Rasika Mundade

Protein arginine methylation of non-histone proteins and its role in diseases

Protein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate arginine residues on histones and other proteins. PRMTs play a crucial role in influencing various cellular functions, including cellular development and tumorigenesis. Arginine methylation by PRMTs is found on both nuclear and cytoplasmic proteins. Recently, there is increasing evidence regarding post-translational modifications of non-histone proteins by PRMTs, illustrating the previously unknown importance of PRMTs in the regulation of various cellular functions by post-translational modifications. In this review, we present the recent developments in the regulation of non-histone proteins by PRMTs.

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.

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.

Regulation of NF-úB: Arginine Methylation Takes the Stage

Nuclear factor κB (NF-κB) is a family of transcription factors that play central roles in multiple biological functions, including immune and inflammatory responses and tumorigenesis. The regulation of the activity of NF-κB is very sophisticated. Post-translational modifications of NF-κB have long been known to play an essential role in the regulation of NF-κB activity. Since the p65 subunit (RelA) is the major subunit of the typical NF-κB heterodimer, more attention has been recently devoted to how p65 is regulated by the post-translational modifications. In this review, we provide an overview of the most recent developments in the regulation of p65 by post-translational modifications.

Abstract 5273: VBIM technology identifies beta-catenin-like protein (BCLP) as a novel negative regulator of NF-kB

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Colon cancer is the second leading cause of cancer related deaths in the United States. The nuclear factor kB (NF-kB) is an important family of transcription factors whose aberrant activation has been found in many types of cancer, including colon cancer. Therefore, understanding the regulation of NF-kB is of ultimate importance for cancer therapy. The purpose of this study is to use a novel validation-based insertional mutagenesis (VBIM) strategy to identify novel regulators of NF-kB, and further evaluate their roles in the regulation of NF-kB signaling in colon cancer cells. We infected D1 cells (293 derived cells with hyper active NF-kB activity) with VBIM virus to cause the overexpression of negative regulators of NF-kB, and then further selected the mutant cells with low NF-kB activity under ganciclovir (GCV) treatment. Targeted gene was then identified by using VBIM specific primers. In a preliminary screen, we identified the novel beta-catenin like protein (BCLP) gene as a negative regulator of NF-kB. Overexpression of BCLP led to decreased NF-kB activity by kB reporter assay, while knocking it down had the opposite effect. Furthermore, we found that overexpression of BCLP in HT29 colon cancer cells greatly reduced both the number and the size of colonies that were formed in a soft agar assay, while sh-RNA mediated knockdown resulted in an opposite effect, confirming that BCLP is a tumor suppressor in HT29 cells. Our future experiments aim to further assess the role of BCLP in colon tumor formation in a mouse xenograft model. In summary, by using the novel VBIM technique, we identified BCLP as a novel negative regulator of NF-kB. This discovery could lead to the establishment of BCLP as a potential biomarker and therapeutic target in colon cancer. Citation Format: Rasika Mundade, Tao Lu. VBIM technology identifies beta-catenin-like protein (BCLP) as a novel negative regulator of NF-kB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5273. doi:10.1158/1538-7445.AM2014-5273