Characterizing the DNA Binding Site Specificity of NF-κB with Protein-Binding Microarrays (PBMs)

Siggers, Trevor; Gilmore, Thomas D.; Barron, Brian; Penvose, Ashley

doi:10.1007/978-1-4939-2422-6_36

Cited by 13 publications

(7 citation statements)

References 34 publications

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“…Transcription levels of NF-κB target genes were analyzed as below. The NF-κB target genes were identified according to database (Siggers et al, 2015 ; Li et al, 2017 ). NF-κB target genes with a P value less than 0.01 were taken into consideration and P values of Log 2 (Fold change) between NF-κB target genes ( P < 0.01) and other genes were calculated by Two-sample Kolmogorov-Smirnov test.…”

Section: Methodsmentioning

confidence: 99%

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

et al. 2019

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Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary cerebrovascular disease caused by a NOTCH3 mutation. However, the underlying cellular and molecular mechanisms remain unidentified. Here, we generated non-integrative induced pluripotent stem cells (iPSCs) from fibroblasts of a CADASIL patient harboring a heterozygous NOTCH3 mutation (c.3226C>T, p.R1076C). Vascular smooth muscle cells (VSMCs) differentiated from CADASIL-specific iPSCs showed gene expression changes associated with disease phenotypes, including activation of the NOTCH and NF-κB signaling pathway, cytoskeleton disorganization, and excessive cell proliferation. In comparison, these abnormalities were not observed in vascular endothelial cells (VECs) derived from the patient's iPSCs. Importantly, the abnormal upregulation of NF-κB target genes in CADASIL VSMCs was diminished by a NOTCH pathway inhibitor, providing a potential therapeutic strategy for CADASIL. Overall, using this iPSCbased disease model, our study identified clues for studying the pathogenic mechanisms of CADASIL and developing treatment strategies for this disease.

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Section: Methodsmentioning

confidence: 99%

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

et al. 2019

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“…Herein, we demonstrate that NF-κB functions downstream of the MELK/EZH2 complex, which opens another exciting pathway to better understand the mechanism of tumorigenesis, beyond the well-established Rel/NF-κB interaction (38, 42). Activation of NF-κB involves a series of sequential events including DNA binding, dimerization, nuclear importation and tyrosine phosphorylation (46, 47). Deeper understanding of molecular mechanisms associated with this pathway will enable development of targeted inhibitors and provide rationale for establishment of the most effective diagnostic and therapeutic applications.…”

Section: Discussionmentioning

confidence: 99%

EZH2 Phosphorylation Promotes Self-Renewal of Glioma Stem-Like Cells Through NF-κB Methylation

Liu

Sun

et al. 2019

Front. Oncol.

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Cancer stem-like cells (CSCs) is a cell population in glioma with capacity of self-renewal and is critical in glioma tumorigenesis. Parallels between CSCs and normal stem cells suggest that CSCs give rise to tumors. Oncogenic roles of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) have been reported to play a crucial role in glioma tumorigenesis. Herein, we focus on mechanistic contributions of downstream molecules to maintaining stemness of glioma stem-like cells (GSCs). Transcriptional factor, NF-κB, co-locates with MELK/EZH2 complex. Clinically, we observe that the proportion of MELK/EZH2/NF-κB complex is elevated in high-grade gliomas, which is associated with poor prognosis in patients and correlates negatively with survival. We describe the interaction between these three proteins. Specifically, MELK induces EZH2 phosphorylation, which subsequently binds to and methylates NF-κB, leading to tumor proliferation and persistence of stemness. Furthermore, the interaction between MELK/EZH2 complex and NF-κB preferentially occurs in GSCs compared with non-stem-like tumor cells. Conversely, loss of this signaling dramatically suppresses the self-renewal capability of GSCs. In conclusion, our findings suggest that the GSCs depend on EZH2 phosphorylation to maintain the immature status and promote self-proliferation through NF-κB methylation, and represent a novel therapeutic target in this difficult to treat malignancy.

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“…Microarray DNA double stranding and basic PBM protocols are as previously described (22,23). All wash steps were carried out in coplin jars on an orbital shaker at 125 rpm.…”

Section: Methodsmentioning

confidence: 99%

NextPBM: a platform to study cell-specific transcription factor binding and cooperativity

Mohaghegh

Bray

Keenan

et al. 2019

Nucleic Acids Research

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High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM ( n uclear ext ract p rotein- b inding m icroarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.

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Characterizing the DNA Binding Site Specificity of NF-κB with Protein-Binding Microarrays (PBMs)

Cited by 13 publications

References 34 publications

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

EZH2 Phosphorylation Promotes Self-Renewal of Glioma Stem-Like Cells Through NF-κB Methylation

NextPBM: a platform to study cell-specific transcription factor binding and cooperativity

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