Chapter 10 Chromatin Jocelyn E Krebs Figure 10

Chapter 10 Chromatin Jocelyn E. Krebs

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 [http: //www. ks. uiuc. edu/research/vmd/plugins/vdna/]

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.

• 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. 1000 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.

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.

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

• • • A nucleosome contains ~200 bp of DNA and two copies of each core histone (H 2 A, H 2 B, H 3, and H 4). 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 H 1). The predicted mass of the nucleosome is 262 k. D.

• Linker histones – A family of histones (such as histone H 1) 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.

A to B: 80 bp Figure 10. 06: Sequences on the DNA that lie on different turns around the nucleosome may be close together.

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.

• The histone octamer has a kernel of an H 32–H 42 tetramer (backbone) associated with two H 2 A–H 2 B dimers. • Each histone is extensively interdigitated with its partner. Figure 10. 08: Histone pairs (H 3 + H 4 or H 2 A + H 2 B) interact to form histone dimers. Figure 10. 09: The crystal structure of the histone core octamer is represented in a ribbon model.

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. • H 1 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.

Figure 10. 11: The histone tails are disordered and exit from both faces of the nucleosome and between turns of the DNA.

H 1; sealing Figure 10. 12: Possible model for the interaction of hitone H 1 with the nucleosome.

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

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

Figure 10. 14: The positive charge on lysine is neutralized upon acetylation, whereas mono-, di- or trimethylation does not eliminate the positive charge.

Figure 10. 15: Acetylation during replication occurs on specific sites in newly synthesized histones before they are incorporated into nucleosomes.

Figure 10. 16: Acetylation associated with gene activation occurs by directly modifying specific sites on histones that are already incorporated into nucleosomes.

Figure 10. 17: Modified sites in histones can have a single type of modification or may be alternatively modified under different conditions.

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 H 3. 3/H 2 A. Z histo. . . Molecular Cell Volume 49, Issue 5 2013 825 - 837 Eliezer Calo , Joanna Wysocka http: //dx. doi. org/10. 1016/j. molcel. 2013. 01. 038 Modification of Enhancer Chromatin: What, How, and Why?

10. 5 Histone Variants Produce Alternative Nucleosomes • All core histones except H 4 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.

Figure? 2 H 2 A. Z Exchange Drives H 4 Acetylation Exchange of H 2 A for H 2 A. Z alters interaction between the N-terminal tail of H 4 and adjacent nucleosomes, exposing the tail to acetylation by Tip 60. The combination of H 2 A. Z exchange and H 4 acetylation function. . . Brendan? D. Price , Alan? D. D? Andrea Chromatin Remodeling at DNA Double-Strand Breaks Cell Volume 152, Issue 6 2013 1344 - 1354 http: //dx. doi. org/10. 1016/j. cell. 2013. 02. 011

Figure 10. 19: 7 -H 2 AX is detected by an antibody (yellow) and appears along the path traced by a laser that produces double strand breaks.

Figure 10. 20: Some histone variants are spread throughout all or most of the chromosome, whereas others show specific distribution patterns

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.

• 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 10. 17 on the nucleosomal surface, which explains the linking number paradox.

Figure 10. 23: The supercoils of the SV 40 minichromosome can be relaxed to generate a circular structure, whose loss of histones then generates supercoils in the free DNA.

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 (H 1) 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.

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

Figure 10. 26: The 30 nm fiber has a coiled structure. Photo courtesy of Barbara Hamkalo, University of California, Irvine Figure 10. 27: The 30 nm fiber is a helical ribbon consisting of two parallel rows of nucleosomes coiled into a solenoid.

Figure 10. 28: Levels of chromatin packaging. Modified courtesy of Karolin Luger and Jeffrey C. Hansen, Colorado State University.

10. 8 Replication of Chromatin Requires Assembly of Nucleosomes • Histone octamers are not conserved during replication, but H 2 A-H 2 B dimers and H 32 -H 42 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. Mc. Knight, UT Southwestern Medical Center at Dallas

10. 8 Replication of Chromatin Requires Assembly of Nucleosomes • Accessory proteins are required to assist the assembly of nucleosomes. • CAF-1 and ASF 1 are histone assembly proteins that are linked to the replication machinery. • A different assembly protein, HIRA, and the histone H 3. 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) • H 3. 3 replaces H 3

Figure 10. 30: Replication fork passage displaces histone octamers from DNA. Adapted from Rocha, W. , and Verreault, A. , FEBS Lett. 582 (2008): 1938 -1949.

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.

• 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.

Nucleosome positioning 1. Intrinsic mechanism : specific sequence recognized 2. 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. Courtesy of Dr. Jocelyn Krebs Figure 10. 33: In the absence of nucleosome positioning, a restriction site lies at all possible locations in different copies of the genome.

• 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.

• 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.

10. 10 DNase Sensitivity Detects Changes in Chromatin Structure Figure 10. 36: The SV 40 minichromosome has a nucleosome gap. Photo courtesy of Moshe Yaniv, Pasteur Institute

• 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.

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. • TH 2 cytokine locus- 120 k. B in Ch 11 and IFN-r in Ch 10 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.

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.

Figure 10. 40: Heterochromatin may spread from a center and then blocks any promoters that it covers.

Figure 10. 41 A: The 87 A and 87 C loci, containing heat shock genes, expand upon heat shock in Drosophila polytene chromosomes. Photo courtesy of Victor G. Corces, Emory University.

The CTCF insulator has numerous roles in vertebrates and may be involved in loop formation. -zw 5 -BEAF-32 Figure 10. 41 B: Specialized chromatin structures that include hypersensitive sites mark the ends of the 87 A 7 domain and insulate genes between them.

Figure 10. FTR 01 A: Position Effect Variegation (PEV) and the Discovery of Insulators

Figure 10. FTR 01 B: Position Effect Variegation (PEV) and the Discovery of Insulators