Eukaryotic Gene Regulation

Volume I

Gerald M. Kolodny

• Publisher: CRC Press
• Year: 2018
• ISBN:
  9781351088831
• Edition: 1
• Topic: Medizin
• Subtopic: Alternativ- & Komplementärmedizin

The cause of cancer and its many manifestations is at present unknown. Since many of its manifestations, including is control of cell division, appear to represent abnormal patterns of gene expression, studies of the regulation of gene expression nwill provide important insights in the understanding and treatment of cancer. This volume attempts to present some of the recent work on regulation of gene expression in eukaryotic cells.

Chapter 1
CHROMATIN STRUCTURE AND GENE REGULATION

R. S. Gilmour

TABLE OF CONTENTS

1. I. Introduction
2. II. The in Vitro Transcription of the Globin Gene from Chromatin
   1. A. Endogenous RNA
   2. B. Transcription with Mercurated Nucleotides
   3. C. Fidelity of In Vitro Transcription
   4. D. In Vitro Transcription of the Globin Gene in Mouse Fetal Liver Chromatin
      1. 1. Transcription of Fetal Liver Chromatin with Hg-UTP
      2. 2. Transcription of Reconstituted Mouse Fetal Liver Chromatin
3. III. Chromatin Reconstitution
   1. A. History
   2. B. Transcription from Reconstituted Chromatin
   3. C. Parameters Affecting Reconstitution
4. IV. Structural Studies on Chromatin Relating to Structure
   1. A. The Nucleosome
   2. B. The Structure of Transcribing Chromatin
5. V. Conclusions
6. References

I. INTRODUCTION

According to current views, the process of selective gene transcription is the predominant mechanism by which tissue-specific functions of differentiated cells are established. A demonstration of selective gene expression requires evidence for a selection of DNA sequences transcribed into RNA. Early investigations using nucleic acid hybridization of the sequence homologies of total cellular RNA isolated from a variety of species demonstrated that qualitatively different RNA molecules are present in different cell types of the same organism; for example, in mouse tissues,¹¹³ in developmental stages of sea urchin embryos⁶³ and embryonic mouse liver,²⁸ in Xenopus oocytes and blastulae,³⁶ and in mouse liver and uterus before and after estrogen stimulation.²⁹ In all of these examples, hybridization conditions were such that only differences in the transcripts from repetitive DNA were detected. In later experiments, transcripts derived from unique DNA sequences were analyzed by hybridizing isolated unique DNA to a large excess of total cellular RNA or, in some cases, nuclear RNA. There again, distinctly different populations of unique sequences are present in the RNAs of different tissues as demonstrated in adult mouse tissues,¹⁷,⁷¹ Dictyostelium developmental stages,⁴⁵ and chick oviduct before and after estrogen stimulation.¹⁰³

The recent use of globin cDNA hybridization probes by a number of workers has extended this conclusion for the particular case of globin raRNA in erythroid vs. non-erythroid cellular RNA.²,⁶²,⁸⁵,⁹⁹,¹⁶²

The recent use of globin cDNA hybridization probes by a number of workers has extended this conclusion for the particular case of globin mRNA in erythroid vs. no-nerythroid cellular RNA.²,⁶²,⁸⁵,⁹⁹,¹⁶²

These results, from a wide variety of sources, attest to the generality and significance of differential gene transcription. Selective sequences of both repetitive and unique DNA are transcribed in vivo in a tissue-specific fashion.

More than 10 years ago the discovery that chromatin can act as a template for in vitro RNA transcription by bacterial RNA polymerase suggested a cell-free approach to studying selective gene expression. Much of this early work was limited technically by the inability to assess the precise qualitative nature of the transcribed RNA. Within recent years, the availability of complementary DNA (cDNA) reverse-transcribed from purified mRNA has made possible the analysis of chromatin transcripts for specific mRNA sequences. In particular, a number of groups reported the in vitro transcription of globin mRNA from erythroid chromatin in a number of tissues and contrasted this with a corresponding lack of these sequences in transcripts from nonerythroid chromatin of the same species.²,⁷,⁵⁹,¹⁶² In analogous studies in other systems, Harris et al.⁷⁶ found ovalbumin sequences in the transcripts from estrogen-stimulated chick oviduct chromatin as compared with unstimulated oviduct chromatin. Likewise, Stein et al.¹⁶⁰ described the in vitro transcription of histone genes in the chromatin from S-phase cells but not in Gl phase chromatin. In a number of these systems, the nature of the mechanism which confers gene specificity has been investigated using chromatin reconstitution techniques.

Recently the validity of experiments involving chromatin transcription and reconstitution has been questioned on a number of grounds. The purpose of this article is to consider these criticisms with particular reference to the in vitro transcription of the globin gene in mouse fetal liver. A discussion of chromatin function would, however, be incomplete without considering also its structural organization; a review of this aspect is included in the hope that together they provide an insight into how selective gene transcription might operate.

II. THE IN VITRO TRANSCRIPTION OF THE GLOBIN GENE FROM CHROMATIN

Reports of in vitro transcription of specific genes has come from a wide variety of tissues. This article, by necessity, will consider results from many of these. However, it must be pointed out that many assumptions have to be made. In addition to chromatin source, there are other variables to consider, as illustrated in the following paragraphs.

For example, there is no absolute definition of chromatin. Empirically speaking, chromatin is a preparation of washed, swollen nuclei. However, investigators have employed a variety of methods to prepare the nuclei and a bewildering battery of buffers of varying ionic strength and composition to wash them. It is generally assumed that within reason these variations have little influence on subsequent experiments; however, this has never been investigated thoroughly.

In most examples to be discussed, chromatin is incubated with bacterial RNA polymerase. The activity of this enzyme on DNA templates is influenced considerably by a number of factors, e.g., manganese ion and salt concentrations. However, a survey of the incubation conditions employed to transcribe chromatin shows considerable variability in nucleoside triphosphate, magnesium ion, manganese ion, salt, and RNA polymerase concentrations. If it is assumed that in each case the different conditions are nevertheless optimal for the system in question, to what extent can data obtained from different systems be compared? Also, to what extent are conditions optimized by the incorporation of labeled nucleoside triphosphates also optimal for the transcription of tissue-specific RNA sequences? To the author’s knowledge, no thorough examination has been carried out on the influence of ionic conditions on sequence specificity of the transcribed product. It is important to bear in mind the implications of these imponderables when comparing results from different systems; however, what is surprising perhaps is the fact that there is a broad area of agreement in the chromatin transcription data obtained from such a wide variation in experimental parameters.

A. Endogenous RNA

The most serious criticism of chromatin transcription experiments is that any in vivo synthesized endogenous RNA isolated along with the chromatin will be indistinguishable from in vitro RNA synthesized by the added bacterial polymerase. Endogenous RNA is tightly bound to chromatin and can only be removed by drastic procedures such as RNAse digestion or total dissociation of the chromatin in cesium chloride.⁶⁰ In addition, contamination from cytoplasmic mRNA can also contribute to the background, depending on the tissue and the method used to prepare chromatin. Despite this fact, many reports show negligible background hybridization in control incubations where chromatin is incubated in the absence of RNA polymerase.

The reasons for this are not clear. It is possible that there are vastly different levels of endogenous RNA in the various systems studied. Variations in the procedures used for isolating and transcribing chromatin and for isolating the in vitro transcript might also contribute. However, in later studies with mouse fetal liver chromatin⁶⁰ and with rabbit bone marrow chromatin,¹⁸² the presence of contaminating endogenous globin RNA was found to affect significantly the estimation of globin-specific sequences in the RNA transcript...

Table of contents

• Cover
• Title Page
• Copyright Page
• FOREWORD
• THE EDITOR
• CONTRIBUTORS
• TABLE OF CONTENTS
• Chapter 1 Chromatin Structure and Gene Regulation
• Chapter 2 The Structure of Transcriptons and The Regulation of Transcription
• Chapter 3 Cascade Regulation a Model of Integrative Control of Gene Expression in Eukaryotic Cells and Organisms
• Chapter 4 Selective Gene De-Repression By De-Repressor RNA
• Chapter 5 The Primer Hypothesis for the Regulation of Eukaryotic Gene Expression
• Index