Chapter Introduction

10: Nucleosomes, Chromatin, and Chromosome Structure

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  • 10.1 Nucleosomes: The Basic Units of DNA Condensation

  • 10.2 Higher-Order Chromosome Structure

  • 10.3 Regulation of Chromosome Structure

MOMENT OF DISCOVERY

C. David Allis

In the early 1990s, there was a huge attempt by many top-notch laboratories to purify an enzyme that would modify histones. In particular, histones were known to be acetylated, but the presumptive “histone acetylase” enzyme that carries out this important task eluded the grasp of biochemists who had tried for years to identify it. Did it even really exist? Was there another way nature devised to modify histones? I didn’t think that would be the case, and my laboratory was one of those dedicated to solving this mystery. Then in 1996 it finally happened, and it was a really neat succession of events that I’ll always remember. Working with my graduate student, Jim Brownell, we isolated a 55,000 dalton protein (p55) that displayed histone acetyltransferase (HAT) activity from enriched extracts of Tetrahymena in an in-gel HAT assay. This was it! Finding this evidence provided the stimulus needed for a really Herculean effort. Brownell grew about 200 L of Tetrahymena culture, and after purification of p55 there was just a couple of gel lanes worth of purified protein. But it was enough for microsequencing of p55 peptides by R. Kobayashi (working at Cold Spring Harbor Laboratory), which provided the information to clone the p55 gene.

I vividly recall the weekend day that Brownell biked to the lab to run all of his precious, most highly purified p55 on a gel, then transferred it to a sequencing-compatible membrane and sent it to Kobayashi. This gel was so precious to us that we both kept watching it every minute to make sure all was going well. As his advisor, I tried to appear calm and collected on the outside; on the inside, I was going crazy. What an important gel this turned out to be! Totally unexpected to us, p55 from Tetrahymena was homologous to Gcn5, a known transcriptional coactivator in yeast. This discovery essentially established that Gcn5/p55 performed its transcriptional function by being a HAT, an enzyme that adds an acetyl group to regulatory lysines on histones. This seminal finding was published in Cell on March 22, 1996, which, remarkably, happened to be my birthday—I couldn’t imagine a better birthday present. With help from Sharon Dent, Brownell expressed and purified the yeast Gcn5, and it, too, was active in the in-gel HAT assay! I remember when Brownell developed the x-ray film of the gel, and upon seeing the positive result, he was barely able to talk: he could only run around wildly, jumping up and down the hallway. I felt the same way. The image of Jim, and the gel result, too, make for a day I will never forget.

—C. David Allis, on establishing that p55 from Tetrahymena is a histone acetylase, as is transcription factor Gcn5

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Eukaryotes contain thousands of times more DNA than do bacteria, and as a result, the DNA condensation problems of eukaryotes—compacting the DNA so that it fits in the cell nucleus—are more complex than those of bacteria. In Chapter 9 we introduced DNA topoisomerases, enzymes that can untwist DNA and keep the long DNA molecules within cellular chromosomes from becoming intertwined. We also discussed the ring-shaped condensins and cohesins that encircle DNA segments to hold them tightly together in loops, thus increasing compaction. This chapter focuses on a specific DNA condensation particle of eukaryotes—the nucleosome—around which DNA is wrapped. Bacteria do not contain nucleosomes, although they have small, basic (positively charged) proteins that are involved in condensing their DNA.

DNA compaction must be dynamic: changes in the degree of condensation must occur quickly and when needed, as the cell passes through the stages of the cell cycle (see Figure 2-10). Furthermore, when in its most highly compacted form, DNA is not accessible to transcription or replication enzymes, so it must be able to rapidly expose regions containing genes that are required at any given moment, and then condense again. Changes in DNA compaction in a cell can occur on a global level (such as during mitosis or replication) or on a local level (such as giving access to specific genes for transcription regulation). To accommodate these essential activities, modification enzymes have evolved that alter the state of DNA condensation by various means, and these enzymes can target their activity to specific regions of the chromosome that must be transcribed or replicated.

In this chapter we explore how nucleosome units are arranged in higher levels of chromosome structure and how the cell manipulates nucleosomes in various ways to change the state of DNA condensation, which affects the regulation of gene expression. We then look at how nucleosomes are modified by enzymes that attach various small chemicals to the nucleosome proteins. These chemical alterations regulate genes and, in fact, are inherited, passing down this information from one cell generation to the next. This is especially important during an organism’s development, to maintain new transcriptional programs of differentiated cell types. Genetic information not coded by the DNA sequence is referred to as epigenetic information, and defects in epigenetic information are associated with cancer.