As the basic structural and functional unit of organisms, the fate of cells is precisely regulated in the process of development and human diseases. Since the launch of the Human Genome Project, scientists' understanding of the mechanisms of gene regulation has changed dramatically. In the 80s of the 20th century, the first enhancers were discovered, which can increase gene expression by 200 times, and since then, enhancers have been widely studied as distal regulatory elements that cooperate with proximal promoters to control the expression of specific genes. With the development of science and technology, the study of chromatin has been extended to the 3D dimension, and the distal enhancement element transmits the activation signal to the promoter through chromosome folding, which further explains the accuracy of gene expression regulation and enriches the transcription model theory.
Originally, enhancers referred only to canonical enhancers (TEs), however, in 2013 Jakob Lovén et al. published a study in Cell (IF: 64.)5) published a paper entitled "Selective inhibition of tumor oncogenes by disruption of super-enhancers" For the first time, the concept of super-enhancers (SEs) was proposed, which are a team of multiple individual regulatory elements that work together to drive very high levels of gene expression, SEs are often regulators of cell identity genes and are associated with complex traits and genetic diseases. Although TE and SE are similar in that they carry the same components, such as transcription factors (TF), cofactors, mediator complexes, and RNA polymerase (POL) complexes, SE carries these factors at an average density of 10 times more than TE. The differences in function and structure between TE and SE are shown in Figure 1.
Fig.1 Comparison of the characteristics of SE and TE (Wang et al.)., 2023）。
SE Introduction. SE is a large cluster of cis-regulatory DNA elements that bind tightly to transcription factors and cofactors and play a key role in defining cell fate and identity. Histone markers such as H3K27AC, H3K4Me1, and the transcription cofactor P300 are commonly used to define SE. Since its discovery, SE has been the focus of a growing body of research as a biomarker to develop novel disease diagnostic tools and establish new directions for clinical** strategies. Therefore, SE can be considered as a new target for disease, and targeting SES is a promising strategy.
TE has been reported to be short, typically spanning 100 to 1000 bp DNA sequences, and containing DNA binding sites (TFBs), which are recognized by sequence-specific TF by different affinities and specificities. SE is a cluster of adjacent enhancers spanning more than 10 kb with high-fold enhancer activity that drives cell type-specific gene expression, including many TF-binding sites (e.g., OCT4, SOX2, nanoG, and KLF4 in embryonic stem cells, as well as MED1 and chromatin modifiers (P300 and BRD4)), and SE is tissue- and cell-specific, which is a major determinant of cell identity (Figure 2). Several studies have shown that even ectopic fragments of SE are able to induce high levels of reporter gene expression in vitro compared to TE.
Fig.2 Schematic diagram of the structure of SE (Yamagata et al.)., 2020）。
Most of the detection methods for SE are Chip-Seq or cut&tag to detect common factors such as major transcription factors, cofactors and BRD4 protein, or the binding sites of H3K27AC and H3K4Me1 in the genome, among which H3K27AC is the most commonly used. Currently, SE identification can be performed in a three-step approach: (1) identification of enhancers based on chip-seq or cut&tag data for cell type-specific master transcription factors;（2）12.The constituent enhancers within 5 kb are spliced together to form a single large sequence;(3) The total background of BRD4, CEBP, MED1, H3K27AC, or other transcription factors suturing enhancers normalizes the ChIP-Seq signal and sorts the remaining individual enhancers to generate a curve with a slope of 1 as a cut-off for separating Se and Te, Se is defined as the region above the curve point, and the remaining enhancer region is considered Te (Figure 3).
Fig. 3 Definition of SE by Chip-Seq data analysis (Yang et al.)., 2023）。
Information about SE in various chip-seq datasets can be found in multiple databases, such as dbsuper, sedb, seanalysis, and sea, which provide resources for studying the relationship between superenhancers, target genes, TF associations, and disease-associated SNPs (Yoshino S and Suzuki H I.)., 2022）。In addition, correlation analysis with transcriptome sequencing data can identify target genes regulated by SE.
SE and the occurrence of disease.
Since 2013, there have been more than 6,000 high-scoring articles on SE, most of which have been conducted in malignant tumors, indicating that it plays an important regulatory role in important biological processes such as malignant tumor development, cell differentiation, and immune response, and the regulated genes include protocarcinoma and tumor suppressor genes, cell identity determining genes, and key genes of inflammatory pathways (Fig. 4). There are several techniques and approaches available for the identification of SE and its function in tumors and regulatory mechanisms. First, to identify SEs, bioinformatics databases** are neededIn recent years, with the improvement of bioinformatics algorithms and high-throughput sequencing technology, the interaction between chromatin sequences can be directly analyzed by 3C, 4C, 5C, or Hi-C technology, and Se-related genes can be identifiedFurthermore, based on CRISPR Cas9 technology, it can be used to study the function of SE;In addition, the use of CRISPR gene editing technology, combined with CHIP-SEQ, ATAC-SEQ, CUT&tag, single-cell sequencing and other technologies, can also be used to further study the transcriptional regulatory elements of SE. Below, let's take a look at a few cases of SE research progress in the field of oncology published in high-scoring journals in 2023
Fig. 4 Role of SE-driven oncogenes in tumors (Wang et al.)., 2023）。
Case 1. In May 2023, Li et al. published a report in Cancer Research (IF: 112) published a research article entitled "LSD1 inhibition disrupts super-enhancer driven oncogenic transcriptional programs in castration-resistant prostate cancer". The team analyzed the gene levels in a series of castration-resistant prostate cancer (CRPC) xenograft mouse models that were sensitive to LSD1 inhibitors** by transcriptomics, and the results showed that tumor growth due to LSD1 inhibition could be attributed to a significant reduction in MYC signaling, and MYC was found to be a consistent target of LSD1. In addition, LSD1 forms a network with BRD4 and FOXA1 and is enriched in the super enhancer (Se) region exhibiting liquid-liquid phase separation. Combining LSD1 inhibitors with BET inhibitors exhibits a potent synergistic effect in disrupting the activity of multiple drivers in CRPC, thereby significantly inhibiting tumor growth. Importantly, the combination of ** has shown superior efficacy over either inhibitor alone in disrupting the newly discovered CRPC-specific SE subsets. These results provide mechanisms and insights into the co-targeting of two key epigenetic factors, and can be rapidly applied to clinical CRPC patients.
Fig.5 Co-targeting LSD1 and BRD4 synergistically disrupted the clinically identified CRPC-specific SE results (Li et al.)., 2023）。
Case 2. In July 2023, Jia et al. published a research article titled "Jun-induced Super-Enhancer RNA Forms R-loop to Promote Nasopharyngeal Carcinoma Metastasis" in Cell Death & Disease (IF:9). Through the analysis of CHIP-SEQ, GRO-SEQ, HIChip and public datasets, the team deeply analyzed the impact of SE and SE-generated RNA (Serna) on nasopharyngeal carcinoma (NPC) metastasis, introduced a novel SerNA-NPCM (a specific SerNA associated with NPC metastasis) involved in NPC metastasis, and proposed a potential molecular mechanism of NPC metastasis. Upon identification, the team found that JUN-mediated Serna-NPCM can form an R loop to regulate the chromatin loop between SE and distal NDRG1 promoters, promoting NPC transfer, and that Serna-NPCM was shown to interact with ACTA1 protein, and further, HNRNPR was identified as a protein that interacts with Serna-NPCM. That is, Serna-NPCM promotes chromatin looping by binding to HNRNPR and ACTA1 proteins, and HNRNPR proteins anchored near NDRG1 and TRIB1 promoters, and part of Serna-NPCM hybridizes to the SE region via the R loop, bringing SE closer to the promoters of NDRG1 and TriB1, thereby regulating their transcription. Eventually, the team proposed that the Serna-NPCM HNRNPR Acta1 NDRG1 signaling axis may be a new potential target for NPC**.
Fig.6 Schematic diagram of the model for regulating the expression of NDRG1 and Trib1 through the formation of the R loop by Serna-NPCM (Jia et al.)., 2023）。
Case 3. In September 2023, Antal et al. published a report in Nature Communications (IF:16.).6) published a research article entitled "A Super-Enhancer-regulated RNA-binding protein Cascade Drives Pancreatic Cancer". The team identified 876 different SEs through unbiased analysis of SEs in 16 different human pancreatic ductal adenocarcinoma (PDAC) cell lines, identifying the key role of HNRNPF, a regulator of alternative splicing, polyadenylation, and RNA stability, and its downstream regulatory gene PRMT1, in tumor growth. At the same time, the results of analysis of published human single-cell (SC) RNA-Seq data showed that HNRNPF was significantly upregulated and upregulated in PDAC cells compared to normal ducts, and its expression increased with tumor stage. Further studies have found that deletion of the HNRNPF gene in SE or knockout of the cell line can effectively slow down the growth of PDAC cells, and the KO orthotopic transplantation mouse model of HNRNPF further confirms the role of HNRNPF in tumor growth. Eventually, the team discovered a MYC, a cancer-associated gene that mutates in a variety of malignancies, coordinates the HNRNPF SE regulatory network that upregulates translation by increasing ribosomal biogenesis to maintain the transformed cancer phenotype. By dissecting a specific SE cascade, it was determined that PRMT1, one of the proteins affected by HNRNPF activation, could be used as a drug target for PDAC to intercept SE-driven malignancies, while potentially avoiding some of the severe toxicities associated with other clinical SE targets.
Fig.7 Schematic diagram of HNRNPF SE regulating tumor growth by regulating HNRNPF expression (Antal et al.)., 2023）。
Summary. The basic theories of SE biology and their functions have been reported over the past few decades. Numerous studies have shown that SE promotes overexpression of oncogenes, and that specific tumor** pathways may include alterations in SE structures and complexes. However, what we know about SE is only the tip of the iceberg. Currently, it is unclear how tumor-suppressive SEs are specifically produced or whether they contribute to tumor suppression in normal cells. Although SE inhibitors have been studied to potentially be used for certain malignancies, due to limited biological knowledge of the different members of the SE complex, the drugs created so far have only exerted a limited effect and show no signs of pharmacological success.
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