ABSTRACT
Overgrowth syndromes such as Perlman syndrome and associated pediatric cancers, including Wilms tumor, arise through genetic and, in certain instances, also epigenetic changes. In the case of the Beckwith-Wiedemann overgrowth syndrome and in Wilms tumor, increased levels of IGF2 have been shown to be causally related to the disease manifestation. In the previous issue of Genes & Development, Hunter and colleagues (pp. 903-908) investigated the molecular mechanisms by which mutations in the gene encoding the RNA degradation component DIS3L2 lead to Perlman syndrome. By analyzing nephron progenitor cells derived from their newly created Dis3l2 mutant mouse lines, the investigators showed that DIS3L2 loss of function leads to up-regulation of IGF2 independently of the let7 microRNA pathway. In a second study in this issue of Genes & Development, Chen and colleagues (pp. 996-1007) show that microRNA processing gene mutations in Wilms tumor lead to an increase in the levels of transcription factor pleomorphic adenoma gene 1 (PLAG1) that in turn activates IGF2 expression. Thus, augmented IGF2 expression seems to be a common downstream factor in both tissue overgrowth and Wilms tumor through several alternative mechanisms.
Subject(s)
Wilms Tumor , Animals , Child , Exoribonucleases/genetics , Female , Fetal Macrosomia , Humans , Insulin-Like Growth Factor II/genetics , Mice , Nephrons , Pregnancy , Stem Cells , Syndrome , Up-RegulationABSTRACT
Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.
Subject(s)
3' Untranslated Regions/physiology , Gene Expression Regulation, Developmental/genetics , RNA, Messenger/genetics , Wilms Tumor/genetics , Wilms Tumor/metabolism , Animals , Cell Line , Down-Regulation , Gene Deletion , Kidney/cytology , Mesoderm/metabolism , Mice , Mouse Embryonic Stem Cells , RNA, Messenger/metabolismABSTRACT
Tumor-suppressor protein Wt1 has been shown to interact with specific proteins that influence its function. These protein interactions have been identified as direct individual interactions but with the potential to exist as a part of a multiprotein complex. In order to obtain the global proteome interaction map of Wt1, an unbiased label-free endogenous immunoprecipitation was performed followed by mass spectrometry to identify protein interactions that are Wt1 centric. This chapter details the different techniques that have been used to identify and characterize Wt1-interacting proteins.
Subject(s)
Protein Interaction Mapping/methods , Protein Interaction Maps , WT1 Proteins/metabolism , Cells, Cultured , Humans , Immunoprecipitation , Mass Spectrometry , Proteomics , Signal Transduction , Two-Hybrid System TechniquesABSTRACT
Differential gene expression analysis has been conventionally performed by microarray techniques; however with the recent advent of next-generation sequencing (NGS) approaches, it has become easier to analyze the coding as well as the noncoding components. Additionally, NGS data analysis also provides information regarding the expression changes of specific isoforms. There are several bioinformatics tools available to analyze NGS data but with different parameters. This chapter provides a comparative insight into these tools by utilizing NGS datasets available from Wt1 knockout and embryonic stem cell line model.
Subject(s)
Computational Biology/methods , Gene Expression Profiling/methods , Mouse Embryonic Stem Cells/cytology , Repressor Proteins/genetics , Animals , Cells, Cultured , Gene Knockout Techniques , High-Throughput Nucleotide Sequencing , Mice , Protein Isoforms/genetics , Sequence Analysis, RNA , WT1 ProteinsABSTRACT
Tumor suppressor protein, Wt1 is a transcription factor that binds to DNA sequence similar to the Early Growth Response gene, EGR1 consensus binding sequence. Biophysical and biochemical validations have shown that the zinc fingers of Wt1 are capable of binding to both DNA and RNA albeit with different binding affinities which potentially is also isoform specific. SELEX based identification of the RNA binding motifs led to the identification of motifs which could not be translated into the in vivo context. With the advent of recent technologies that allow cross-linking of RNA and protein and high throughput sequencing techniques, it is now possible to analyze the in vivo RNA binding interactome of Wt1. This chapter outlines the initial studies that were aimed at addressing the Wt1 RNA interactome and also provides a detailed overview of some of the recent techniques used.
