression of cell-type-specific genes,and their functions in immune mechanisms have gradually been discovered and studied.Among them,the identification of enhancers is a hot and difficult point of research in this field.There are fews reports on the study of porcine enhancers and their related biological processes at the whole genome level.In this study,Poly(I:C)was used to stimulate the porcine alveolar macrophages,combined with STARR-seq technology,to identify and validate the enhancers associated with the disease infection model in pigs at the whole genome level,in order to clarify the enhancers in the disease infection model The regulatory relationship with target genes provides a theoretical basis for analyzing the mechanism of porcine viral infection.The main results of the paper are as follows:(1)An experimental platform for cell STARR-seq was constructed in this research group:through multiple experiments and optimization of the experimental process,the quality of subsequent sequencing data was ensured.In this study,the electrotransfection parameters of 3D4/21 were optimized,and improvements were made in three aspects: the type of electroporation fluid,the exponential wave,and the different voltages of the square wave.The results showed that when the electroporation fluid was DPBS,the waveform was exponential and the voltage was at200 V,3D4/21 has the highest electrotransfection efficiency and lower mortality.(2)After the completion of the construction of the STARR screening library,the library was verified from three perspectives: the detection of the length of the inserted fragments in the screening library,the detection of restriction enzyme digestion of the screening library,and the detection of the length of the DNA fragments after one-strand reverse transcription.It was detected that the fragment length and size were consistent with expectations,that is,the STARR screening library was constructed successfully.(3)In the whole genome,the enhancer elements of porcine alveolar macrophages 3D4/21 were identified: 14,677 enhancers were identified in normal cells,and there are16,542 enhancers in cells stimulated by Poly(I:C),and there are 13,255 enhancers in common,most of the identified enhancers are located in the intergenic region.(4)In the study,the correlation analysis between STARR-seq data and ChIP-seq data was carried out.The coefficients of H3K27 ac and H3K4me1 with histone modification were 0.58 and 0.48 respectively,which were positive correlations,and jointly verified the reliability of STARR-seq technology.(5)In this study,STARR-seq data and ChIA-PET data were compared and analyzed.ChIA-PET captures the interactions between chromatin based on RNAPII and CTCF proteins,maps enhancers to these interaction regions,and overlaps with IGV pairs.The data was visualized,and it was found that the enhancer sites overlap with the anchor points of these proteins,indicating that the identified enhancers play an important role in 3D4/21.(6)The expression levels of IFIT,MALAT1 and GBP genes showed an upward trend after Poly(I:C)stimulation.The signal intensity of STARR-seq enhancer,ChIP-seq apparent modification,and ChIA-PET chromatin interaction increased.These results provide a theoretical basis for the STARR-seq enhancer as a pharmacological target of Poly(I:C)and other double-stranded RNA virus analogs.