Nucleosomes

Chapter 29: Nucleosomes • The nucleosome is the basic structural subunit of chromatin – consisting of ~200 bp of DNA and an octamer of histone proteins. • Histones are conserved DNA-binding proteins that – form the basic subunit of chromatin in eukaryotes. – Histones H2A, H2B, H3, H4 form an octameric core around which DNA coils to form a nucleosome. – Histone H1 is external to the nucleosome. • A nonhistone is – any structural protein found in a chromosome except one of the histones. • Nucleosomes are the “beads” of the “beads on a string” description of Chromatin 29.2 The Nucleosome Is the Subunit of All Chromatin Individual nucleosomes are released by digestion of chromatin with micrococcal nuclease. • A nucleosome contains – ~200 bp of DNA, – two copies of each core histone (H2A, H2B, H3, H4) = the protein octamer – and one copy of H1. • DNA is wrapped around the outside surface of the protein octamer. • Micrococcal nuclease is an endonuclease that cleaves DNA • Micrococcal nuclease, in chromatin, cleaves DNA preferentially between nucleosomes – releases individual nucleosomes from chromatin as 11S particles. 1 29.3 DNA Is Coiled in Arrays of Nucleosomes • >95% of the DNA is recovered in nucleosomes or multimers when micrococcal nuclease cleaves chromatin. • The length of DNA per nucleosome varies for individual tissues in a range from 154–260 bp. 2 29.4 Nucleosomes Have a Common Structure • Nucleosomal DNA is divided into – the core DNA – and linker DNA – depending on its susceptibility to micrococcal nuclease. • Micrococcal 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. is associated with linker DNA – may lie at the point where DNA enters and leaves the nucleosome. • The core particle is – a digestion product of the nucleosome that retains the histone octamer and has 146 bp of DNA; its structure is similar to that of the nucleosome itself. • Core DNA is – the 146 bp of DNA contained on a core particle that is generated by cleaving the DNA on a nucleosome with micrococcal nuclease. • Linker DNA is – all DNA contained on a nucleosome in excess of the 146 bp core DNA. – Its length varies from 8–114 bp, – it is cleaved by micrococcal nuclease to leave the core DNA. 29.7 Organization of the Core Particle In a symmetrical model for the nucleosome, the H32-H42 tetramer provides a kernel for the shape. • The histone fold is – a motif found in all four core histones in which three α-helices are connected by two loops. • The histone octamer has – a kernel of a H32 · H42 tetramer associated with two H2A · H2B dimers. • Each histone is extensively interdigitated with its partner. • All core histones have the structural motif of the histone fold. • N-terminal tails extend out of the nucleosome. 3 The crystal structure of the histone core octamer is represented in a space-filling model with the H32-H42 tetramer shown in light blue and the H2A-H2B dimers shown in dark blue. The crystal structure of the histone core octamer is represented in a space-filling model with the H32-H42 tetramer shown in light blue and the H2A-H2B dimers shown in dark blue. The potential path of the DNA is shown in the side view by the parallel lines in a 20 Å wide bundle. 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). All four core histones contact DNA. The N-terminal histone tails are disordered and exit from the nucleosome between turns of the DNA. The structure of a "half nucleosome" shows the contacts with one turn of DNA. 4 29.8 The Path of Nucleosomes in the Chromatin Fiber The 10 nm fiber is a continuous string of nucleosomes. • 10 nm chromatin fibers are – consist of a string of nucleosomes. – a linear array of nucleosomes – generated by unfolding from the natural condition of chromatin (the 30nm fiber). • The 30 nm fiber is – the basic level of organization of nucleosomes in chromatin. – is a coiled coil of nucleosomes. – 30 nm fibers have six nucleosomes/turn, organized into a solenoid. – Histone H1 is required for formation of the 30 nm fiber. The 30 nm fiber is a helical ribbon consisting of two parallel rows of nucleosomes coiled into a solenoid. 29.9 Reproduction of Chromatin Requires Assembly of Nucleosomes • Histone octamers are not conserved during replication • H2A · H2B dimers and H32 · H42 tetramers are conserved. • There are different pathways for the assembly of nucleosomes during replication and independently of replication. • Accessory proteins are required to assist the assembly of nucleosomes. • CAF-1 is an assembly protein that is linked to the PCNA subunit of the replisome; – it is required for deposition of H32 · H42 tetramers following replication. • A different assembly protein and a variant of histone H3 may be used for replication-independent assembly. 5 Replication fork passage displaces histone octamers from DNA. They disassemble into H3-H4 tetramers and H2A-H2B dimers. Newly synthesized histones are assembled into H3-H4 tetramers and H2AH2B dimers. The old and new tetramers and dimers are assembled with the aid of CAF-1 at random into new nucleosomes immediately behind the replication fork. 29.10 Do Nucleosomes Lie at Specific Positions? • Nucleosomes may form at specific positions as the result either of the local structure of DNA or of proteins that interact with specific sequences. • The most common cause of nucleosome positioning is the binding of proteins to DNA to establish a boundary. • Nucleosome positioning describes the placement of nucleosomes at defined sequences of DNA instead of at random locations with regards to sequence. – Positioning may affect which regions of DNA are in the linker and which face of DNA is exposed on the nucleosome surface. Nucleosome positioning places a specific restriction site at unique position relative to the linker sites cleaved by micrococcal nuclease in all copies of the genome. If nucleosome positioning did not exist, a restriction site would lie at all possible locations in different copies of the genome. In other words, a piece of DNA is wound the same way around the nucleosomes in all copies of the gene in an organism. 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 (black). 6 29.11 Histone Octamers Are Displaced by Transcription • Nucleosomes are found at the same frequency when transcribed genes or nontranscribed genes are digested with micrococcal nuclease. • Some heavily transcribed genes appear to be exceptional cases that are devoid of nucleosomes. • RNA polymerase displaces histone octamers during transcription in a model system, but octamers reassociate with DNA as soon as the polymerase has passed. 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. • Nucleosomes are reorganized when transcription passes through a gene. 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. Histone octamers are disassembled ahead of transcription to remove nucleosomes. They reform following transcription. Release of H2A·H2B dimers probably initiates the disassembly process. 7 29.12 DNase Hypersensitive Sites Change Chromatin Structure • A hypersensitive site is – a short region of chromatin detected by its extreme sensitivity to cleavage by DNAase I and other nucleases – it is an area from which nucleosomes are excluded. • Hypersensitive sites are found at the promoters and enhancers of expressed genes. • They are generated by the binding of transcription factors that displace histone octamers. A domain containing a transcribed gene is defined by increased sensitivity to degradation by DNase I. Sensitivity to DNase I can be measured by determining the rate of disappearance of the material hybridizing with a particular probe. The SV40 gap includes hypersensitive sites, sensitive regions, and a protected region of DNA. The hypersensitive site of a chicken β-globin gene comprises a region that is susceptible to several nucleases. In adult erythroid cells, the adult β-globin gene is highly sensitive to DNAase I digestion, the embryonic β-globin gene is partially sensitive (probably due to spreading effects), but ovalbumin is not sensitive. 8 29.14 Insulators Block the Actions of Enhancers and Heterochromatin • An insulator is a sequence that prevents an activating or inactivating effect passing from one side to the other. • Insulators are able to block passage of any activating or inactivating effects from enhancers or silencers. • Insulators may provide barriers against the spread of heterochromatin. • Two insulators can protect the region between them from all external effects. Specialized chromatin structures (SCS) that include hypersensitive sites mark the ends of a domain in the D. melanogaster genome and insulate genes between them from the effects of surrounding sequences. 9 29.15 An LCR May Control a Domain • The locus control region (LCR) is – required for the expression of several genes in a domain. • A globin domain is • marked by hypersensitive sites at either end. • The group of sites at the 5' side constitutes • the LCR and • is essential for the function of all genes in the cluster. • An LCR is – located at the 5’ end of the domain and consists of several hypersensitive sites. • Domains may possess three types of sites: •insulators to prevent effects from spreading between domains; •MARs to attach the domain to the nuclear matrix; •and LCRs that are required for initiation of transcription. • An enhancer may act on more than one promoter within the domain. 10