Chromatrap Publications

Here is a list of our citations, where you will find the source of the information by clicking on each individual link. 

Peritoneal Modulators of EZH2-miR-155 Cross-Talk in Endometriosis

Abstract: 

Activation of trimethylation of histone 3 lysine 27 (H3K27me3) by EZH2, a component of the Polycomb repressive complex 2 (PRC2), is suggested to play a role in endometriosis. However, the mechanism by which this complex is dysregulated in endometriosis is not completely understood. Here, using eutopic and ectopic tissues, as well as peritoneal fluid (PF) from IRB-approved and consented patients with and without endometriosis, the expression of PRC2 complex components, JARID2, miR-155 (known regulators of EZH2), and a key inflammatory modulator, FOXP3, was measured. A higher expression of EZH2, H3K27me3, JARID2, and FOXP3 as well as miR-155 was noted in both the patient tissues and in endometrial PF treated cells. Gain-or-loss of function of miR-155 showed an effect on the PRC2 complex but had little effect on JARID2 expression, suggesting alternate pathways. Chromatin immunoprecipitation followed by qPCR showed differential expression of PRC2 complex proteins and its associated binding partners in JARID2 vs. EZH2 pull down assays. In particular, endometriotic PF treatment increased the expression of PHF19 (p = 0.0474), a gene silencer and co-factor that promotes PRC2 interaction with its targets. Thus, these studies have identified the potential novel crosstalk between miR-155-PRC2 complex-JARID2 and PHF19 in endometriosis, providing an opportunity to test other epigenetic targets in endometriosis.

 

Key words: 

Kit 500189, Ishikawa cell line, EZH2 , JAIRD2, endometriosis 

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Functional Characterization of a Dual Enhancer/Promoter Regulatory Element Leading Human CD69 Expression

Redondo-Anton et al, 2020

Abstract:

The CD69 gene encodes a C-type lectin glycoprotein with immune regulatory properties which is expressed on the cell surfaces of all activated hematopoietic cells. CD69 activation kinetics differ by developmental stage, cell linage and activating conditions, and these differences have been attributed to the participation of complex gene regulatory networks. An evolutionarily conserved regulatory element, CNS2, located 4kb upstream of the CD69 gene transcriptional start site, has been proposed as the major candidate governing the gene transcriptional activation program. To investigate the function of human CNS2, we studied the effect of its endogenous elimination via CRISPR-Cas9 on CD69 protein and mRNA expression levels in various immune cell lines. Even when the entire promoter region was maintained, CNS2-/- cells did not express CD69, thus indicating that CNS2 has promoter-like characteristics. However, like enhancers, inverted CNS2 sustained transcription, although at a diminished levels, thereby suggesting that it has dual promoter and enhancer functions. Episomal luciferase assays further suggested that both functions are combined within the CNS2 regulatory element. In addition, CNS2 directs its own bidirectional transcription into two different enhancer-derived RNAs molecules (eRNAs) which are transcribed from two independent transcriptional start sites in opposite directions. This eRNA transcription is dependent on only the enhancer sequence itself, because in the absence of the CD69 promoter, sufficient RNA polymerase II levels are maintained at CNS2 to drive eRNA expression. Here, we describe a regulatory element with overlapping promoter and enhancer functions, which is essential for CD69 gene transcriptional regulation.

 

Key words: 

Kit 500189, H3K4me3, CNS2, immune regulation, CD69, Jurkat T Cells, immortalised cell lines

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IL-6 regulates CCR5 expression and immunosuppressive capacity of MDSC in murine melanoma

Weber et al, 2020 

Abstract: 

Background: Myeloid-derived suppressor cells (MDSC) play a major role in the immunosuppressive melanoma microenvironment. They are generated under chronic inflammatory conditions characterized by the constant production of inflammatory cytokines, chemokines and growth factors, including IL-6. Recruitment of MDSC to the tumor is mediated by the interaction between chemokines and chemokine receptors, in particular C–C chemokine receptor (CCR)5. Here, we studied the mechanisms of CCR5 upregulation and increased immunosuppressive function of CCR5+ MDSC.

Methods: The immortalized myeloid suppressor cell line MSC-2, primary immature myeloid cells and in vitro differentiated MDSC were used to determine factors and molecular mechanisms regulating CCR5 expression and immunosuppressive markers at the mRNA and protein levels. The relevance of the identified pathways was validated on the RET transgenic mouse melanoma model, which was also used to target the identified pathways in vivo.

Results: IL-6 upregulated the expression of CCR5 and arginase 1 in MDSC by a STAT3-dependent mechanism. MDSC differentiated in the presence of IL-6 strongly inhibited CD8+ T cell functions compared with MDSC differentiated without IL-6. A correlation between IL-6 levels, phosphorylated STAT3 and CCR5 expression in tumor-infiltrating MDSC was demonstrated in the RET transgenic melanoma mouse model. Surprisingly, IL-6 overexpressing tumors grew significantly slower in mice accompanied by CD8+ T cell activation. Moreover, transgenic melanoma-bearing mice treated with IL-6 blocking antibodies showed significantly accelerated tumor development.

Conclusion: Our in vitro and ex vivo findings demonstrated that IL-6 induced CCR5 expression and a strong immunosuppressive activity of MDSC, highlighting this cytokine as a promising target for melanoma immunotherapy. However, IL-6 blocking therapy did not prove to be effective in RET transgenic melanoma-bearing mice but rather aggravated tumor progression. Further studies are needed to identify particular combination therapies, cancer entities or patient subsets to benefit from the anti-IL-6 treatment.

 

Key words: 

Kit 500189, C57BL/6 mice, Mouse cells, MDSC, pSTAT3, IL-6, CCR5 regulation, cancer immunotherapy

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HMTase Inhibitors as a Potential Epigenetic-Based Therapeutic Approach for Friedreich’s Ataxia

Sherzai et al, 2020

Abstract:

Friedreich’s ataxia (FRDA) is a progressive neurodegenerative disorder caused by a homozygous GAA repeat expansion mutation in intron 1 of the frataxin gene (FXN), which instigates reduced transcription. As a consequence, reduced levels of frataxin protein lead to mitochondrial iron accumulation, oxidative stress, and ultimately cell death; particularly in dorsal root ganglia (DRG) sensory neurons and the dentate nucleus of the cerebellum. In addition to neurological disability, FRDA is associated with cardiomyopathy, diabetes mellitus, and skeletal deformities. Currently there is no effective treatment for FRDA and patients die prematurely. Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, we investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin protein levels was observed. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies.

 

Key words: 

Kit 500189, primary fibroblasts, H3K9Ac, H3K9me3, H3K27me3, FXN gene, Freidrich's Ataxia

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Bcl-3 promotes multi-modal tumour cell migration via NF-κB1 mediated regulation of Cdc42

Turnham et al, 2020

Abstract: 

A key challenge in the implementation of anti-metastatics as cancer therapies is the multi-modal nature of cell migration, which allows tumour cells to evade the targeted inhibition of specific cell motility pathways. The nuclear factor-kappaB (NF-κB) co-factor B-cell lymphoma 3 (Bcl-3) has been implicated in breast cancer cell migration and metastasis, yet it remains to be determined exactly which cell motility pathways are controlled by Bcl-3 and whether migrating tumour cells are able to evade Bcl-3 intervention. Addressing these questions and the mechanism underpinning Bcl-3’s role in this process would help determine its potential as a therapeutic target. Here we identify Bcl-3 as an upstream regulator of the two principal forms of breast cancer cell motility, involving collective and single-cell migration. This was found to be mediated by the master regulator Cdc42 through binding of the NF-κB transcription factor p50 to the Cdc42 promoter. Notably, Bcl-3 depletion inhibited both stable and transitory motility phenotypes in breast cancer cells with no evidence of migratory adaptation. Overexpression of Bcl-3 enhanced migration and increased metastatic tumour burden of breast cancer cells in vivo, whereas overexpression of a mutant Bcl-3 protein, which is unable to bind p50, suppressed cell migration and metastatic tumour burden suggesting that disruption of Bcl-3/NF-κB complexes is sufficient to inhibit metastasis. These findings identify a novel role for Bcl-3 in intrinsic and adaptive multi-modal cell migration mediated by its direct regulation of the Rho GTPase Cdc42 and identify the upstream Bcl-3:p50 transcription complex as a potential therapeutic target for metastatic disease.

 

Key words: 

Immortallised breast cancer cell lines, p50, NF-κB, neoplasm metastasis 

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Direct monitoring of breast and endometrial cancer cell epigenetic response to DNA methyltransferase and histone deacetylase inhibitors.

Teixeira et al, 2019

Abstract: 

DNA methylation and histone deacetylation are key epigenetic processes involved in normal cellular function and tumorigenesis. Therapeutic strategies based on DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors are currently in use and under development for the treatment of cancers. Genome-wide DNA methylation profiling has been proposed for use in disease diagnosis, and histone modification profiling for disease stratification will follow suit. However, whether epigenome sequencing technologies will be feasible for rapid clinic diagnosis and patient treatment monitoring remains to be seen, and alternative detection technologies will almost certainly be needed. Here we used electrochemical impedance spectroscopy (EIS) employing a graphene-based screen-printed electrode system to directly measure global DNA methylation and histone H3 acetylation to compare non-cancer and breast cancer cell lines. We demonstrated that whilst global methylation was not useful as a differential marker in the cellular systems tested, histone H3 acetylation was effective at higher chromatin levels. Using breast and endometrial cancer cell models, EIS was then used to monitor cellular responses to the DNMT and HDAC inhibitors 5-Aza-2′-deoxycytidine and suberoylanilide hydroxamic acid in vitro, and proved very effective at detecting global cellular responses to either treatment, indicating that this approach could be useful in following treatment response to epigenetic drugs. Moreover, this work reports the first combined analysis of two epigenetic markers using a unified graphene-based biosensor platform, demonstrating the potential for multiplex analysis of both methylation and acetylation on the same sample.

 

Key words: 

Acetylated H3, Hec50 cell line, MCF7 cell line, MCF12A cell line, Histone acetylation, cancer detection 

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Olive-Oil-Derived Polyphenols Effectively Attenuate Inflammatory Responses of Human Keratinocytes by Interfering with the NF-

Aparicio-Soto et al, 2019 

Abstract: 

Scope: Extra virgin olive oil (EVOO) is rich in phenolic compounds, including hydroxytyrosol (HTy) and hydroxytyrosyl acetate (HTy-Ac), which have presented multiple beneficial properties. Their impact on inflammatory responses in human keratinocytes and modes of action have not been addressed yet.

Methods and results: Primary human keratinocytes are pretreated with HTy-Ac or HTy for 30 min and stimulated with IL-1β or Toll-like receptor 3 ligand (TLR3-l). Thymic stromal lymphopoietin (TSLP), measured by ELISA, is attenuated by both polyphenols in a dose-dependent manner. The expression of several inflammation-related genes, including distinct TSLP isoforms and IL-8, are assessed by quantitative RT-PCR and likewise inhibited by HTy-Ac/HTy. Mechanistically, EVOO phenols counteracts IκB degradation and translocation of NF-κB to the nucleus, a transcription factor of essential significance to TSLP and IL-8 transcriptional activity; this is evidenced by immunoblotting. Accordingly, NF-κB recruitment to critical binding sites in the TSLP and IL-8 promoter is impeded in the presence of HTy-Ac/HTy, as demonstrated by chromatin immunoprecipitation. Promoter reporter assays finally reveal that the neutralizing effect on NF-κB induction has functional consequences, resulting in reduced NF-κB-directed transcription.

Conclusion: EVOO phenols afford protection from inflammation in human keratinocytes by interference with the NF-κB pathway.

 

Key words:

Kit 500165, Enzymatic shearing, primary human keratinocytes, NF-κB, inflammation 

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Metformin Regulates the Expression of CD133 Through the AMPK-CEBPβ Pathway in Hepatocellular Carcinoma Cell Lines1

Maehara et al, 2019

Abstract: 

CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin, one of the biguanides used for the treatment of diabetes, is also known to reduce the risk of cancer development and cancer stem-like cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling but not the mammalian target of rapamycin (mTOR). AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CCAAT/enhancer-binding protein beta (CEBPβ) was upregulated by metformin and that two isoforms of CEBPβ reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a three dimensional (3D) culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-fluorouracil (5-FU) treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPβ signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.

 

Key words: 

Kit 500192, Hepatocellular carcinoma cell lines, CD133, cancer stem cells, gene regulation

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Thymic stromal lymphopoietin production induced by skin irritation results from concomitant activation of protease‐activated receptor 2 and interleukin 1 pathways

Redhu et al, 2019

Abstract: 

Background: Thymic stromal lymphopoietin (TSLP) mediates proallergic T helper 2-type responses by acting on leucocytes. Endogenous pathways regulating TSLP production are poorly defined.

Objectives: To uncover the mechanisms by which skin barrier disruption elicits TSLP production and to delineate the level at which individual mechanistic components may converge.

Methods: A combination of primary keratinocytes, skin explants and in vivo strategies was employed. Murine skin was tape stripped in the presence of neutralizing antibodies or antagonists. Cells and explants were stimulated with interleukin (IL)-1 and protease-activated receptor 2 agonist (PAR-2-Ag). TSLP levels were quantified by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction. Chromatin immunoprecipitation and promoter reporter assays were used to examine recruitment and functional activity of nuclear factor kappa B (NF-κB) at the TSLP promoter.

Results: TSLP induction in mouse skin occurred in a PAR-2- and IL-1-dependent manner. This scenario was duplicated by exogenous IL-1 plus PAR-2-Ag vs. each stimulus alone. Joint activity of PAR-2 and IL-1 was also observed in human keratinocytes. The TSLP promoter was identified as the target of PAR-2/IL-1, whereby PAR-2 activation augmented the recruitment of NF-κB and transcriptional activation over IL-1 alone. Combined treatment showed activity at concentrations of IL-1 unable to elicit NF-κB activity on their own.

Conclusions: Skin barrier disruption activates the IL-1 and the PAR-2 pathways, which act in concert to activate the TSLP promoter and possibly other inflammatory genes. Awareness of this combined activity may permit a more flexible clinical management by selective targeting of either pathway individually or collectively.

 

Key words: 

Kit 500165, enzymatic shearing kit, NF-κB, TSLP promoter,  primary keratinocytes, inflammation 

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LUMAN/CREB3 Plays a Dual Role in Stress Responses as a Cofactor of the Glucocorticoid Receptor and a Regulator of Secretion

Penney et al, 2018

Abstract: 

LUMAN/CREB3, originally identified through its interaction with a cell cycle regulator HCFC1, is a transcription factor involved in the unfolded protein response during endoplasmic reticulum stress. Previously using gene knockout mouse models, we have shown that LUMAN modulates the glucocorticoid (GC) response leading to enhanced glucocorticoid receptor (GR) activity and lower circulating GC levels. Consequently, the stress response is dysregulated, leading to a blunted stress response in the Luman-deficient mice. One question that remained was how LUMAN deficiency affected the stress response at the cellular level leading to the changes in the physiological stress response. Here, we found that LUMAN interacts with GR through a putative nuclear receptor box site and can activate GR in the absence of a ligand. Further investigation showed that, when activated, LUMAN binds to the glucocorticoid response element (GRE), increasing the activity of GR exponentially compared to GR-ligand binding alone. On the other hand, we also found that in the absence of LUMAN, cells were more sensitive to cellular stress, exhibiting decreased secretory capacity. Hence our current data suggest that LUMAN may function both as a transcriptional cofactor of GR and a hormone secretion regulator, and through this, plays a role in stress sensitivity and reactivity to stress.

 

Key words: 

Kit 500189, m-Hippo-E14 cells, CREB3 transcription factor, neurology

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Prostate Derived Ets Factor (PDEF) inhibits metastasis by inducing epithelial/luminal phenotype in prostate cancer cells

Wang et al, 2018 

Abstract: 

Metastasis is the primary cause of prostate cancer morbidity and mortality. Our previous studies revealed that Sam pointed domain ETS transcription factor, a.k.a. prostate-derived ETS factor (SPDEF/PDEF), inhibits prostate cancer metastasis. However, the mechanism is still unclear. In this study, using microarray and gene set enrichment analysis, we discovered that PDEF upregulated epithelial/luminal differentiation-related genes while it suppressed stemness and epithelial-to-mesenchymal transition–related genes, especially Twist1. We also observed loss of PDEF and gain of Twist1 expression during prostate cancer progression in the TRAMP mouse model. Moreover, Twist1 knockdown resulted in upregulation of PDEF expression, suggesting a reciprocal regulation between PDEF and Twist1. Mechanistically, our ChIP-seq analysis revealed that PDEF directly regulated cytokeratin 18 (CK18) transcription through the GGAT motif within its putative promoter region. CK18 knockdown resulted in increased expression of Twist1, suggesting that PDEF regulated Twist1 in part via CK18. Our analysis of multiple clinical prostate cancer cohorts revealed an inverse relationship between PDEF expression and tumor grade, tumor metastasis, and poor patient survival. Furthermore, a two-gene signature of low PDEF and high Twist1 can better predict poor survival in prostate cancer patients than either gene alone. Collectively, our findings demonstrate PDEF inhibits prostate tumor progression, in part, by directly regulating transcription of CK18, and that PDEF/Twist1 expression could help distinguish between lethal and indolent prostate cancer.

 

Key words: 

PDEF, Prostate cancer, transgenic mice, tumour xenografts

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Compensatory expression of Nur77 and Nurr1 regulates NF-κB-dependent inflammatory signaling in astrocytes

Popichak et al, 2018

Abstract: 

Inflammatory activation of glial cells promotes loss of dopaminergic neurons in Parkinson's disease (PD). The transcription factor, Nuclear Factor-kappa B (NF-κB), regulates the expression of multiple neuroinflammatory cytokines and chemokines in activated glial cells that are damaging to neurons. Thus, inhibition of NF-κB signaling in glial cells could be a promising therapeutic strategy for the prevention of neuroinflammatory injury. Nuclear orphan receptors in the NR4A family, including NR4A1 (Nur77) and NR4A2 (Nurr1), can inhibit the inflammatory effects of NFκB but there are no approved drugs that target these receptors. Therefore, we postulated that a novel NR4A receptor ligand, 1,1-bis (3'-indolyl) -1-(p-methoxyphenyl) methane (C-DIM5), would suppress NF-κB-dependent inflammatory gene expression in astrocytes following treatment with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and the inflammatory cytokines, IFN-γ and TNF-α. C-DIM5 increased expression of Nur77 mRNA and suppressed expression of multiple neuroinflammatory genes. C-DIM5 also inhibited the expression of NF-κB-regulated inflammatory and apoptotic genes in qPCR array studies and effected p65 binding to unique genes in ChIP-seq experiments but did not prevent p65 translocation to the nucleus, suggesting a nuclear-specific mechanism. C-DIM5 prevented nuclear export of Nur77 in astrocytes induced by MPTP treatment and simultaneously recruited Nurr1 to the nucleus, consistent with known transrepressive properties of this receptor. Combined RNAi knockdown of Nur77 and Nurr1 inhibited anti-inflammatory activity of C-DIM5, demonstrating that C-DIM5 requires these receptors to inhibit NF-κB. Collectively; these data demonstrate that NR4A1/Nur77 and NR4A2/Nurr1 dynamically regulated inflammatory gene expression in glia by modulating the transcriptional activity of NF-κB.

 

Key words: 

Kit 500189, primary glia cells, p65, NF-kB, neuroinflammation

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