Declassify the world's first ATAC-seq and CUT-Tag joint application to reveal a new mechanism for tumor immune escape.

Title: the phosphotase PAC1 acts as a T cell suppressor and attenuates host antitumor immunity ❖ Journal: Nature Immunology (if: 23.53) ❖ Author and setting: Liu Liang, associate researcher and postdoctoral fellow of School of basic medicine of Peking University, is the co first author of the paper, and Yin Yuxin, Professor of the Institute of Systems Biomedicine, Peking University, is the corresponding author. On January 13, 2020, Yin Yuxin, a research group of the Institute of Systems Biomedicine of Peking University, published the title "the Phosphotase PAC1 acts as a T cell suppressor and attenuates host antagonist immunity revealed the mechanism of phosphatase PAC1 acting as a T cell suppressor and attenuates host antagonist immunity.inhibition of PAC1 pathway can activate the defense function of T lymphocytes.this study provides a potential new drug target for tumor treatment and has great significance.Figure 1: the role model of PAC1 in ROS mediated tumor infiltrating lymphocyte dysfunction. In this study, Yin Yuxin's research team systematically analyzed the inflammatory factors and metabolites in the tumor microenvironment, and found that the tumor produced superoxide ROS could maintain the high expression of PAC1 gene in peripheral infiltrating T lymphocytes.PAC1 reduces chromatin openness (ATAC SEQ test), histone acetylation, and the binding ability of chd4 to DNA (cut & amp; tag test) by recruiting nucleosome reconstruction and deacetylation (NuRD) complex, and specifically inhibits T lymphocyte defense function, resulting in T cell failure and cancer immune escape.this is the first article in the world that ATAC SEQ and cut & amp; tag are combined to study the mechanism of tumor.ATAC SEQ data can obtain the dynamic changes of chromatin openness. Combined with cut & amp; tag or chip SEQ (gold standard for DNA protein interaction research), we can further explore the key factors (including cis regulatory elements and transfer factors) regulating a certain biological process.chip SEQ experiment needs formaldehyde fixation, ultrasonic breaking, and other operations, which requires a high amount of cells. At the same time, some target proteins fixed by formaldehyde will change the antigen epitopes, resulting in antibody binding failure, and eventually lead to the failure of the experiment.as a revolutionary technology of DNA protein interaction research, cut & tag can detect the binding of target protein and chromosome in situ without formaldehyde fixation and ultrasonic fragmentation.finally, the researchers successfully obtained the data of chd4 binding to chromatin by using cut & amp; tag technology.research ideas and results 1. The researchers of gene screening first analyzed the data in the cancer genome map (TCGA), and found that PAC1 was significantly up-regulated in function depleted T lymphocytes, and the prognosis of tumor patients with high expression of PAC1 was poor.after locking the research gene, the next step is to explore whether the upregulation of PAC1 is associated with the depletion of T lymphocyte function.2. Function verification of PAC1. In order to study the function of PAC1, researchers used cell model to conduct in vitro experiments and mouse model experiments, and found that PAC1 gene has inhibitory function on T cell proliferation and effect.by further using mouse model to induce colitis related cancer, it was found that PAC1 can promote the development of inflammation related tumors.finally, using PAC1 gene knockout mice and tumor bearing mice models, the researchers found that the deletion of PAC1 enhanced the effector function of CD8 + T cells, reduced the expression of PD-1 on the surface of T cells, and promoted the host anti-tumor immune response, thus inhibiting tumor progression and metastasis.3. After obtaining the information that PAC1 is indeed related to tumor escape, the researchers studied the mechanism.using the systematic analysis of inflammatory factors and metabolites in tumor microenvironment, it was found that the tumor produced superoxide ROS can maintain the high expression of PAC1 gene in peripheral infiltrating T lymphocytes. Furthermore, ROS promoted the transcription and expression of PAC1 by activating transcription factor EGR1. in terms of regulatory mechanism, researchers found that PAC1 can remodel the chromatin openness of T cells by recruiting nucleosome remodeling and deacetylation (NuRD) complex, specifically inhibit the expression of downstream effector genes, and finally promote the formation of depleted T lymphocytes. in the study of the regulatory mechanism of PAC1, the researchers used immunoprecipitation and mass spectrometry to find that there were protein interactions between PAC1 and several components of the nucleosome deacetylation complex NuRD. the overexpression of PAC1 selectively inhibited the acetylation of histones H3 and H4. because histone acetylation can affect nucleosome configuration and chromatin accessibility, the researchers used ATAC SEQ Technology (vazyme ා td501) to study chromatin openness, and found that the loss of PAC1 increased chromatin openness. and h3k27ac (chip SEQ) data were consistent with ATAC SEQ data (see Figure 2). Figure 2: IGV view analysis of ATAC SEQ coverage of gzmB locus and peaks enrichment of h3k27ac and chd4; Tag technology (vazyme ා S603) further studied the binding spectrum of chd4, the core component of NuRD complex, with DNA. It was found that the deletion of PAC1 weakened the binding function of chd4 to T cell effector related gene sites (see Figure 2), and the enrichment of chd4 was mainly concentrated in the transcription initiation site and its attachment (see Figure 3). Fig. 3: enrichment of reads near TSS. To sum up, PAC1, as an important suppressor of T lymphocyte function, can weaken host immune surveillance function and promote tumor immune escape. inhibition of PAC1 pathway can activate the defense function of T lymphocytes. This study provides a potential new drug target for tumor immunotherapy. vazyme product support Figure 4: the ATAC SEQ experimental scheme research team used trueprep DNA library prep kit V2 for Illumina (vazyme) in the ATAC SEQ study #Td501) DNA transposase library Kit (the kit has the characteristics of high efficiency and convenience, which can capture the regulatory sequences of all active transcripts in the genome at the DNA level). By comparing the chromatin openness data of WT and Ko groups, it was found that compared with the wild-type CD8, the open chromatin data of WT and Ko groups were higher than that of wild-type CD8+ In T cells, the increase of accessible gene sites induced by PAC1 deficiency is closely related to the development of memory T cells. --- Figure 5: Cut & amp; tag experimental scheme. The team used cut & amp; tag technology to analyze the binding of chd4, the core component of NuRD complex, with DNA when studying the biological function of PAC1 in NuRD complex. by comparing the difference of the binding level of chd4 and DNA in WT and Ko groups, it was found that PAC1 knockout weakened the binding function of chd4 to T cell effector related gene sites. Compared with chip SEQ, cut & amp; tag has the advantages of high signal-to-noise ratio, low initial cell volume and simple operation. in the cut & amp; tag experiment, the team used hyperactive pa-tn5 transferase adapter complex (vazyme ා S603). --- this article is the first technical application article about cut & amp; tag published worldwide after the introduction of cut & amp; tag by nature communication. novozan always insists on serving customers with high-quality products, and hopes to join hands with more scientific research workers to reach the summit in all fields of life science. if you want to get more cut & amp; tag hard core knowledge, you can take part in novozan online lecture. Full of dry goods are ready for you to increase your knowledge at home. If you have any questions, please contact your novozan people. novozan will make unremitting efforts to provide you with better service. If you have any needs, you can call 400-600-9335 at any time. Click to read the original to apply for limited trial! Reference Lu D, Liu L, sun y, et al. The phosphotase PAC1 acts as a T cell suppressor and attenuates host antitumor immunity [J]. Nature Immunology, 2020: 1-11. Plate maker: Pinellia ternata