1. Chapter 10
Chromatin
Jocelyn E. Krebs

2. Figure 10.CO: A three-dimensional model of chromatin showing one possible arrangement for the nucleosomes in the 30 nm fiber.
Photo courtesy of Thomas Bishop, Tulane University, using VDNA plug-in for VMD [

3. 10.1 Introduction
nucleosome – The basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins.
histone tails – Flexible amino- or carboxy-terminal regions of the core histones that extend beyond the surface of the nucleosome.
Histone tails are sites of extensive posttranslational modification.

4. 30 nm fiber – A coil of nucleosomes. 1000 fold compact
10 nm fiber – A linear array of nucleosomes, generated by unfolding from the natural condition of chromatin. (40 fold compact)
30 nm fiber – A coil of nucleosomes fold compact
It is the basic level of organization of nucleosomes in chromatin.
Heterochromatin is compact as mitotic chromatin
Protein, DNA, and RNA (attached transcript)
Nonhistone – Any structural protein found in a chromosome except one of the histones.

5. 10.2 The Nucleosome Is the Subunit of All Chromatin
MNase (micrococcal nuclease) cleaves linker DNA and releases individual nucleosomes from chromatin.
>95% of the DNA is recovered in nucleosomes or multimers when MNase cleaves DNA in chromatin.
The length of DNA per nucleosome varies for individual tissues in a range from 154 to 260 bp.
Figure 10.02: Chromatin spilling out of lysed nuclei consists of a compactly organized series of particles. The bar is 100 nm.

6. Figure 10.03: Micrococcal nuclease digests chromatin in nuclei into a multimeric series of DNA bands that can be separated by gel electrophoresis.
Photo courtesy of Markus Noll, Universität Zürich

7. A nucleosome contains ~200 bp of DNA and two copies of each core histone (H2A, H2B, H3, and H4).
DNA is wrapped around the outside surface of the protein octamer.
The histone octamer is a complex of two copies each of the four different core histones.
Figure 10.04: The nucleosome consists of approximately equal masses of DNA and histones (including H1). The predicted mass of the nucleosome is 262 kD.

8. Diameter: 11 nm Hieght: 6 nm 1 ¾ turn
Linker histones – A family of histones (such as histone H1) that are not components of the nucleosome core.
Linker histones bind nucleosomes and/or linker DNA and promote 30 nm fiber formation.
External particle
Diameter: 11 nm
Hieght: 6 nm
1 ¾ turn
Figure 10.05: The nucleosome is roughly cylindrical, with DNA organized into 1 3/4 turns around the surface.

9. A to B: 80bp
Figure 10.06: Sequences on the DNA that lie on different turns around the nucleosome may be close together.

10. 10.3 Nucleosomes Have a Common Structure
Nucleosomal DNA is divided into the core DNA and linker DNA depending on its susceptibility to micrococcal nuclease.
The core DNA is the length of 146 bp that is found on the core particles produced by prolonged digestion with micrococcal nuclease.
Linker DNA is the region of 8 to 114 bp that is susceptible to early cleavage by the enzyme.
Figure 10.07: Micrococcal nuclease initially cleaves between nucleosomes.

11. Each histone is extensively interdigitated with its partner.
The histone octamer has a kernel of an H32–H42 tetramer (backbone) associated with two H2A–H2B dimers.
Each histone is extensively interdigitated with its partner.
Figure 10.09: The crystal structure of the histone core octamer is represented in a ribbon model.
Figure 10.08: Histone pairs (H3 + H4 or H2A + H2B) interact to form histone dimers.

12. 10.3 Nucleosomes Have a Common Structure
All core histones have the structural motif of the histone fold. N- and C-terminal tails extend out of the nucleosome.
H1 is associated with linker DNA and may lie at the point where DNA enters or leaves the nucleosome.
Figure 10.10: The histone fold domains of the histones are located in the core of the nucleosome.

13. Figure 10.11: The histone tails are disordered and exit from both faces of the nucleosome and between turns of the DNA.

14. H1; sealing
Figure 10.12: Possible model for the interaction of hitone H1 with the nucleosome.

15. 10.4 Nucleosomes Are Covalently Modified
Histones are modified by methylation, acetylation, phosphorylation, and other modifications.
Combinations of specific histone modifications define the function of local regions of chromatin; this is known as the histone code.
Figure 10.13: The histone tails (and sometimes the core regions) can be acetylated, methylated, phosphorylated, and ubiquitylated at numerous sites.
Adapted from The Scientist 17 (2003): p. 27

16. Figure 10.13: The histone tails (and sometimes the core regions) can be acetylated, methylated, phosphorylated, and ubiquitylated at numerous sites.
Adapted from The Scientist 17 (2003): p. 27

17. Figure 10.14: The positive charge on lysine is neutralized upon acetylation, whereas mono-, di- or trimethylation does not eliminate the positive charge.

18. Figure 10.15: Acetylation during replication occurs on specific sites in newly synthesized histones before they are incorporated into nucleosomes.

19. Figure 10.16: Acetylation associated with gene activation occurs by directly modifying specific sites on histones that are already incorporated into nucleosomes.

20. Figure 10.17: Modified sites in histones can have a single type of modification or may be alternatively modified under different conditions.

21. Figure?1 Epigenetic Features of Active, Primed, and Poised Enhancers (A) Schematic representation of the major chromatin features found at active enhancers. Enhancers are associated with incorporation of hypermobile nucleosomes containing H3.3/H2A.Z histo...
Molecular Cell Volume 49, Issue
Eliezer Calo , Joanna Wysocka
Modification of Enhancer Chromatin: What, How, and Why?

26. 10.5 Histone Variants Produce Alternative Nucleosomes
All core histones except H4 are members of families of related variants.
Histone variants can be closely related or highly divergent from canonical histones.
Different variants serve different functions in the cell.
Figure 10.18: The major core histones contain a conserved histone-fold domain.

27. Figure?2 H2A.Z Exchange Drives H4 Acetylation Exchange of H2A for H2A.Z alters interaction between the N-terminal tail of H4 and adjacent nucleosomes, exposing the tail to acetylation by Tip60. The combination of H2A.Z exchange and H4 acetylation function...
Brendan?D. Price , Alan?D. D?Andrea
Chromatin Remodeling at DNA Double-Strand Breaks
Cell Volume 152, Issue

28. Figure 10.19: 7-H2AX is detected by an antibody (yellow) and appears along the path traced by a laser that produces double strand breaks.

30. Figure 10.20: Some histone variants are spread throughout all or most of the chromosome, whereas others show specific distribution patterns

31. 10.6 DNA Structure Varies on the Nucleosomal Surface
Dnase I, II-> single strand nick formarion
10-13 bp interval
B-form DNA cleavage site is determined by DNA conformation
Figure 10.21: Sites for nicking lie at regular intervals along core DNA, as seen in a DNAase I digest of nuclei.
Figure 10.22: 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.)
Photo courtesy of Leonard C. Lutter, Henry Ford Hospital, Detroit, MI.

32. DNA is wrapped 1.65 times around the histone octamer.
The structure of the DNA is altered so that it has an increased number of base pairs/turn in the middle, but a decreased number at the ends.
Approximately 0.6 negative turns of DNA are absorbed by the change in bp/turn from 10.5 in solution to an average of on the nucleosomal surface, which explains the linking number paradox.

33. Figure 10.23: 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.

34. 10.7 The Path of Nucleosomes in the Chromatin Fiber
10 nm chromatin fibers are unfolded from 30 nm fibers and consist of a string of nucleosomes.
30 nm fibers consist of 10 nm fibers coiled into a two-start helix.
Linker histones (H1) promote the formation of the 30 nm fiber.
Figure 10.25: The 10 nm fiber is a continuous string of nucleosomes.
Figure 10.27: The 30 nm fiber is a helical ribbon consisting of two parallel rows of nucleosomes coiled into a solenoid.

35. Figure 10.24: The 10 nm fiber in partially unwound state can be seen to consist of a string of nucleosomes.
Photo courtesy of Barbara Hamkalo, University of California-Irvine

36. Figure 10.26: The 30 nm fiber has a coiled structure.
Figure 10.27: The 30 nm fiber is a helical ribbon consisting of two parallel rows of nucleosomes coiled into a solenoid.
Photo courtesy of Barbara Hamkalo, University of California, Irvine

37. Figure 10.28: Levels of chromatin packaging.
Modified courtesy of Karolin Luger and Jeffrey C. Hansen, Colorado State University.

38. 10.8 Replication of Chromatin Requires Assembly of Nucleosomes
Histone octamers are not conserved during replication, but H2A-H2B dimers and H32-H42 tetramers are conserved.
There are different pathways for the assembly of nucleosomes during replication and independently of replication.
Figure 10.29: Replicated DNA is immediately incorporated into nucleosomes.
Photo courtesy of Steven L. McKnight, UT Southwestern Medical Center at Dallas

39. 10.8 Replication of Chromatin Requires Assembly of Nucleosomes
Accessory proteins are required to assist the assembly of nucleosomes.
CAF-1 and ASF1 are histone assembly proteins that are linked to the replication machinery.
A different assembly protein, HIRA, and the histone H3.3 variant are used for replication-independent assembly.
HIRA is required for decondensation of the sperm nucleus (protamines are replaced by histones for replication after fertilization)
H3.3 replaces H3

40. Figure 10.30: Replication fork passage displaces histone octamers from DNA.
Adapted from Rocha, W., and Verreault, A., FEBS Lett. 582 (2008):

41. 10.9 Do Nucleosomes Lie at Specific Positions?
Nucleosomes may form at specific positions as the result of either the local structure of DNA or proteins that interact with specific sequences.
indirect end labeling – A technique for examining the organization of DNA by making a cut at a specific site and identifying all fragments containing the sequence adjacent to one side of the cut.
It reveals the distance from the cut to the next break(s) in DNA.

42. A common cause of nucleosome positioning is when proteins binding to DNA establish a boundary.
Positioning may affect which regions of DNA are in the linker and which face of DNA is exposed on the nucleosome surface.
Figure 10.31: Nucleosome positioning places restrictive sites at unique positions relative to the linker sites cleaved by micrococcal nuclease.

43. Nucleosome positioning
Intrinsic mechanism : specific sequence recognized
Extrinsic mechanism: the first nucleosome determine assembly
TATA box is covered by nucleosome
Figure 10.32: An MNase map of nucleosome positions in an inactive gene.
Figure 10.33: In the absence of nucleosome positioning, a restriction site lies at all possible locations in different copies of the genome.
Courtesy of Dr. Jocelyn Krebs

44. translational positioning – The location of a histone octamer at successive turns of the double helix, which determines which sequences are located in linker regions.
Figure 10.34: Translational positioning describes the linear position of DNA relative to the histone octamer.

45. rotational positioning – The location of the histone octamer relative to turns of the double helix, which determines which face of DNA is exposed on the nucleosome surface.
Figure 10.35: Rotational positioning describes the exposure of DNA on the surface of the nucleosome.

46. Figure 10.36: The SV40 minichromosome has a nucleosome gap.
DNase Sensitivity Detects Changes in Chromatin Structure
Figure 10.36: The SV40 minichromosome has a nucleosome gap.
Photo courtesy of Moshe Yaniv, Pasteur Institute

47. Hypersensitive sites are found at the promoters of expressed genes.
Hypersensitive sites are generated by the binding of transcription factors that exclude histone octamers.
A domain containing a transcribed gene is defined by increased sensitivity to degradation by DNase I.
Figure 10.37: In adult erythroid cells, the adult b-globin gene is highly sensitive to DNase I digestion; the embryonic b-globin gene is partially sensitive, but ovalbumin is not sensitive.
Photos courtesy of Harold Weintraub, Fred Hutchinson Cancer Research Center. Used with permission of Mark Groudine.

48. 10.11 An LCR May Control a Domain
A locus control region (LCR) is located at the 5′ end of a chromosomal domain and typically consist of multiple DNAse hypersensitive sites.
LCRs regulate gene clusters.
TH2 cytokine locus- 120 kB in Ch11 and IFN-r in Ch10
Figure 10.38: The globin domain is marked by hypersensitive sites at either end. The group of sites at the 5' side consititues the LCR and is essential for function of all genes in the cluster.

49. 10.12 Insulators Define Independent Domains
Insulators are able to block passage of any activating or inactivating effects from enhancers, silencers, and LCRs.
Insulators can provide barriers against the spread of heterochromatin.
Insulators are specialized chromatin structures that have hypersensitive sites. Two insulators can protect the region between them from all external effects.
Figure 10.39: An enhancer activates a promoter in its vicinity, but may be blocked from doing so by an insulator located between them.

50. Figure 10.40: Heterochromatin may spread from a center and then blocks any promoters that it covers.

51. Figure 10.41A: The 87A and 87C loci, containing heat shock genes, expand upon heat shock in Drosophila polytene chromosomes.
Photo courtesy of Victor G. Corces, Emory University.

52. The CTCF insulator has numerous roles in vertebrates and may be involved in loop formation.
zw5
BEAF-32
Figure 10.41B: Specialized chromatin structures that include hypersensitive sites mark the ends of the 87A7 domain and insulate genes between them.

56. Figure 10.FTR01A: Position Effect Variegation (PEV) and the Discovery of Insulators

57. Figure 10.FTR01B: Position Effect Variegation (PEV) and the Discovery of Insulators