Chapter 10 Chromatin

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.

10.1 Introduction 10 nm fiber – A linear array of nucleosomes, generated by unfolding from the natural condition of chromatin. 30 nm fiber – A coil of nucleosomes. It is the basic level of organization of nucleosomes in chromatin. 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.

10.2 The Nucleosome Is the Subunit of All Chromatin 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. Figure 10.05: The nucleosome is roughly cylindrical, with DNA organized into 1 3/4 turns around the surface.

10.2 The Nucleosome Is the Subunit of All Chromatin 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.

10.2 The Nucleosome Is the Subunit of All Chromatin 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.

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.

10.3 Nucleosomes Have a Common Structure The histone octamer has a kernel of an H32–H42 tetramer 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. Protein Data Bank 1AOI.

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.

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

10.4 Nucleosomes Are Covalently Modified 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.

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.

10.6 DNA Structure Varies on the Nucleosomal Surface 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.

10.6 DNA Structure Varies on the Nucleosomal Surface 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.

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 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.

10.7 The Path of Nucleosomes in the Chromatin Fiber 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 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

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.

10.8 Replication of Chromatin Requires Assembly of Nucleosomes 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.

10.9 Do Nucleosomes Lie at Specific Positions? 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.

10.9 Do Nucleosomes Lie at Specific Positions? 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.

10.9 Do Nucleosomes Lie at Specific Positions? Figure 10.35: Rotational positioning describes the exposure of DNA on the surface of the nucleosome. 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.

10.10 DNase Sensitivity Detects Changes in Chromatin Structure 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. Figure 10.38: The globin domain is marked by hypersensitive sites at either end. The group of sites at the 5' side constitues 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.

10.12 Insulators Define Independent Domains Figure 10.40: Heterochromatin may spread from a center and then blocks any promoters that it covers. Figure 10.41: Left panel: The 87A and 87C loci, containing heat shock genes, expand upon heat shock in Drosophila polytene chromosomes. (Top chromosome, no heat shock, bottom chromosome, after heat shock) Right panel: Specialized chromatin structures that include hypersensitive sites mark the ends of the 87A7 domain and insulate genes between them from the effects of surrounding sequences. Photo courtesy of Victor G. Corces, Emory University.

10.12 Insulators Define Independent Domains The CTCF insulator has numerous roles in vertebrates and may be involved in loop formation.