Lakshmi Prabhu

Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond

Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and epigenetic silencing etc. depend on the crucial interactions between cellular proteins and DNA. Chromatin immunoprecipitation (ChIP) is an important experimental technique for studying interactions between specific proteins and DNA in the cell and determining their localization on a specific genomic locus. In recent years, the combination of ChIP with second generation DNA-sequencing technology (ChIP-seq) allows precise genomic functional assay. This review addresses the important applications of ChIP-seq with an emphasis on its role in genome-wide mapping of transcription factor binding sites, the revelation of underlying molecular mechanisms of differential gene regulation that are governed by specific transcription factors, and the identification of epigenetic marks. Furthermore, we also describe the ChIP-seq data analysis workflow and a perspective for the exciting potential advancement of ChIP-seq technology in the future.

Role of Novel Serine 316 Phosphorylation of the p65 Subunit of NF-κB in Differential Gene Regulation.

Background: Post-translational modification is an important approach to regulate NF-κB activity. Results: Serine 316 (Ser-316) is a novel phosphorylation site on p65. Conclusion: Phosphorylation of Ser-316 on p65 is essential for NF-κB activation and its related biological functions. Significance: Our data shed light on how NF-κB transcriptional specificity is achieved through site-specific phosphorylation. Nuclear factor κB (NF-κB) is a central coordinator in immune and inflammatory responses. Constitutive NF-κB is often found in some types of cancers, contributing to oncogenesis and tumor progression. Therefore, knowing how NF-κB is regulated is important for its therapeutic control. Post-translational modification of the p65 subunit of NF-κB is a well known approach for its regulation. Here, we reported that in response to interleukin 1β, the p65 subunit of NF-κB is phosphorylated on the novel serine 316. Overexpression of S316A (serine 316 → alanine) mutant exhibited significantly reduced ability to activate NF-κB and decreased cell growth as compared with wtp65 (wild type p65). Moreover, conditioned media from cells expressing the S316A-p65 mutant had a considerably lower ability to induce NF-κB than that of wtp65. Our data suggested that phosphorylation of p65 on Ser-316 controls the activity and function of NF-κB. Importantly, we found that phosphorylation at the novel Ser-316 site and other two known phosphorylation sites, Ser-529 and Ser-536, either individually or cooperatively, regulated distinct groups of NF-κB-dependent genes, suggesting the unique role of each individual phosphorylation site on NF-κB-dependent gene regulation. Our novel findings provide an important piece of evidence regarding differential regulation of NF-κB-dependent genes through phosphorylation of different p65 serine residues, thus shedding light on novel mechanisms for the pathway-specific control of NF-κB. This knowledge is key to develop strategies for prevention and treatment of constitutive NF-κB-driven inflammatory diseases and cancers.

Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer

Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-κB-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared with WT-YBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-κB activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer.

Role of post-translational modification of the Y box binding protein 1 in human cancers

Y box binding protein-1 (YBX1) belongs to a DNA- and RNA-binding family of transcription factors, containing the highly conserved cold shock domain (CSD). YBX1 is involved in a number of cellular functions including transcription, translation, DNA damage repair etc., and it is upregulated during times of environmental stress. YBX1 is localized in both the cytoplasm and the nucleus. There, its nuclear translocation is observed in a number of cancers and is associated with poor prognosis and disease progression. Additionally, YBX1 expression is upregulated in a variety of cancers, pointing towards its role as a potential oncogene. Under certain circumstances, YBX1 also promotes the expression of multidrug resistance 1 (MDR1) gene, which is involved in the development of drug resistance. Thus, it is critical to understand the mechanism of YBX1 regulation and its downstream effects on promoting cancer development. A number of recent studies have highlighted the mechanisms of YBX1 regulation. Mass spectrometric analyses have reported several post-translational modifications that possibly play an important role in modulating YBX1 function. Phosphorylation is the most widely occurring post-translational modification in YBX1. In vivo analyses of sites like S102 and more recently, S165 illustrate the relationship of post-translational regulation of YBX1 in promoting cell proliferation and tumor growth. This review provides a comprehensive and up-to-date account of post-translational modifications identified in YBX1. This knowledge is a key in allowing us to better understand the mechanism of YBX1 regulation, which will aid in development of novel therapeutic strategies to target YBX1 in many types of cancer in the future.

Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer

Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer.

Adapting AlphaLISA high throughput screen to discover a novel small-molecule inhibitor targeting protein arginine methyltransferase 5 in pancreatic and colorectal cancers

Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are notoriously challenging for treatment. Hyperactive nuclear factor κB (NF-κB) is a common culprit in both cancers. Previously, we discovered that protein arginine methyltransferase 5 (PRMT5) methylated and activated NF-κB. Here, we show that PRMT5 is highly expressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression, while shRNA knockdown showed an opposite effect. Using an innovative AlphaLISA high throughput screen, we discovered a lead compound, PR5-LL-CM01, which exhibited robust tumor inhibition effects in both cancers. An in silico structure prediction suggested that PR5-LL-CM01 inhibits PRMT5 by binding with its active pocket. Importantly, PR5-LL-CM01 showed higher anti-tumor efficacy than the commercial PRMT5 inhibitor, EPZ015666, in both PDAC and CRC. This study clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC, and establishes PR5-LL-CM01 as a promising basis for new drug development in the future.

NF-κB: Its Role in Colorectal Cancer

Colorectal cancer (CRC) is a major worldwide health problem and is the second leading cause of cancer-related deaths in the United States. Despite considerable progress in diagnosis and treatment, a high mortality rate persists, largely due to the complications associated with metastatic incidences. The pro-inflammatory transcription factor nuclear factor κB (NF-κB) is a central player in inflammatory responses and tumor progression. In CRC, constitutively activated NF-κB has been observed in the majority of patients. NF-κB significantly affects the process of tumorigenesis by promoting many aspects including tumor growth, proliferation, invasiveness, and angiogenesis. Importantly, the critical contribution of NF-κB to inflammation and tumorigenesis is due to its control of the expression of a large variety of target genes, many of which, when aberrantly expressed, help to orchestrate and promote CRC malignant potential. These NF-κB target genes include those vital to cell cycle regulation, cell proliferation, metastasis, and cell survival. Additionally, activation of NF-κB in both cancerous cells and inflammatory cells and subsequent induction of cytokines/chemokines within the tumor microenvironment also contribute to CRC cell malignancy in both autocrine and paracrine manners. These evidences implicate inhibition of NF-κB as an important approach for CRC therapy. Several recent combinatorial approaches using classical chemotherapeutics with NF-κB inhibitors seem to have resulted in very promising outcomes.