Super-enhancer Research Articles

Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma

Multiple myeloma is a hematological malignancy arising from immunoglobulin-secreting plasma cells. It remains poorly understood how chromatin rewiring of regulatory elements contributes to tumorigenesis and therapy resistance in myeloma. Here we generate a high-resolution contact map of myeloma-associated super-enhancers by integrating H3K27ac ChIP-seq and HiChIP from myeloma cell lines, patient-derived myeloma cells and normal plasma cells. Our comprehensive transcriptomic and phenomic analyses prioritize candidate genes with biological and clinical implications in myeloma. We show that myeloma cells frequently acquire SE that transcriptionally activate an oncogene PPP1R15B, which encodes a regulatory subunit of the holophosphatase complex that dephosphorylates translation initiation factor eIF2α. Epigenetic silencing or knockdown of PPP1R15B activates pro-apoptotic eIF2α-ATF4-CHOP pathway, while inhibiting protein synthesis and immunoglobulin production. Pharmacological inhibition of PPP1R15B using Raphin1 potentiates the anti-myeloma effect of bortezomib. Our study reveals that myeloma cells are vulnerable to perturbation of PPP1R15B-dependent protein homeostasis, highlighting a promising therapeutic strategy.

Abstract We084: Deciphering the Molecular Code Underlining Postnatal Cardiomyocyte Maturation by an RNA Splicing Factor RBFox1

Mammalian hearts undergo major changes after birth during the perinatal period. While several extrinsic factors such as mechanical load, electrical stimulation, hormones, and nutrients have been implicated in this process, the intrinsic regulatory circuit governing cardiomyocyte postnatal maturation is largely unknown. Importantly, stem-cell derived cardiomyocytes commonly exhibit immaturity which is a major limiting factor for their application as disease models or cell therapy agents. Therefore, uncovering the intrinsic regulatory mechanism of mammalian cardiomyocyte maturation has significant implications in both basic cardiac biology and cell-based therapy and disease modeling. In our recent analysis of the global transcriptome transition from neonatal to adult rat hearts, we found RNA splicing among the top changed pathways. Additionally, we found that expression of RBFox1, a cardiac enriched RNA splicing regulator, was significantly increased during the postnatal transition to adolescence. We further demonstrate that RBFox1 mediated post-transcriptional regulation has a potent effect to promote neonatal and stem-cell derived cardiomyocyte maturation. RNA-seq followed by Gene Ontology enrichment analysis showed that Rbfox1 led to differentially expressed genes enriched in cardiac contraction and conduction, sarcomere organization, K + ion import and muscle filament sliding, as well as differential isoform switches enriched in cardiac contraction and sarcomere structure. An evolutionarily conserved, temporal specific super enhancer exists upstream of cardiac Rbfox1 , which is occupied by active epigenetic markers in the postnatal period. We have identified the tissue and temporal transcriptional activity of this super enhancer in vitro and in vivo . Several binding motifs of transcription factors are predicted within it and a key DNA fragment was identified to be essential for enhancer activity. A transgenic mouse strain has been established to facilitate the tracing of myocyte maturation in vivo by constructing a reporter system driven by a 4xSuperEnhancer-Rbfox1Promoter-tdTomato construct. In conclusion, we propose a mechanism that RBFox1 is an intracellular regulator with tissue- and temporal-specific expression patterns that regulates cardiac maturation by promoting alternative splicing that fine tunes the genetic code.

The BET PROTAC inhibitor GNE-987 displays anti-tumor effects by targeting super-enhancers regulated gene in osteosarcoma

BackgroundOsteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism.MethodsWe evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism.ResultsIn this study, we found that extremely low concentrations of GNE-987(2–10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development.ConclusionsThis research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.

Epigenomic landscapes during prefrontal cortex development and aging in rhesus.

The prefrontal cortex (PFC) is essential for higher-level cognitive functions. How epigenetic dynamics participates in PFC development and aging is largely unknown. Here, we profiled epigenomic landscapes of rhesus monkey PFCs from prenatal to aging stages. The dynamics of chromatin states, including higher-order chromatin structure, chromatin interaction and histone modifications are coordinated to regulate stage-specific gene transcription, participating in distinct processes of neurodevelopment. Dramatic changes of epigenetic signals occur around the birth stage. Notably, genes involved in neuronal cell differentiation and layer specification are pre-configured by bivalent promoters. We identified a cis-regulatory module and the transcription factors (TFs) associated with basal radial glia development, which was associated with large brain size in primates. These TFs include GLI3, CREB5 and SOX9. Interestingly, the genes associated with the basal radial glia (bRG)-associated cis-element module, such as SRY and SOX9, are enriched in sex differentiation. Schizophrenia-associated single nucleotide polymorphisms are more enriched in super enhancers (SEs) than typical enhancers, suggesting that SEs play an important role in neural network wiring. A cis-regulatory element of DBN1 is identified, which is critical for neuronal cell proliferation and synaptic neuron differentiation. Notably, the loss of distal chromatin interaction and H3K27me3 signal are hallmarks of PFC aging, which are associated with abnormal expression of aging-related genes and transposon activation, respectively. Collectively, our findings shed light on epigenetic mechanisms underlying primate brain development and aging.

SMC3 contributes to heart development by regulating super-enhancer associated genes

Abnormal cardiac development has been observed in individuals with Cornelia de Lange syndrome (CdLS) due to mutations in genes encoding members of the cohesin complex. However, the precise role of cohesin in heart development remains elusive. In this study, we aimed to elucidate the indispensable role of SMC3, a component of the cohesin complex, in cardiac development and its underlying mechanism. Our investigation revealed that CdLS patients with SMC3 mutations have high rates of congenital heart disease (CHD). We utilized heart-specific Smc3-knockout (SMC3-cKO) mice, which exhibit varying degrees of outflow tract (OFT) abnormalities, to further explore this relationship. Additionally, we identified 16 rare SMC3 variants with potential pathogenicity in individuals with isolated CHD. By employing single-nucleus RNA sequencing and chromosome conformation capture high-throughput genome-wide translocation sequencing, we revealed that Smc3 deletion downregulates the expression of key genes, including Ets2, in OFT cardiac muscle cells by specifically decreasing interactions between super-enhancers (SEs) and promoters. Notably, Ets2-SE-null mice also exhibit delayed OFT development in the heart. Our research revealed a novel role for SMC3 in heart development via the regulation of SE-associated genes, suggesting its potential relevance as a CHD-related gene and providing crucial insights into the molecular basis of cardiac development.

Super-enhancer-driven IRF2BP2 enhances ALK activity and promotes neuroblastoma cell proliferation.

Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB). The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing. The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2, and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB. Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of the NB susceptibility gene ALK.

SP1 undergoes phase separation and activates RGS20 expression through super-enhancers to promote lung adenocarcinoma progression

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related death worldwide, but the underlying molecular mechanisms remain largely unclear. The transcription factor (TF) specificity protein 1 (SP1) plays a crucial role in the development of various cancers, including LUAD. Recent studies have indicated that master TFs may form phase-separated macromolecular condensates to promote super-enhancer (SE) assembly and oncogene expression. In this study, we demonstrated that SP1 undergoes phase separation and that its zinc finger 3 in the DNA-binding domain is essential for this process. Through Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using antibodies against SP1 and H3K27ac, we found a significant correlation between SP1 enrichment and SE elements, identified the regulator of the G protein signaling 20 (RGS20) gene as the most likely target regulated by SP1 through SE mechanisms, and verified this finding using different approaches. The oncogenic activity of SP1 relies on its phase separation ability and RGS20 gene activation, which can be abolished by glycogen synthase kinase J4 (GSK-J4), a demethylase inhibitor. Together, our findings provide evidence that SP1 regulates its target oncogene expression through phase separation and SE mechanisms, thereby promoting LUAD cell progression. This study also revealed an innovative target for LUAD therapies through intervening in SP1-mediated SE formation.

TWEAK/Fn14 signalling driven super-enhancer reprogramming promotes pro-metastatic metabolic rewiring in triple-negative breast cancer

Triple Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype suffering from limited targeted treatment options. Following recent reports correlating Fibroblast growth factor-inducible 14 (Fn14) receptor overexpression in Estrogen Receptor (ER)-negative breast cancers with metastatic events, we show that Fn14 is specifically overexpressed in TNBC patients and associated with poor survival. We demonstrate that constitutive Fn14 signalling rewires the transcriptomic and epigenomic landscape of TNBC, leading to enhanced tumour growth and metastasis. We further illustrate that such mechanisms activate TNBC-specific super enhancers (SE) to drive the transcriptional activation of cancer dependency genes via chromatin looping. In particular, we uncover the SE-driven upregulation of Nicotinamide phosphoribosyltransferase (NAMPT), which promotes NAD+ and ATP metabolic reprogramming critical for filopodia formation and metastasis. Collectively, our study details the complex mechanistic link between TWEAK/Fn14 signalling and TNBC metastasis, which reveals several vulnerabilities which could be pursued for the targeted treatment of TNBC patients.

The chromatin accessibility and transcriptomic landscape of the aging mice cochlea and the identification of potential functional super-enhancers in age-related hearing loss

BackgroundPresbycusis, also referred to as age-related hearing loss (ARHL), is a condition that results from the cumulative effects of aging on an individual's auditory capabilities. Given the limited understanding of epigenetic mechanisms in ARHL, our research focuses on alterations in chromatin-accessible regions.MethodsWe employed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in conjunction with unique identifier (UID) mRNA-seq between young and aging cochleae, and conducted integrated analysis as well as motif/TF-gene prediction. Additionally, the essential role of super-enhancers (SEs) in the development of ARHL was identified by comparative analysis to previous research. Meanwhile, an ARHL mouse model and an aging mimic hair cell (HC) model were established with a comprehensive identification of senescence phenotypes to access the role of SEs in ARHL progression.ResultsThe control cochlear tissue exhibited greater chromatin accessibility than cochlear tissue affected by ARHL. Furthermore, the levels of histone 3 lysine 27 acetylation were significantly depressed in both aging cochlea and aging mimic HEI-OC1 cells, highlighting the essential role of SEs in the development of ARHL. The potential senescence-associated super-enhancers (SASEs) of ARHL were identified, most of which exhibited decreased chromatin accessibility. The majority of genes related to the SASEs showed obvious decreases in mRNA expression level in aging HCs and was noticeably altered following treatment with JQ1 (a commonly used SE inhibitor).ConclusionThe chromatin accessibility in control cochlear tissue was higher than that in cochlear tissue affected by ARHL. Potential SEs involved in ARHL were identified, which might provide a basis for future therapeutics targeting SASEs related to ARHL.

Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

MICAL2 Is a Super Enhancer Associated Gene that Promotes Pancreatic Cancer Growth and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers and thus identifying more effective therapies is a major unmet need. In this study we characterized the super enhancer (SE) landscape of human PDAC to identify novel, potentially targetable, drivers of the disease. Our analysis revealed that MICAL2 is a super enhancer-associated gene in human PDAC. MICAL2 is a flavin monooxygenase that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin related transcription factors (MRTF-A and MRTF-B). We found that MICAL2 is overexpressed in PDAC and correlates with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, we observed that MICAL2 promotes both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 expression also inhibited macropinocytosis. Through in vitro phenotypic analyses, we show that MICAL2, MRTF-A and MRTF-B influence PDAC cell proliferation, migration and promote cell cycle progression. Importantly, we demonstrate that MICAL2 is essential for in vivo tumor growth and metastasis. Interestingly, we find that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo . This study highlights the multiple ways in which MICAL2 impacts PDAC biology and suggests that its inhibition may impede PDAC progression. Our results provide a foundation for future investigations into the role of MICAL2 in PDAC and its potential as a target for therapeutic intervention.

BRD4 plays an antiaging role in the senescence of renal tubular epithelial cells.

Age-related kidney failure is often induced by a decrease in the bioavailability of tubular epithelial cells in elderly chronic kidney disease (CKD) patients. BRD4, an epigenetic regulator and a member of the bromodomain and extraterminal (BET) protein family, acts as a super-enhancer (SE) organizing and regulating genes expression during embryogenesis and cancer development. But the physiological function of BRD4 in normal cells has been less studied. This study aimed to research certain biological roles of BRD4 in the process of normal cell aging and discuss the potential mechanisms. In this study, we investigated the biological functions of BRD4 proteins in the aging of renal tubular cells. At first, we used a D-galactose (D-gal) and BRD4 inhibitor (Abbv-075) to replicate kidney senescence in vivo. D-gal and Abbv-075 were then used to measure the aging-related changes, such as changes in cell cycle, β-galactosidase activity, cell migration, and p16 protein expression in vitro. At last, we knocked down and over-expressed BRD4 to investigate the aging-related physiological phenomena in renal tubular cells. In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing β-galactosidase activity as well as up-regulating p16 protein expression in primary human tubular epithelial cells. In the aging mice model, D-gal significantly induced renal function impairment and attenuated BRD4 protein expression. At the same time, the BRD4 inhibitor (Abbv-075) was able to mimic D-gal-induced cell senescence. In vivo, Abbv-075 also decreased kidney function and up-regulated p21 protein expression. When we knocked down the expression of BRD4, the senescence-associated β-galactosidase (SA-β-gal) activity increased dramatically, cell migration was inhibited, and the proportion of cells in the G0/G1 phase increased. Additionally, the knockdown also promoted the expression of the senescence-related proteins p16. When the renal tubular cells were overexpressed with BRD4, cell aging-related indicators were reversed in the D-gal-induced cell aging model. BRD4 appears to have an active role in the aging of renal tubular cells in vivo and in vitro. The findings also suggest that BRD4 inhibitors have potential nephrotoxic effects for oncology treatment. BRD4 may be a potential therapeutic biomarker and drug target for aging-related kidney diseases, which warrants additional studies.

A specific super-enhancer actuated by berberine regulates EGFR-mediated RAS–RAF1–MEK1/2–ERK1/2 pathway to induce nasopharyngeal carcinoma autophagy

Nasopharyngeal carcinoma (NPC), primarily found in the southern region of China, is a malignant tumor known for its highly metastatic characteristics. The high mortality rates caused by the distant metastasis and disease recurrence remain unsolved clinical problems. In clinic, the berberine (BBR) compound has widely been in NPC therapy to decrease metastasis and disease recurrence, and BBR was documented as a main component with multiple anti-NPC effects. However, the mechanism by which BBR inhibits the growth and metastasis of nasopharyngeal carcinoma remains elusive. Herein, we show that BBR effectively inhibits the growth, metastasis, and invasion of NPC via inducing a specific super enhancer (SE). From a mechanistic perspective, the RNA sequencing (RNA-seq) results suggest that the RAS–RAF1–MEK1/2–ERK1/2 signaling pathway, activated by the epidermal growth factor receptor (EGFR), plays a significant role in BBR-induced autophagy in NPC. Blockading of autophagy markedly attenuated the effect of BBR-mediated NPC cell growth and metastasis inhibition. Notably, BBR increased the expression of EGFR by transcription, and knockout of EGFR significantly inhibited BBR-induced microtubule associated protein 1 light chain 3 (LC3)-II increase and p62 inhibition, proposing that EGFR plays a pivotal role in BBR-induced autophagy in NPC. Chromatin immunoprecipitation sequencing (ChIP-seq) results found that a specific SE existed only in NPC cells treated with BBR. This SE knockdown markedly repressed the expression of EGFR and phosphorylated EGFR (EGFR-p) and reversed the inhibition of BBR on NPC proliferation, metastasis, and invasion. Furthermore, BBR-specific SE may trigger autophagy by enhancing EGFR gene transcription, thereby upregulating the RAS–RAF1–MEK1/2–ERK1/2 signaling pathway. In addition, in vivo BBR effectively inhibited NPC cells growth and metastasis, following an increase LC3 and EGFR and a decrease p62. Collectively, this study identifies a novel BBR-special SE and established a new epigenetic paradigm, by which BBR regulates autophagy, inhibits proliferation, metastasis, and invasion. It provides a rationale for BBR application as the treatment regime in NPC therapy in future.

MDB-60. CDK7 INHIBITION DISRUPTS ACQUIRED RESISTANCE MECHANISMS IN MYC-MB

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for 20% of all pediatric brain tumors. Medulloblastoma manifests as a clinically heterogeneous tumor with varying prognoses depending on the molecular subgroup. Group 3 tumors have the worst overall prognosis, and are associated with MYC gene amplification, higher rates of metastasis, and increased likelihood of recurrence. Our prior work demonstrated that WEE1 is upregulated in all MB subtypes and is a critical mediator of MB cell viability. We have established that Myc-driven MB is particularly sensitive to the WEE1 inhibitor AZD1775. Although AZD1775 showed promising results in combination with other genotoxic agents, tumor cells frequently acquire resistance to small molecule inhibitors. Thus, to identify regulators of resistance in Myc-MB we performed a Kinome scale RNAi screen in isogenic parental and resistant cells and identified CDK7 as a key mediator of AZD1775 resistance that restored sensitivity to WEE1 inhibition when depleted. CDK7 controls transcriptional initiation by phosphorylating the c-terminal domain of RNA Pol II. CDK7 has been implicated in several Myc-associated malignancies and is frequently enriched at Super Enhancers. Enhancer-promoter interactions (EPIs) are dynamic and can facilitate resistance mechanisms by inducing additional enhancer elements to promote transcription. Here, we investigated 3D chromatin interactions by HiChIP and further assessed CTCF and H3K27ac patterns after treatment with the CDK7 inhibitor, THZ1, by Cut and Run. Our analysis suggests that AZD1775-resistant cells are reorganized to establish alternate cis-acting elements that reinforce transcription of DNA repair and proliferation genes, circumventing AZD1775 treatment. We further examine cisplatin-resistant MB cells to determine if the mechanism is consistent in Myc-MB cell lines or therapy specific. Overall, we suggest that reorganized chromatin structure aids resistance development and that inhibition of CDK7 intervenes in this process, restoring sensitivity to standard therapies.

Phase-separated super-enhancers confer an innate radioresistance on genomic DNA.

Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.

Identification and experimental validation of immune-related gene PPARG is involved in ulcerative colitis

BackgroundThe pathophysiology of ulcerative colitis (UC) is believed to be heavily influenced by immunology, which presents challenges for both diagnosis and treatment. The main aims of this study are to deepen our understanding of the immunological characteristics associated with the disease and to identify valuable biomarkers for diagnosis and treatment. MethodsThe UC datasets were sourced from the GEO database and were analyzed using unsupervised clustering to identify different subtypes of UC. Twelve machine learning algorithms and Deep learning model DNN were developed to identify potential UC biomarkers, with the LIME and SHAP methods used to explain the models' findings. PPI network is used to verify the identified key biomarkers, and then a network connecting super enhancers, transcription factors and genes is constructed. Single-cell sequencing technology was utilized to investigate the role of Peroxisome Proliferator Activated Receptor Gamma (PPARG) in UC and its correlation with macrophage infiltration. Furthermore, alterations in PPARG expression were validated through Western blot (WB) and immunohistochemistry (IHC) in both in vitro and in vivo experiments. ResultBy utilizing bioinformatics techniques, we were able to pinpoint PPARG as a key biomarker for UC. The expression of PPARG was significantly reduced in cell models, UC animal models, and colitis models induced by dextran sodium sulfate (DSS). Interestingly, overexpression of PPARG was able to restore intestinal barrier function in H2O2-induced IEC-6 cells. Additionally, immune-related differentially expressed genes (DEGs) allowed for efficient classification of UC samples into neutrophil and mitochondrial metabolic subtypes. A diagnostic model incorporating the three disease-specific genes PPARG, PLA2G2A, and IDO1 demonstrated high accuracy in distinguishing between the UC group and the control group. Furthermore, single-cell analysis revealed that decreased PPARG expression in colon tissue may contribute to the polarization of M1 macrophages through activation of inflammatory pathways. ConclusionIn conclusion, PPARG, a gene related to immunity, has been established as a reliable potential biomarker for the diagnosis and treatment of UC. The immune response it controls plays a key role in the progression and development of UC by enabling interaction between characteristic biomarkers and immune infiltrating cells.

Mechanistic insights into super-enhancer-related genes as prognostic signatures in colon cancer.

Colon cancer (CC) is the most frequently occurring digestive system malignancy and is associated with a dismal prognosis. While super-enhancer (SE) genes have been identified as prognostic markers in several cancers, their potential as practical prognostic markers for CC patients remains unexplored. We obtained super-enhancer-related genes (SERGs) from the Human Super-Enhancer Database (SEdb). Transcriptome and relevant clinical data for colon cancer (CC) were sourced from the Gene Expression Omnibus (GEO) database. Subsequently, we identified up-regulated SERGs by the Weighted Gene Co-expression Network Analysis (WGCNA). Prognostic signatures were constructed via univariate and multivariate Cox regression analysis. We then delved into the mechanisms of these predictive genes by examining immune infiltration. We also assessed differential sensitivities to chemotherapeutic drugs between high- and low-SERGs risk patients. The critical gene was further validated using external datasets and finally confirmed by qRT PCR. We established a ten-gene risk score prognostic model (S100A11, LZTS2, CYP2S1, ZNF552, PSMG1, GJC1, NXN, and DCBLD2), which can effectively predict patient survival rates. This model demonstrated effective prediction capabilities in survival rates at 1, 3, and 5 years and was successfully validated using external datasets. Furthermore, we detected significant differences in immune cell infiltration between high- and low-SERGs risk groups. Notably, high-risk patients exhibited heightened sensitivity to four chemotherapeutic agents, suggesting potential benefits for precision therapy in CC patients. Finally, qRT-PCR validation revealed a significant upregulation of LZTS2 mRNA expression in CC cells. These findings reveal that the SERGs model could effectively predict the prognosis of CC.

#2607 Generation of a tissue resident macrophage depleted reporter rat to examine cardiovascular homeostasis and kidney disease

Abstract Cardiovascular disease (CVD) and chronic kidney disease (CKD) are currently the 1st and 10th leading cause of global mortality, respectively. By 2050, CKD is predicted to be the 5th leading cause, with most of those individuals dying of CVD. Recent single cell RNA sequencing data has highlighted the plasticity of macrophage populations in both renal and cardiovascular injury and repair. Many of these subpopulations of macrophages remain uncharacterized. We have generated the Red FIRE rat, which is a cross between the Csf1r-mApple reporter rat and the super-enhancer FIRE knockout rat. The Csf1r-mApple reporter rat introduces a fluorescent signal to cells of the monocyte-macrophage lineage and the KO of FIRE (fms-intronic regulatory element), while the FIRE rat has fms-intronic regulatory element (FIRE), which is the superenhancer in the Csf1r locus knocked out, resulting in depletion of tissue resident macrophages. These red FIRE rats will allow us to visualize all Csf1r expressing cells (macrophages) and in the FIRE rats deplete populations of tissue resident macrophages (TRM) (including those in the kidney and heart). This will enable us to visualize and study TRM populations in homeostasis and injury. We hypothesise that the red FIRE rats will show a more severe phenotype in injury and have altered vascular function (local and systemic). Male and female (8–10-week-old) rats underwent a 6- or 12-week 5/6 subtotal nephrectomy (5/6 STNx) in groups of 4-8. Baseline, midpoint and endpoint urine and serum were collected for analysis, as well as blood pressure measurements and echocardiograms to monitor kidney and heart health/decline. At the end of the experiment, kidneys and the heart were collected for flow cytometry, immunofluorescence, RT-qPCR, and histological staining. The thoracic aorta, mesentery, and common renal arteries were collected for functional analysis using isometric (wire)myography. The knockout of the FIRE super enhancer resulted in depletion of kidney TRMs (Fig. 1) as visualised by immunofluorescent staining of both wild type and macrophage-depleted (red FIRE) rat tissue. Figure 1A shows the composite image and individual stains; DAPI, IBa1 (pan macrophage marker), and Csf1r-mApple (tissue resident macrophages) on a naïve heart. Representative composite images also compare STNx hearts (Fig. 1B) and kidneys (Fig. 1C) in both wild type and macrophage-depleted rats. This allowed for comparison of these rats in both injury and homeostasis. Figure 1: Immunofluorescent staining of the uninjured heart of a FIRE wild type (WT), mApple positive rat (A), the injured kidney (B) and heart (C) of mApple positive, FIRE wild type (Control) or mApple positive, FIRE knock out (FIRE) rats post 12-week 5/6 subtotal nephrectomy (5/6 STNx). DAPI: nuclei, IBa1: all macrophages, csf1r (mApple): tissue resident macrophages. Representative imagining, figure made using FIJI. Our preliminary data suggests previously undescribed roles for tissue-resident macrophages in rats i.e. ion handling, vascular homeostasis and roles in CKD and repair. These results could help reveal new specific druggable targets that could be exploited for treatments which could halt progression of disease and thus reduce end stage renal failure patient numbers, healthcare costs and premature CVD-related deaths.

Enhancer selectivity across cell types delineates three functionally distinct enhancer-promoter regulation patterns

BackgroundMultiple enhancers co-regulating the same gene is prevalent and plays a crucial role during development and disease. However, how multiple enhancers coordinate the same gene expression across various cell types remains largely unexplored at genome scale.ResultsWe develop a computational approach that enables the quantitative assessment of enhancer specificity and selectivity across diverse cell types, leveraging enhancer-promoter (E-P) interactions data. We observe two well-known gene regulation patterns controlled by enhancer clusters, which regulate the same gene either in a limited number of cell types (Specific pattern, Spe) or in the majority of cell types (Conserved pattern, Con), both of which are enriched for super-enhancers (SEs). We identify a previously overlooked pattern (Variable pattern, Var) that multiple enhancers link to the same gene, but rarely coexist in the same cell type. These three patterns control the genes associating with distinct biological function and exhibit unique epigenetic features. Specifically, we discover a subset of Var patterns contains Shared enhancers with stable enhancer-promoter interactions in the majority of cell types, which might contribute to maintaining gene expression by recruiting abundant CTCF.ConclusionsTogether, our findings reveal three distinct E-P regulation patterns across different cell types, providing insights into deciphering the complexity of gene transcriptional regulation.

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.