1. Chapter 19 Nucleosomes

2. 19.1 Introduction 19.2 The nucleosome is the subunit of all chromatin 19.3 DNA is coiled in arrays of nucleosomes 19.4 Nucleosomes have a common structure 19.5 DNA structure varies on the nucleosomal surface 19.6 Supercoiling and the periodicity of DNA 19.7 The path of nucleosomes in the chromatin fiber 19.8 Organization of the histone octamer 19.9 Histones are modified 19.10 Reproduction of chromatin requires assembly of nucleosomes 19.11 Do nucleosomes lie at specific positions? 19.12 Are transcribed genes organized in nucleosomes? 19.13 Histone octamers are displaced by transcription 19.14 DNAase hypersensitive sites change chromatin structure 19.15 Domains define regions that contain active genes 19.16 Heterochromatin propagates from a nucleation event 19.17 Heterochromatin depends on interactions with histones 19.18 X chromosomes undergo global changes 19.19 Chromosome condensation is caused by condensins 19.20 Methylation is perpetuated by a maintenance methylase 19.21 Methylation is responsible for imprinting 19.22 Epigenetic effects can be inherited 19.23 Yeast prions show unusual inheritance 19.24 Prions cause diseases in mammals

3. Histones are conserved DNA-binding proteins of eukaryotes that form the nucleosome, the basic subunit of chromatin. Nucleosome is the basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins. 19.1 Introduction

4. Figure 18.9 The sister chromatids of a mitotic pair each consist of a fiber (~30 nm in diameter) compactly folded into the chromosome. Photograph kindly provided by E. J. DuPraw. 19.1 Introduction

5. Micrococcal nuclease is an endonuclease that cleaves DNA; in chromatin, DNA is cleaved preferentially between nucleosomes. 19.2 The nucleosome is the subunit of all chromatin

6. Figure 19.1 Chromatin spilling out of lysed nuclei consists of a compactly organized series of particles. The bar is 100 nm. Photograph kindly provided by Pierre Chambon. 19.2 The nucleosome is the subunit of all chromatin

7. Figure 19.2 Individual nucleosomes are released by digestion of chromatin with micrococcal nuclease. The bar is 100 nm. Photograph kindly provided by Pierre Chambon. 19.2 The nucleosome is the subunit of all chromatin

8. Figure 19.3 The nucleosome consists of approximately equal masses of DNA and histones (including H1). The predicted mass of the nucleosome is 262 kD. 19.2 The nucleosome is the subunit of all chromatin

9. Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface. 19.2 The nucleosome is the subunit of all chromatin

10. Figure 19.5 The two turns of DNA on the nucleosome lie close together. 19.2 The nucleosome is the subunit of all chromatin

11. Figure 19.6 Sequences on the DNA that lie on different turns around the nucleosome may be close together. 19.2 The nucleosome is the subunit of all chromatin

12. Core DNA is the 146 bp of DNA contained on a core particle. Core particle is a digestion product of the nucleosome that retains the histone octamer and has 146 bp of DNA; its structure appears similar to that of the nucleosome itself. Linker DNA is all DNA contained on a nucleosome in excess of the 146 bp core DNA. 19.3 DNA is coiled in arrays of nucleosomes

13. Figure 19.7 Micrococcal nuclease digests chromatin in nuclei into a multimeric series of DNA bands that can be separated by gel electrophoresis. Photograph kindly provided by Markus Noll. 19.3 DNA is coiled in arrays of nucleosomes

14. Figure 19.8 Each multimer of nucleosomes contains the appropriate number of unit lengths of DNA. Photograph kindly provided by John Finch. 19.3 DNA is coiled in arrays of nucleosomes

15. Figure 19.9 Micrococcal nuclease reduces the length of nucleosome monomers in discrete steps. Photograph kindly provided by Roger Kornberg. 19.3 DNA is coiled in arrays of nucleosomes

16. Figure 19.10 Microccocal nuclease initially cleaves between nucleosomes. Mononucleosomes typically have ~200 bp DNA. End-trimming reduces the length of DNA first to ~165 bp, and then generates core particles with 146 bp. 19.3 DNA is coiled in arrays of nucleosomes

17. Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface. 19.3 DNA is coiled in arrays of nucleosomes

18. Figure 19.11 Nicks in double-stranded DNA are revealed by fragments when the DNA is denatured to give single strands. If the DNA is labeled at (say) 5 ends, only the 5 fragments are visible by autoradiography. The size of the fragment identifies the distance of the nick from the labeled end. 19.4 DNA structure varies on the nucleosomal surface

19. Figure 2.4 When restriction fragments are identified by their possession of a labeled end, each fragment directly shows the distance of a cutting site from the end. Successive fragments increase in length by the distance between adjacent restriction sites. 19.4 DNA structure varies on the nucleosomal surface

20. Figure 19.12 Sites for nicking lie at regular intervals along core DNA, as seen in a DNAase I digest of nuclei. Photograph kindly provided by Leonard Lutter. 19.4 DNA structure varies on the nucleosomal surface

21. Figure 19.13 Two numbering schemes divide core particle DNA into 10 bp segments. Sites may be numbered S1 to S13 from one end; or taking S7 to identify coordinate 0 of the dyad symmetry, they may be numbered -7 to +7. 19.4 DNA structure varies on the nucleosomal surface

22. Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface. 19.4 DNA structure varies on the nucleosomal surface

23. Figure 19.14 The most exposed positions on DNA recur with a periodicity that reflects the structure of the double helix. (For clarity, sites are shown for only one strand.) 19.4 DNA structure varies on the nucleosomal surface

24. Figure 19.15 High resolution analysis shows that each site for DNAase I consists of several adjacent susceptible phosphodiester bonds as seen in this example of sites S4 and S5 analyzed in end-labeled core particles. Photograph kindly provided by Leonard Lutter. 19.4 DNA structure varies on the nucleosomal surface

25. Linking number paradox describes the discrepancy between the existence of -2 supercoils in the path of DNA on the nucleosome compared with the measurement of -1 supercoil released when histones are removed. Minichromosome of SV40 or polyoma is the nucleosomal form of the viral circular DNA. 19.5 Supercoiling and the periodicity of DNA

26. Figure 19.16 The supercoils of the SV40 minichromosome can be relaxed to generate a circular structure, whose loss of histones then generates supercoils in the free DNA. 19.5 Supercoiling and the periodicity of DNA

27. Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface. 19.5 Supercoiling and the periodicity of DNA

28. Figure 19.17 The 10 nm fiber in partially unwound state can be seen to consist of a string of nucleosomes. Photograph kindly provided by Barbara Hamkalo. 19.6 The path of nucleosomes in the chromatin fiber

29. Figure 19.18 The 10 nm fiber is a continuous strong of nucleosomes. 19.6 The path of nucleosomes in the chromatin fiber

30. Figure 19.19 The 30 nm fiber has a coiled structure. Photograph kindly provided by Barbara Hamkalo. 19.6 The path of nucleosomes in the chromatin fiber

31. Figure 19.20 The 30 nm fiber may have a helical coil of 6 nucleosomes per turn, organized radially. 19.6 The path of nucleosomes in the chromatin fiber

32. Figure 19.21 In a symmetrical model for the nucleosome, the H32- H42 tetramer provides a kernel for the shape. One H2A-H2B dimer can be seen in the top view; the other is underneath. 19.7 Organization of the histone octamer

33. Figure 19.22 The crystal structure of the histone core octamer is represented in a space-filling model with the H32-H42 tetramer shown in white and the H2A-H2B dimers shown in blue. Only one of the H2A-H2B dimers is visible in the top view, because the other is hidden underneath. The potential path of the DNA is shown in the top view as a narrow tube (one quarter the diameter of DNA), and in the side view by the parallel lines in a 20 ? wide bundle. Photographs kindly provided by Evangelos Moudrianakis. 19.7 Organization of the histone octamer

34. Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface. 19.7 Organization of the histone octamer

35. Figure 19.23 Histone positions in a top view show H3-H4 and H2A-H2B pairs in a half nucleosome; the symmetrical organization can be seen in the superimposition of both halves. 19.7 Organization of the histone octamer

36. Figure 19.24 The globular bodies of the histones are localized in the histone octamer of the core particle, but the locations of the N-terminal tails, which carry the sites for modification, are not known, and could be more flexible. 19.7 Organization of the histone octamer

37. Figure 19.25 Acetylation of lysine or phosphorylation of serine reduces the overall positive charge of a protein. 19.7 Organization of the histone octamer

38. Figure 19.26 Replicated DNA is immediately incorporated into nucleosomes. Photograph kindly provided by S. MacKnight. 19.8 Reproduction of chromatin requires assembly of nucleosomes

39. Figure 19.27 In vitro, DNA can either interact directly with an intact (crosslinked) histone octamer or can assemble with the H32-H42 tetramer, after which two H2A-H2B dimers are added. 19.8 Reproduction of chromatin requires assembly of nucleosomes

40. Figure 19.28 If histone octamers were conserved, old and new octamers would band at different densities when replication of heavy octamers occurs in light amino acids (part 1); but actually the octamers band diffusely between heavy and light densities, suggesting disassembly and reassembly (part 2). 19.8 Reproduction of chromatin requires assembly of nucleosomes

41. Figure 19.29 Nucleosome positioning places restriction sites at unique positions relative to the linker sites cleaved by micrococcal nuclease. 19.9 Do nucleosomes lie at specific positions?

42. Figure 19.30 In the absence of nucleosome positioning, a restriction site lies at all possible locations in different copies of the genome. Fragments of all possible sizes are produced when a restriction enzyme cuts at a target site (red) and micrococcal nuclease cuts at the junctions between nucleosomes (green). 19.9 Do nucleosomes lie at specific positions?

43. Figure 19.31 Translational positioning describes the linear position of DNA relative to the histone octamer. Displacement of the DNA by 10 bp changes the sequences that are in the more exposed linker regions, but does not alter which face of DNA is protected by the histone surface and which is exposed to the exterior. DNA is really coiled around the nucleosomes, and is shown in linear form only for convenience. 19.9 Do nucleosomes lie at specific positions?

44. Figure 19.32 Rotational positioning describes the exposure of DNA on the surface of the nucleosome. Any movement that differs from the helical repeat (~10.2 bp/turn) displaces DNA with reference to the histone surface. Nucleotides on the inside are more protected against nucleases than nucleotides on the outside. 19.9 Do nucleosomes lie at specific positions?

45. Figure 19.33 The extended axis of an rDNA transcription unit alternates with the only slightly less extended non-transcribed spacer. Photograph kindly provided by Charles Laird. 19.10 Are transcribed genes organized in nucleosomes?

46. Figure 19.34 An SV40 minichromo some can be transcribed. Photograph kindly provided by Pierre Chambon. 19.10 Are transcribed genes organized in nucleosomes?

47. Figure 19.35 RNA polymerase is comparable in size to the nucleosome and might encounter difficulties in following the DNA around the histone octamer. 19.10 Are transcribed genes organized in nucleosomes?

48. Figure 19.36 A protocol to test the effect of transcription on nucleosomes shows that the histone octamer is displaced from DNA and rebinds at a new position. 19.10 Are transcribed genes organized in nucleosomes?

49. Figure 19.37 RNA polymerase displaces DNA from the histone octamer as it advances. The DNA loops back and attaches (to polymerase or to the octamer) to form a closed loop. As the polymerase proceeds, it generates positive supercoiling ahead. This displaces the octamer, which keeps contact with DNA and/or polymerase, and is inserted behind the RNA polymerase. 19.10 Are transcribed genes organized in nucleosomes?

50. Figure 19.38 The URA3 gene has translationally positioned nucleosomes before transcription. When transcription is induced, nucleosome positions are randomized. When transcription is repressed, the nucleosomes resume their particular positions. Photograph kindly provided by Fritz Thoma. 19.10 Are transcribed genes organized in nucleosomes?

51. Figure 19.39 Indirect end-labeling identifies the distance of a DNAase hypersensitive site from a restriction cleavage site. The existence of a particular cutting site for DNAase I generates a discrete fragment, whose size indicates the distance of the DNAase I hypersensitive site from the restriction site. 19.11 DNAase hypersensitive sites change chromatin structure

52. Figure 19.40 The SV40 minichromosome has a nucleosome gap. Photograph kindly provided by Moshe Yaniv. 19.11 DNAase hypersensitive sites change chromatin structure

53. Figure 19.41 The SV40 gap includes hypersensitive sites, sensitive regions, and a protected region of DNA. The hypersensitive site of a chicken b- globin gene comprises a region that is susceptible to several nucleases. 19.11 DNAase hypersensitive sites change chromatin structure

54. Domain of a chromosome may refer either to a discrete structural entity defined as a region within which supercoiling is independent of other domains; or to an extensive region including an expressed gene that has heightened sensitivity to degradation by the enzyme DNAase I. 19.12 Domains define regions that contain active genes

55. Figure 19.42 Sensitivity to DNAase I can be measured by determining the rate of disappearance of the material hybridizing with a particular probe. 19.12 Domains define regions that contain active genes

56. Figure 19.43 In adult erythroid cells, the adult b-globin gene is highly sensitive to DNAase I digestion, the embryonic b-globin gene is partially sensitive (probably due tIn adult erythroid cells, the adult b-globin gene is highly sensitive to DNAase I digestion, the embryonic b-globin gene is partially sensitive (probably due to spreading effects), but ovalbumin is not sensitive. Data kindly provided by Harold Weintraub. 19.12 Domains define regions that contain active genes

57. Epigenetic changes influence the phenotype without altering the genotype. They consist of changes in the properties of a cell that are inherited but that do not represent a change in genetic information. 19.13 Heterochromatin depends on interactions with histones

58. Figure 19.44 Position effect variegation in eye color results when the white gene is integrated near heterochromatin. Cells in which white is inactive give patches of white eye, while cells in which white is active give red paPosition effect variegation in eye color results when the white gene is integrated near heterochromatin. Cells in which white is inactive give patches of white eye, while cells in which white is active give red patches. The severity of the effect is determined by the closeness of the integrated gene to heterochromatin. Photograph kindly provided by Steve Henikoff. 19.13 Heterochromatin depends on interactions with histones

59. Figure 19.45 Extension of heterochromatin inactivates genes. The probability that a gene will be inactivated depends on its distance from the heterochromatin region. 19.13 Heterochromatin depends on interactions with histones

60. Figure 19.46 Formation of heterochromatin is initiated when RAP1 binds to DNA. SIR3/4 bind to RAP1 and also to histones H3/H4. The complex polymerizes along chromatin and may connect telomeres to the nuclear matrix. 19.13 Heterochromatin depends on interactions with histones

61. Constitutive heterochromatin describes the inert state of permanently nonexpressed sequences, usually satellite DNA. Dosage compensation describes mechanisms employed to compensate for the discrepancy between the presence of two X chromosomes in one sex but only one X chromosome in the other sex. Facultative heterochromatin describes the inert state of sequences that also exist in active copies-for example, one mammalian X chromosome in females. Single X hypothesis describes the inactivation of one X chromosome in female mammals. 19.14 Global changes in X chromosomes

62. Figure 19.47 Different means of dosage compensation are used to equalize X chromosome expression in male and female. 19.14 Global changes in X chromosomes

63. Figure 19.48 X-linked variegation is caused by the random inactivation of one X chromosome in each precursor cell. Cells in which the + allele is on the active chromosome have wild phenotype; but cells in which the - allele is on the active chromosome have mutant phenotype. 19.14 Global changes in X chromosomes

64. Figure 19.49 X- inactivation involves stabilization of XIST RNA, which coats the inactive chromosome. 19.14 Global changes in X chromosomes

65. Imprinting describes a change in a gene that occurs during passage through the sperm or egg with the result that the paternal and maternal alleles have different properties in the very early embryo. May be caused by methylation of DNA. 19.15 Methylation is responsible for imprinting

66. Figure 19.50 The state of methylated sites could be perpetuated by an enzyme that recognizes only hemimethylated sites as substrates. 19.15 Methylation is responsible for imprinting

67. Figure 19.51 The state of methylation is controlled by three enzymes. 19.15 Methylation is responsible for imprinting

68. Figure 19.52 The parental alleles of Igf2 are differentially methylated in the early embryo, but the patterns of methylation are reset when gametes are formed by the adult. 19.15 Methylation is responsible for imprinting

69. Prion is a proteinaceous infectious agent, which behaves as an inheritable trait, although it contains no nucleic acid. Examples are PrPSc, the agent of scrapie in sheep and bovine spongiform encephalopathy, and Psi, which confers an inherited state in yeast. 19.16 Epigenetic effects can be inherited

70. Figure 19.50 The state of methylated sites could be perpetuated by an enzyme that recognizes only hemimethylated sites as substrates. 19.16 Epigenetic effects can be inherited

71. Figure 19.53 What happens to protein complexes on chromatin during replication? 19.16 Epigenetic effects can be inherited

72. Figure 19.45 Extension of heterochromatin inactivates genes. The probability that a gene will be inactivated depends on its distance from the heterochromatin region. 19.16 Epigenetic effects can be inherited

73. Figure 19.54 Acetylated cores are conserved and distributed at random to the daughter chromatin fibers at replication. Each daughter fiber has a mixture of old (acetylated) cores and new (unacetylated) cores. 19.16 Epigenetic effects can be inherited

74. Figure 19.55 The state of the Sup35 protein determines whether termination of translation occurs. 19.17 Yeast prions show unusual inheritance

75. Figure 19.56 Newly synthesized Sup35 protein is converted into the [PSI+] state by the presence of pre-existing [PSI+] protein. 19.17 Yeast prions show unusual inheritance

76. Figure 19.57 Purified protein can convert the[psi-] state of yeast to [PSI+]. 19.17 Yeast prions show unusual inheritance

77. Scrapie is a infective agent made of protein. 19.18 Prions cause diseases in mammals

78. Figure 19.58 A PrpSc protein can only infect an animal that has the same type of endogenous PrPC protein. 19.18 Prions cause diseases in mammals

79. 1. All eukaryotic chromatin consists of nucleosomes. 2. The path of DNA around the histone octamer creates 3. Nucleosomes are organized into a fiber of 30 nm diameter which has 6 nucleosomes per turn and a packing ratio of 40. 4. RNA polymerase displaces histone octamers during transcription. 5. Two types of changes in sensitivity to nucleases are associated with gene activity. 6. Formation of heterochromatin may be initiated at certain sites and then propagated for a distance that is not precisely determined. 19.19 Summary

80. 7. Inactive chromatin at yeast telomeres and silent mating type loci appears to have a common cause, and involves the interaction of certain proteins with the N-terminal tails of histones H3 and H4. 8. Inactivation of one X chromosome in female (eutherian) mammals occurs at random. 9. Methylation of DNA is inherited epigenetically. 10. Prions are proteinaceous infectious agents that are responsible for the disease ofscrapie in sheep and for related diseases in man. 19.19 Summary