Robert E. Kingston

Preparation and Analysis of RNA

T he ability to isolate clean intact RNA from cells is essential for experiments that measure transcript levels, for cloning of intact cDNAs, and for functional analysis of RNA metabolism. RNA isolation procedures frequently must be performed on numerous different cell samples, and therefore are designed to allow processing of multiple samples simultaneously. This chapter begins by describing several methods commonly used to isolate RNA, and concludes with methods used to analyze RNA expression levels, RNA synthesis rates, and genome-wide location of RNAs.

Introduction of DNA into Mammalian Cells

After cloning a gene, many researchers wish to analyze its characteristics by reintroducing normal and mutant variants into various cell types. There are many reasons to do this. The genetic elements responsible for regulation of expression can be determined by mutating regulatory sequences and subsequently examining activity under a variety of physiological conditions. The effects that the gene has on cellular growth can be ascertained by characterizing the phenotypes of cell lines that either overexpress the gene, or that express mutant forms of the gene. Overexpressing cell lines can also be used for purification of the product for biochemical characterization, or large-scale production of a product for use as a drug. All these goals require the ability to introduce DNA into a cell efficiently. The protocols in this chapter cover methods that are used to introduce genes into mammalian cells, as well as techniques used to investigate their regulation, to express the gene products, and to delete the endogenous form of the gene.

Chapter 16 Control of Class II Gene Transcription during in Vitro Nucleosome Assembly

Publisher Summary This chapter discusses the control of class II gene transcription during in vitro nucleosome assembly and whether the competitive nucleosome assembly pathway creates conditions under which the regulatory activity of particular factors is more apparent. In vitro functional and structural studies of nucleosome and chromatin reconstitution should allow an investigation into the individual roles of multiple factors regulating a single transcription unit. Functional studies can address the role of a factor in establishing the transcriptional potential of a promoter in chromatin and in the initiation and elongation of transcription. Binding studies can be used to address the ability of a factor to bind to nucleosomal DNA and the fate of the nucleosome upon such binding. These in vitro approaches provide assays for the function of chromosomal structural proteins in transcription. Several laboratories have used in vitro approaches to investigate the involvement of nucleosomes in class II gene transcription..

DNA‐Protein Interactions

F or several decades, DNA-binding proteins have been studied because of their involvement in cellular processes such as replication, recombination, viral integration, and transcription. In recent years, the number of workers interested in the study of these proteins has greatly increased as the advent of recombinant DNA technology has led to the isolation of numerous biologically important genes. Many investigators are interested in how transcription of these genes is controlled in response to environmental or developmental signals, and have started to characterize the DNA sequences responsible for this regulation. This analysis has naturally led to the detection, isolation, and characterization of the proteins that bind to these regulatory sequence elements. This chapter summarizes the techniques currently used to characterize DNA-protein interactions and to isolate DNA-binding proteins.

Molecular biology: Specifying transcription

To switch on the right genes at the right times, our cells rely on many different proteins. Some help to remodel the architecture of the genome, and are remarkably choosy about which other proteins they work with.

Chromatin Assembly and Analysis

M any nuclear processes require that DNA be recognized by sequence-specific DNAbinding proteins. The packaging of DNA into chromatin inhibits DNA binding by most proteins, and also influences the efficiency of events such as transcription, replication, recombination, and DNA repair. These considerations have led to an increased interest in characterizing chromatin structure in general and the precise structural changes that can occur over specific chromosomal domains.

Identifying candidate ncRNAs that direct changes in chromatin structure

Background Long non-coding RNAs (ncRNAs) are increasingly recognized as important regulators of genomic processes and cell specification. Many IncRNAs are hypothesized to localize to and regulate chromatin, functionally impacting epigenetic state through interactions with chromatinmodifying proteins’. Numerous IncRNAs have been reported to play a role in the activity or recruitment of the epigenetic factors the Polycomb group (PcG) proteins to genomic sites 2,3 . However, identification and func

Chromatin: Assembly, remodelled

Biochemical assays reveal that nucleosome maturation and chromatin remodelling by the motor protein Chd1 are distinct, separable enzymatic activities.

The structure of Polycomb-repressed chromatin in the bithorax complex

The 300 kb of the Drosophila bithorax complex (BX-C) is the original model for studying Hox gene repression by Polycomb group proteins. Decades of genetic experiments have led to the hypothesis that Polycomb organizes BX-C chromatin differently in each of the abdominal parasegments, and the structure of the chromatin is critical for maintaining cell identity. Studying the molecular organization of chromatin in individual parasegments has been technically difficult. We solved this problem by developing a sorted nuclei ChIP-seq pipeline. In this system, transgenic embryos produce tagged nuclei in single parasegments. Using FACS, we sorted these tagged nuclei and performed small-scale ChIP-seq. Initial results show that a histone mark catalyzed by Polycomb group proteins, trimethylated histone H3 at lysine 27 (H3K27me3), covers less of the BX-C as we move from PS5 to PS7. The boundaries of the H3K27 methylation correlate precisely with previously identified CTCF binding sites, a protein known to act as a chromatin barrier. Correspondingly, a mark that correlates with gene activation, H3K4me3, appears over the transcription start sites of BX-C genes that are not associated with Polycomb histone methyltransferase activity. Going forward, we will assay the localization of Polycomb group proteins and many other chromatin regulatory proteins to gain a complete picture of the chromatin environment that regulates Hox gene expression in the developing embryo. Still, the current results offer a compelling first look at BX-C chromatin in vivo, and provide molecular support to a long-standing genetic hypothesis.

Identification of regions in the HOX cluster that can confer repression in a Polycomb-dependent manner

Polycomb Group (PcG)-mediated repression is critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We have identified and characterized two new potential PcG-dependent regulatory elements within the human HOXB and HOXC clusters. Their repressive activities are similar to a previously identified element in the HOXD cluster. The PcG proteins BMI1 and SUZ12 are recruited to a reporter construct in mesenchymal stem cells and confer repression that was dependent upon PcG expression. In addition, JARID2 was observed to localize to these three elements. Interestingly, the requirement for JARID2 is variable at the different regions despite its localization. We conclude that distinct regions of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression and that JARID2 plays a role in the recruitment of PRC2.