3. How Genomes FunctionIn order for the cell to utilize the biological information contained within its genome, groups of genes, each gene representing a single unit of information, have to be expressed in a coordinated manner. This coordinated gene expression determines the make-up of the transcriptome, which in turn specifies the nature of the proteome and defines the activities that the cell is able to carry out.In Part 3 of Genomes we examine the events that result in the transfer of biological information from genome to proteome. Our knowledge of these events was initially gained through studies of individual genes, often as ‘naked' DNA in test-tube experiments. These experiments provided an interpretation of gene expression that in recent years has been embellished by more sophisticated studies that have taken greater account of the fact that, in reality, it is the genome that is expressed, not individual genes, and that this expression occurs in living cells rather than in a test tube.We begin our investigation of genome expression in Chapter 8, by examining the substantial and important impact that the nuclear environment has on the utilization of the biological information contained in the genomes of eukaryotes, the accessibility of that information being dependent on the way in which the DNA is packaged into chromatin and being responsive to processes that can silence or inactivate part or all of a chromosome.Chapter 9 describes the events involved in initiation of transcription, and emphasizes the critical role the DNA-binding proteins play during the early stages of genome expression. The synthesis of transcripts and their subsequent processing into functional RNAs is dealt with in Chapter 10, and Chapter 11 covers the equivalent events that lead to synthesis of the proteome.As you read Chapters 8–11 you will discover that control over the composition of the transcriptome and of the proteome can be exerted at various stages during the overall chain of events that make up genome expression. These regulatory threads will be drawn together in Chapter 12, where we examine how genome activity changes in response to extracellular signals during differentiation and development.

8. Accessing the Genome 9. Assembly of the Transcription Initiation Complex 10. Synthesis and Processing of RNA 11. Synthesis and Processing of the Proteome 12. Regulation of Genome Activity

8. Accessing the Genome

Learning outcomes

When you have read Chapter 8, you should be able to:1.Explain how chromatin structure influences genome expression 2.Describe the internal architecture of the eukaryotic nucleus 3.Distinguish between the terms ‘constitutive heterochromatin', ‘facultative heterochromatin' and ‘euchromatin' 4.Discuss the key features of functional domains, insulators, and locus control regions, and describe the experimental evidence supporting our current knowledge of these structures 5.Describe the various types of chemical modification that can be made to histone proteins, and link this information to the concept of the ‘histone code' 6.State why nucleosome positioning is important in gene expression and give details of a protein complex involved in nucleosome remodeling 7.Explain how DNA methylation is carried out and describe the importance of methylation in silencing the genome 8.Give details of the involvement of DNA methylation in genomic imprinting and X inactivation

8.1. Inside the Nucleus 8.2. Chromatin Modifications and Genome Expression

8.1. Inside the Nucleus

Figure 8.1. The internal architecture of the eukaryotic nucleus. (A) Transmission electron micrograph showing the nuclear matrix of a cultured human HeLa cell. Cells were treated with a non-ionic detergent to remove membranes, digested with a deoxyribonuclease to degrade most of the DNA, and extracted with ammonium sulfate to remove histones and other chromatin-associated proteins. From Molecular Cell Biology, by H Lodish, A Berk, SL Zipursky, P Matsudaira, D Baltimore and J Darnell. ©1986, 1990, 1995, 2000 by WH Freeman and Company. Used with permission. (B) and (C) Images of living nuclei containing fluorescently labeled proteins (see Technical Note 8.1). In (B), the nucleolus is shown in blue and Cajal bodies in yellow. The purple areas in (C) indicate the positions of proteins involved in RNA splicing. B and C from Misteli, Science, 291, 843–847. Copyright 2000 American Association for the Advancement of Science.

Figure 8.2. A scheme for organization of DNA in the nucleus. The nuclear matrix is a fibrous protein-based structure whose precise composition and arrangement in the nucleus has not been described. Euchromatin, predominantly in the form of the 30 nm chromatin fiber (see Figure 2.6 ) is thought to be attached to the matrix by AT-rich sequences called matrix-associated or scaffold attachment regions (MARs or SARs)

Figure 8.3. A functional domain in a DNase I sensitive region.

Figure 8.4. Insulator sequences in the fruit-fly genome. The diagram shows the region of the Drosophila genome containing the two hsp70 genes. The insulator sequences scs and scs are either side of the gene pair. The arrows below the two ′genes indicate that they lie on different strands of the double helix and so are transcribed in opposite directions.

Figure 8.5. The positional effect. (A) A cloned gene that is inserted into a region of highly packaged chromatin will be inactive, but one inserted into open chromatin will be expressed. (B) The results of cloning experiments without (red) and with (blue) insulator sequences. When insulators are absent, the expression level of the cloned gene is variable, depending on whether it is inserted into packaged or open chromatin. When flanked by insulators, the expression level is consistently high because the insulators establish a functional domain at the insertion site.

Figure 8.6. Insulators maintain the independence of a functional domain. (A) When placed between a gene and its upstream regulatory modules, an insulator sequence prevents the regulatory signals from reaching the gene. (B) In their normal positions, insulators prevent cross-talk between functional domains, so the regulatory modules of one gene do not influence expression of a gene in a different domain. For more details about regulatory modules, see Box 9.6

Figure 8.7. DNase I hypersensitive sites indicate the position of the locus control region for the human β-globin gene cluster. A series of hypersensitive sites are located in the 20 kb of DNA upstream of the start of the β-globin gene cluster. These sites mark the position of the locus control region. Additional hypersensitive sites are seen immediately upstream of each gene, at the position where RNA polymerase attaches to the DNA. These hypersensitive sites are specific to different developmental stages, being seen only during the phase of development when the adjacent gene is active. The 60 kb region shown here represents the entire β-globin functional domain. See Figure 2.14 for more information on the developmental regulation of expression of the β-globin gene cluster.

Technical Note 8.1. Fluorescence recovery after photobleaching (FRAP)

8.2. Chromatin Modifications and Genome Expression

Figure 8.8. Two ways in which chromatin structure can influence gene expression. A region of unpackaged chromatin in which the genes are accessible is flanked by two more compact segments. Within the unpackaged region, the positioning of the nucleosomes influences gene expression. On the left, the nucleosomes have regular spacing, as displayed by the typical ‘beads-on-a-string' structure. On the right, the nucleosome positioning has changed and a short stretch of DNA, approximately 300 bp, is exposed. See Figures 2.5 and 2.6 for more details on nucleosomes.

Figure 8.9. Two views of the nucleosome core octamer. The view on the left is downwards from the top of the barrel-shaped octamer; the view on the right is from the side. The two strands of the DNA double helix wrapped around the octamer are shown in brown and green. The octamer comprises a central tetramer of two histone H3 (blue) and two histone H4 (bright green) subunits plus a pair of H2A (yellow)–H2B (red) dimers, one above and one below the central tetramer. Note the N-terminal tails of the histone proteins protruding from the core octamer. Reprinted with permission from Luger et al., Nature, 389, 251–260. Copyright 1997 Macmillan Magazines Limited

Figure 8.10. Nucleosome remodeling, sliding and transfer

Figure 8.11. Maintenance methylation and de novo methylation

Figure 8.12. A model for the link between DNA methylation and genome expression. Methylation of the CpG island upstream of a gene provides recognition signals for the methyl-CpG-binding protein (MeCP) components of a histone deacetylase complex (HDAC). The HDAC modifies the chromatin in the region of the CpG island and hence inactivates the gene. Note that the relative positions and sizes of the CpG island and the gene are not drawn to scale.

Figure 8.13. A pair of imprinted genes on human chromosome 11. Igf2 is imprinted on the chromosome inherited from the mother, and H19 is imprinted on the paternal chromosome. The drawing is not to scale: the two genes are approximately 90 kb apart.

Research Briefing 8.1. Discovery of the mammalian