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  • The following are the supplementary

    2019-07-24

    The following are the supplementary data related to this article.
    Transparency document
    Introduction Complicated and differentiated multi-cellular organisms exploit the integrated binding of transcription factors (TFs). These TFs usually bind to distally cis-regulatory elements, also known as enhancers. These events spatiotemporally facilitate exact programs of the gene expression during cancer progression or development [1,2]. In the cancer progression or development, enhancers exhibit stereotypical and complicated patterns in chromatin. These modification and organization of enhancers are difference among cell types and are conserved across the same species [1,3]. These distally cis-regulatory elements are bound by multi-transcription factors and the complexes of coactivators, and are flanked by specific histone modifications. In the enhancer regions, there are the acetylation of histone H3 on lysine 27 (H3K27ac) and mono-methylation of histone H3 on lysine 4 (H3K4me1) [4,5]. Although in the cells, the histone modification markers are loyally correlated with the enhancer activity and states, their precise function on enhancer or neighbors' promoter remain unclear. Recently, the enzymes worked for writing the functional enhancer modifications have been found. The MLL/COMPASS family was identified as the histone writer for enhancer. MLL3 (also known as KMT2C) and MLL4 (also known as KMT2D) belong to the MLL/COMPASS family, and have been identified as the major H3K4 mono-methyltransferase for enhancer [6]. The MLL3 and MLL4 are the huge molecular weight proteins, and both of their C-terminals contained SET domains responsible for the H3K4me1 catalysis. Several groups found that both MLL3 and MLL4 associate with some genes' enhancers and facilitate their transcriptions. Furthermore, they recruit other coactivators, like p300, which acetylates at H3K27 [7]. It has been reported that knocking down of MLL3 and MLL4 decreased the activity of enhancer, and the H3K4me1 served as an important histone marker to label the enhancer activity [2,8]. However, the precise regulation of MLL/COMPASS family in the enhancer remains less understand. In the immune cells, multiple inhibition pathways have been reported to attenuate immune responses. The immune feedback systems mainly are ligand-receptor-binding manners. For example, the antigen-presenting Diphenylterazine (DTZ) (APCs) connect with T cells using the receptors (like programmed death 1 (PD1)) or ligands (like programmed death ligand 1(PD-L1, also known as CD274)) [9]. PD1 is belonging to a family of the CD28/CTLA-4. The interaction between PD1 and PD-L1 triggers an inhibitory signal which inhibits the activated T cells [[10], [11], [12]]. In the cancer cells, this PD1 and PD-L1 signaling inhibits immune response of anti-tumor process [[13], [14], [15]]. There are two types of T-cells, including a subset of CD4+CD25+ T cells and a subset of CD8+CD122+ T cells. Both of subsets of T cells play important roles in maintaining self-tolerance and escaping from autoimmunity [16]. Tumor cells expressing PD-L1 evade immune attacks [14,17].
    Results
    Discussion Cancer cells utilize the high expression of PD-L1 to escape T-cell-controlled immunosurveillance [14,17]. In prostate cancer, the regulation of PD-L1 still needs to be elucidated. For other regulated manner of PD-L1, CMTM6 could maintain PD-L1 in the endosome and membrane to block its degradation in the lysosome [13,15]. This is the post-transcription regulation for PD-L1. In the transcriptional level, HIF-1α and IRF-1 were identified as the major transcription factors in the promoter of PD-L1 [25,26]. To the best of our knowledge, there is no enhancer for PD-L1 in cancer cells. In this study, we found a novel enhancer region for PD-L1. The enhancer was functional because the H3K4me1 level was related to the PD-L1 mRNA level. H3K4me1 is the major marker for the enhancer. It is noted that the level of H3K4me1 is related to the activity of enhancer [5]. There are several methyltransferases in the cells. MLL family was a major group for H3K4 methylation in the enhancer region [2]. In the study, we identified that MLL3 was the major mediator of PD-L1 but not other MLLs. MLL2 slightly mediated the PD-L1 expression (Fig. 1). However, the effect was not as robust as MLL3. We also demonstrated that MLL3 bound to the enhancer and promoter of PD-L1. The luciferase assay showed that only promoter had less transcript activity than enhancer plus promoter. The global Hi-C and 4C seq databases demonstrated that the new enhancer had interaction with PD-L1 promoter in liver tissues, MCF7 and LNCaP cells. Our 3C data also demonstrated that the new enhancer had interaction with PD-L1 promoter in PC3 cell. Therefore, we defined a new enhancer and a regulator for PD-L1, thus extending the understanding of regulation for immune evasion.