Effects of super-enhancers in cancer metastasis: mechanisms and therapeutic targets.

Metastasis remains the principal cause of cancer-related lethality despite advancements in cancer treatment. Dysfunctional epigenetic alterations are crucial in the metastatic cascade. Among these, super-enhancers (SEs), emerging as new epigenetic regulators, consist of large clusters of regulatory elements that drive the high-level expression of genes essential for the oncogenic process, upon which cancer cells develop a profound dependency. These SE-driven oncogenes play an important role in regulating various facets of metastasis, including the promotion of tumor proliferation in primary and distal metastatic organs, facilitating cellular migration and invasion into the vasculature, triggering epithelial-mesenchymal transition, enhancing cancer stem cell-like properties, circumventing immune detection, and adapting to the heterogeneity of metastatic niches. This heavy reliance on SE-mediated transcription delineates a vulnerable target for therapeutic intervention in cancer cells. In this article, we review current insights into the characteristics, identification methodologies, formation, and activation mechanisms of SEs. We also elaborate the oncogenic roles and regulatory functions of SEs in the context of cancer metastasis. Ultimately, we discuss the potential of SEs as novel therapeutic targets and their implications in clinical oncology, offering insights into future directions for innovative cancer treatment strategies.

Pervasive structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs.

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.

LINC00887 Acts as an Enhancer RNA to Promote Medullary Thyroid Carcinoma Progression by Binding with FOXQ1.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare but aggressive endocrine malignancy that originates from the parafollicular C cells of the thyroid gland. Enhancer RNAs (eRNAs) are non-coding RNAs transcribed from enhancer regions, which are critical regulators of tumorigenesis. However, the roles and regulatory mechanisms of eRNAs in MTC remain poorly understood. This study aims to identify key eRNAs regulating the malignant phenotype of MTC and to uncover transcription factors involved in the regulation of key eRNAs. METHODS: GSE32662 and GSE114068 were used for the identification of differentially expressed genes, eRNAs, enhancers and enhancer-regulated genes in MTC. Metascape and the transcription factor affinity prediction method were used for gene function enrichment and transcription factor prediction, respectively. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to assess the binding of histone H3 lysine 27 acetylation (H3K27ac)-enriched regions to anti- H3K27ac. RIP-qPCR was used to detect the binding between FOXQ1 and LINC00887. CCK8 and Transwell were performed to measure the proliferation and invasion of MTC cells, respectively. Intracellular reactive oxygen species (ROS) levels were quantified using a ROS assay kit. RESULTS: Four eRNAs (H1FX-AS1, LINC00887, MCM3AP-AS1 and A1BG-AS1) were screened, among which LINC00887 was the key eRNA promoting the proliferation and invasion of MTC cells. A total of 135 genes controlled by LINC00887-regulated enhancers were identified; among them, BCL2, PRDX1, SFTPD, TPO, GSS, RAD52, ZNF580, and ZFP36L1 were significantly enriched in the "ROS metabolic process" term. As a transcription factor regulating genes enriched in the "ROS metabolic process" term, FOXQ1 could recruit LINC00887. Overexpression of FOXQ1 restored LINC00887 knockdown-induced downregulation of GSS and ZFP36L1 transcription in MTC cells. Additionally, FOXQ1 overexpression counteracted the inhibitory effects of LINC00887 knockdown on the proliferation and invasion of MTC cells and the promotion of intracellular ROS accumulation induced by LINC00887 knockdown. CONCLUSION: LINC00887 was identified as a key eRNA promoting the malignant phenotype of MTC cells. The involvement of FOXQ1 was essential for LINC00887 to play a pro-tumorigenic role in MTC. Our findings suggest that the FOXQ1/LINC00887 axis is a potential therapeutic target for MTC.

CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex.

CUX1 is a homeodomain-containing transcription factor that is essential for the development and differentiation of multiple tissues. CUX1 is recurrently mutated or deleted in cancer, particularly in myeloid malignancies. However, the mechanism by which CUX1 regulates gene expression and differentiation remains poorly understood, creating a barrier to understanding the tumor-suppressive functions of CUX1. Here, we demonstrate that CUX1 directs the BAF chromatin remodeling complex to DNA to increase chromatin accessibility in hematopoietic cells. CUX1 preferentially regulates lineage-specific enhancers, and CUX1 target genes are predictive of cell fate in vivo. These data indicate that CUX1 regulates hematopoietic lineage commitment and homeostasis via pioneer factor activity, and CUX1 deficiency disrupts these processes in stem and progenitor cells, facilitating transformation.

Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.

MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.

Identification of drug responsive enhancers by predicting chromatin accessibility change from perturbed gene expression profiles.

Individual may response to drug treatment differently due to their genetic variants located in enhancers. These variants can alter transcription factor's (TF) binding strength, affect enhancer's chromatin activity or interaction, and eventually change expression level of downstream gene. Here, we propose a computational framework, PERD, to Predict the Enhancers Responsive to Drug. A machine learning model was trained to predict the genome-wide chromatin accessibility from transcriptome data using the paired expression and chromatin accessibility data collected from ENCODE and ROADMAP. Then the model was applied to the perturbed gene expression data from Connectivity Map (CMAP) and Cancer Drug-induced gene expression Signature DataBase (CDS-DB) and identify drug responsive enhancers with significantly altered chromatin accessibility. Furthermore, the drug responsive enhancers were related to the pharmacogenomics genome-wide association studies (PGx GWAS). Stepping on the traditional drug-associated gene signatures, PERD holds the promise to enhance the causality of drug perturbation by providing candidate regulatory element of those drug associated genes.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and spatial proximity.

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.

Estrogen receptor 1 chromatin profiling in human breast tumors reveals high inter-patient heterogeneity with enrichment of risk SNPs and enhancer activity at most-conserved regions.

Estrogen Receptor 1 (ESR1; also known as ERα, encoded by ESR1 gene) is the main driver and prime drug target in luminal breast cancer. ESR1 chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ESR1 chromatin action, along with its biological implications. Here, we use a large set of ESR1 ChIP-seq data from 70 ESR1+ breast cancers to explore inter-patient heterogeneity in ESR1 DNA binding to reveal a striking inter-tumor heterogeneity of ESR1 action. Of note, commonly shared ESR1 sites show the highest estrogen-driven enhancer activity and are most engaged in long-range chromatin interactions. In addition, the most commonly shared ESR1-occupied enhancers are enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ESR1 and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we can confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ESR1-chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ESR1 landscape, with the most common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.

Aberrant Super-Enhancer Landscape in Enzalutamide-Resistant Prostate Cancer Cells.

Background: Castration-resistant prostate cancer (CRPC), which has developed resistance to next-generation antiandrogens, such as enzalutamide (Enz), is a lethal disease. Furthermore, transcriptional regulation by super enhancers (SEs) is crucial for the growth and spread of prostate cancer, as well as drug resistance. The functions of SEs, a significant class of noncoding DNA cis-regulatory elements, have been the subject of numerous recent studies in the field of cancer research. Materials and Methods: The goal of this research was to identify SEs associated with Enz resistance in C4-2B cells using chromatin immunoprecipitation sequencing and cleavage under targets and tagmentation (CUT&Tag). Using HOMER analysis to predict protein/gene-binding motifs, we identified master transcription factors (TFs) that may bind to SE sites. Using small interfering RNA, WST-1 assays, and qRT-PCR, we then confirmed the associations between TFs of SEs and Enz resistance. Results: A total of 999 SEs were screened from C4-2B EnzR cells in total. Incorporating analysis with RNA-seq data revealed 41 SEs to be strongly associated with the promotion of Enz resistance. In addition, we finally predicted that master TFs bind to SE-binding regions. Subsequently, we selected zinc finger protein 467 (ZFP467) and SMAD family member 3 to confirm the functional connections of master TFs with Enz resistance through SEs (ZNF467). Conclusions: In this study, SMAD3 and ZNF467 were found to be closely related to Enz-resistant CRPC. Our research uncovered a sizable group of SEs linked to Enz resistance in prostate cancer, dissected the mechanisms underlying SE Enz resistance, and shed light on potential clinical uses for SEs.

Identification and genetic validation of leukemia inhibitory factor super-enhancers in acute respiratory distress syndrome and lung cancer.

Super-enhancers play prominent roles in driving robust pathological gene expression, but they are hidden in human genome at noncoding regions, making them difficult to explore. Leukemia inhibitory factor (LIF) is a multifunctional cytokine crucially involved in acute respiratory distress syndrome (ARDS) and lung cancer progression. However, the mechanisms governing LIF regulation in disease contexts remain largely unexplored. In this study, we observed elevated levels of LIF in the bronchoalveolar lavage fluid (BALF) of patients with sepsis-related ARDS compared to those with nonsepsis-related ARDS. Furthermore, both basal and LPS-induced LIF expression were under the control of super-enhancers. Through analysis of H3K27Ac ChIP-seq data, we pinpointed three potential super-enhancers (LIF-SE1, LIF-SE2, and LIF-SE3) located proximal to the LIF gene in cells. Notably, genetic deletion of any of these three super-enhancers using CRISPR-Cas9 technology led to a significant reduction in LIF expression. Moreover, in cells lacking these super-enhancers, both cell growth and invasion capabilities were substantially impaired. Our findings highlight the critical role of three specific super-enhancers in regulating LIF expression and offer new insights into the transcriptional regulation of LIF in ARDS and lung cancer.

Copper-binding proteins and exonic splicing enhancers and silencers.

Eukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of constitutively or alternatively spliced exons in mature transcripts. This "auxiliary" splicing code has been characterized as exonic splicing enhancers and silencers (ESE and ESS). The exact interplay between protein and splicing codes is, however, poorly understood. Here, we show that exons encoding copper-coordinating amino acids in human cuproproteins lack ESEs and/or have an excess of ESSs, yet RNA sequencing and expressed sequence tags data show that they are more efficiently included in mature transcripts by the splicing machinery than average exons. Their largely constitutive inclusion in messenger RNA is facilitated by stronger splice sites, including polypyrimidine tracts, consistent with an important role of the surrounding intron architecture in ensuring high expression of metal-binding residues during evolution. ESE/ESS profiles of codons and entire exons that code for copper-coordinating residues were very similar to those encoding residues that coordinate zinc but markedly different from those that coordinate calcium. Together, these results reveal how the traditional and auxiliary splicing motifs responded to constraints of metal coordination in proteins.

TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene.

Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors: Kit, Kdr, Pdgfra. We used genome editing in mice to delete the TADs boundaries at the Kit locus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that although Kit is highly active in both mast cells and melanocytes, deletion of the TAD boundary between the Kit and Kdr genes results in ectopic activation only in melanocytes. Thus, the epigenetic landscape, namely the mutual arrangement of enhancers and actively transcribing genes, is important for predicting the consequences of the TAD boundaries removal. We also found that mice without a TAD border between the Kit and Kdr genes have a phenotypic manifestation of the mutation - a lighter coloration. Thus, the data obtained shed light on the principles of interaction between the 3D chromatin organization and epigenetic marks in the regulation of gene activity.

Super enhancer lncRNAs: a novel hallmark in cancer.

Super enhancers (SEs) consist of clusters of enhancers, harboring an unusually high density of transcription factors, mediator coactivators and epigenetic modifications. SEs play a crucial role in the maintenance of cancer cell identity and promoting oncogenic transcription. Super enhancer lncRNAs (SE-lncRNAs) refer to either transcript from SEs locus or interact with SEs, whose transcriptional activity is highly dependent on SEs. Moreover, these SE-lncRNAs can interact with their associated enhancer regions in cis and modulate the expression of oncogenes or key signal pathways in cancers. Inhibition of SEs would be a promising therapy for cancer. In this review, we summarize the research of SE-lncRNAs in different kinds of cancers so far and decode the mechanism of SE-lncRNAs in carcinogenesis to provide novel ideas for the cancer therapy.

Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD).

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Cis-regulatory control of transcriptional timing and noise in response to estrogen.

Cis-regulatory elements control transcription levels, temporal dynamics, and cell-cell variation or transcriptional noise. However, the combination of regulatory features that control these different attributes is not fully understood. Here, we used single-cell RNA-seq during an estrogen treatment time course and machine learning to identify predictors of expression timing and noise. We found that genes with multiple active enhancers exhibit faster temporal responses. We verified this finding by showing that manipulation of enhancer activity changes the temporal response of estrogen target genes. Analysis of transcriptional noise uncovered a relationship between promoter and enhancer activity, with active promoters associated with low noise and active enhancers linked to high noise. Finally, we observed that co-expression across single cells is an emergent property associated with chromatin looping, timing, and noise. Overall, our results indicate a fundamental tradeoff between a gene's ability to quickly respond to incoming signals and maintain low variation across cells.

CDK5RAP3 is a novel super-enhancer-driven gene activated by master TFs and regulates ER-Phagy in neuroblastoma.

Super enhancers (SEs) are genomic regions comprising multiple closely spaced enhancers, typically occupied by a high density of cell-type-specific master transcription factors (TFs) and frequently enriched in key oncogenes in various tumors, including neuroblastoma (NB), one of the most prevalent malignant solid tumors in children originating from the neural crest. Cyclin-dependent kinase 5 regulatory subunit-associated protein 3 (CDK5RAP3) is a newly identified super-enhancer-driven gene regulated by master TFs in NB; however, its function in NB remains unclear. Through an integrated study of publicly available datasets and microarrays, we observed a significantly elevated CDK5RAP3 expression level in NB, associated with poor patient prognosis. Further research demonstrated that CDK5RAP3 promotes the growth of NB cells, both in vitro and in vivo. Mechanistically, defective CDK5RAP3 interfered with the UFMylation system, thereby triggering endoplasmic reticulum (ER) phagy. Additionally, we provide evidence that CDK5RAP3 maintains the stability of MEIS2, a master TF in NB, and in turn, contributes to the high expression of CDK5RAP3. Overall, our findings shed light on the molecular mechanisms by which CDK5RAP3 promotes tumor progression and suggest that its inhibition may represent a novel therapeutic strategy for NB.

Allele-Specific Regulation of the Candidate Autism Liability Gene RAI1 by the Enhancer Variant rs4925102 (C/G).

Retinoic acid-induced 1 (RAI1) is a dosage-sensitive gene that causes autistic phenotypes when deleted or duplicated. Observations from clinical cases and animal models also suggest that changes of RAI1 expression levels contribute to autism. Previously, we used a bioinformatic approach to identify several single nucleotide polymorphisms (SNPs) located within the 5'-region of RAI1 that correlate with RAI1 mRNA expression in the human brain. In particular, the SNP rs4925102 was identified as a candidate cis-acting regulatory variant, the genotype of which may affect the binding of transcription factors that influence RAI1 mRNA expression. In this study, we provide experimental evidence based on reporter gene, chromatin immunoprecipitation (ChIP), and chromatin conformation capture (3C) assays that rs4925102 regulates RAI1 mRNA expression in an allele-specific manner in human cell lines, including the neuroblastoma-derived cell line SH-SY5Y. We also describe a statistically significant association between rs4925102 genotype and autism spectrum disorder (ASD) diagnosis in a case-control study and near-statistically significant association in an Autism Genome Project (AGP) transmission disequilibrium (TDT) study using Caucasian subjects.

Emergence of enhancers at late DNA replicating regions.

Enhancers are fast-evolving genomic sequences that control spatiotemporal gene expression patterns. By examining enhancer turnover across mammalian species and in multiple tissue types, we uncover a relationship between the emergence of enhancers and genome organization as a function of germline DNA replication time. While enhancers are most abundant in euchromatic regions, enhancers emerge almost twice as often in late compared to early germline replicating regions, independent of transposable elements. Using a deep learning sequence model, we demonstrate that new enhancers are enriched for mutations that alter transcription factor (TF) binding. Recently evolved enhancers appear to be mostly neutrally evolving and enriched in eQTLs. They also show more tissue specificity than conserved enhancers, and the TFs that bind to these elements, as inferred by binding sequences, also show increased tissue-specific gene expression. We find a similar relationship with DNA replication time in cancer, suggesting that these observations may be time-invariant principles of genome evolution. Our work underscores that genome organization has a profound impact in shaping mammalian gene regulation.