Gene IX - Chapter 30

Controlling Chromatin Structure CHAPTER OUTLINE Introduction Chromatin Can Have Alternative States • Chromatin structure is stable and cannot be changed by altering the equilibrium of transcription factors and histones. Chromatin Remodeling Is an Active Process • There are several chromatin remodeling complexes that use energy provided by hydrolysis of ATP. • The SWI/SNF, RSC, and NURF complexes all are very large, and they share some common subunits. • Aremodeling complex does not itself have specificity for any particular target site, but must be recruited by a compo­ nent of the transcription apparatus. Nucleosome Organization May Be Changed at the Promoter • Remodeling complexes are recruited to promoters by sequence-specific activators. • The factor may be released once the remodeling complex has bound. • The MMTV promoter requires a change in rotational position­ ing of a nucleosome to allow an activator to bind to DNA on the nucleosome. Histone Modification Is a Key Event • Histones are modified by methylation, acetylation, and phosphorylation. Histone Acetylation Occurs in Two Circumstances • Histone acetylation occurs transiently at replication. • Histone acetylation is associated with activation of gene expression.. Acetylases Are Associated with Activators • Deacetylated chromatin may have a more condensed structure. • Transcription activators are associated with histone acety­ lase activities in large complexes. • Histone acetylases vary in their target specificity. • Acetylation could affect transcription in a quantitative or qualitative way. Deacetylases Are Associated with Repressors • Deacetylation is associated with repression of gene activity. • Deacetylases are present in complexes with repressor activity. Methylation of Histones and DNA Is Connected • Methylation of both DNA and histones is a feature of inac­ tive chromatin. • The two types of methylation event may be connected. Chromatin States Are Interconverted by Modification • Acetylation of histones is associated with gene activation. • Methylation of DNA and of histones is associated with heterochromatin. Promoter Activation Involves an Ordered Series of Events • The remodeling complex may recruit the acetylating complex. • Acetylation of histones may be the event that maintains the complex in the activated state. Histone Phosphorylation Affects Chromatin Structure • At least two histones are targets for phosphorylation, possi­ bly with opposing effects. Some Common Motifs Are Found in Proteins That Modify Chromatin • The chromo domain is found in several chromatin proteins that have either activating or repressing effects on gene expression. • The SET domain is part of the catalytic site of protein methyltra nsferases. • The bromo domain is found in a variety of proteins that interact with chromatin and is used to recognize acetylated sites on histones. Summary 796 III Introduction When transcription is treated in terms of inter­ actions involving DNA and individual transcrip­ tion factors and RNA polymerases, we get an accurate description of the events that occur in vitro, but this lacks an important feature of tran­ scription in vivo. The cellular genome is or­ ganized as nucleosomes, but initiation of transcription generally is prevented if the pro­ moter region is packaged into nucleosomes. In this sense, his tones function as generalized repressors of transcription (a rather old idea), although we see in this chapter that they are also involved in more specific interactions. Acti­ vation of a gene requires changes in the state of chromatin: The essential issue is how the tran­ scription factors gain access to the promoter DNA. Local chromatin structure is an integral part of controlling gene expression. Genes may exist in either of two structural conditions. Genes are found in an "active" state only in the cells in which they are expressed. The change of struc­ ture precedes the act of transcription, and indi­ cates that the gene is "transcribable." This suggests that acquisition of the "active" struc­ ture must be the first step in gene expression. Active genes are found in domains of euchro­ matin with a preferential susceptibility to nucle­ ases (see Section 29.19, Domains Define Regions That Contain Active Genes). Hypersensitive sites are created at promoters before a gene is acti­ vated (see Section 29.18, DNAase Hypersensi­ tive Sites Change Chromatin Structure). More recently it has turned out that there is an intimate and continuing connection between initiation of transcription and chro­ matin structure. Some activators of gene tran­ scription directly modify histones; in particular, acetylation of histones is associated with gene activation. Conversely, some repressors of tran­ scription function by deacetylating histones. Thus a reversible change in histone structure in the vicinity of the promoter is involved in the control of gene expression. This may be part of the mechanism by which a gene is main­ tained in an active or inactive state. The mechanisms by which local regions of chromatin are maintained in an inactive (silent) state are related to the means by which an indi­ vidual promoter is repressed. The proteins involved in the formation of heterochromatin act on chromatin via the histones, and modifi­ cations of the histones may be an important feature in the interaction. Once established, such changes in chromatin may persist through cell divisions, creating an epigenetic state in which the properties of a gene are determined by the self-perpetuating structure of chromatin. The name epigenetic reflects the fact that a gene may have an inherited condition (it may be active or may be inactive) that does not depend on its sequence. Yet a further insight into epi­ genetic properties is given by the self-perpetu­ ating structures of prions (proteinaceous infectious agents). liB Chromatin Can Have Alternative States Key concept • Chromatin structure is stable and cannot be changed by altering the equilibrium of transcription factors and histones. Two types of models have been proposed to explain how the state of expression of DNA is changed: equilibrium and discontinuous change-of-state. FIGURE 30.1 shows the equilibrium model. Here the only pertinent factor is the concentra­ tion of the repressor or activator protein, which drives an equilibrium between free form and DNA-bound form. When the concentration of the protein is high enough, its DNA-binding site is occupied, and the state of expression of the Target site is free when protein concentration is low .. . . . . .. ..~ ~ ~ High concentration of protein results in binding to DNA target g- 50%i . o 100 0 %[Z]g •Protein concentration FIGURE 30.1 In an equilibrium model, the state of a binding site on DNA depends on the con­ centration of the protein that binds to it. 30.2 Chromatin Can Have Alternative States 797 DNA is affected. (Binding might either repress or activate any particular target sequence.) This type of model explains the regulation of tran­ scription in bacterial cells, where gene expres­ sion is determined exclusively by the actions of individual repressor and activator proteins (see Chapter 12, The Operon). Whether a bacterial gene is transcribed can be predicted from the sum of the concentrations of the various fac­ tors that either activate or repress the individ­ ual gene. Changes in these concentrations at any time will change the state of expression accordingly. In most cases, the protein binding is cooperative, so that once the concentration becomes high enough, there is a rapid associa­ tion with DNA, resulting in a switch in gene expression. A different situation applies with eukary­ otic chromatin. Early in vitro experiments showed that either an active or inactive state can be established, but this is not affected by the subsequent addition of other components. The transcription factor TFmA, which is required for RNA polymerase III to transcribe 55 rRNA genes, cannot activate its target genes in vitro if they are complexed with histones. If the factor is presented with free DNA, though, it forms a transcription complex, and then the addition of histones does not prevent the gene from RNA polymerase and factors {­ cannot get access to DNA /f tJ Histone octamers cannot {­ get access to DNA /f FIGURE 30.2 If nucleosomes form at a promoter, transcrip­ tion factors (and RNA poLymerase) cannot bind. If tran­ scription factors (and RNA poLymerase) bind to the promoter to estabLish a stabLe compLex for initiation, his­ tones are excluded. remaining active. Once the factor has bound, it remains at the site; this allows a succession of RNA polymerase molecules to initiate transcrip­ tion. Whether the factor or histones get to the control site first may be the critical factor. FIGURE 30.2 illustrates the two types of con­ dition that can exist at a eukaryotic promoter. In the inactive state, nucleosomes are present, and they prevent basal factors and RNA poly­ merase from binding. In the active state, the basal apparatus occupies the promoter, and his­ tone octamers cannot bind to it. Each type of state is stable. A similar situation is seen with the TFnD complex at promoters for RNA polymerase II. A plasmid containing an adenovirus promoter can be transcribed in vitro by RNA polymerase II in a reaction that requires TFnD and other transcription factors. The template can be assem­ bled into nucleosomes by the addition of his­ tones. If the histones are added before the TFnD, transcription cannot be initiated. If the TFnD is added first, though, the template still can be transcribed in its chromatin form. Thus TFnD can recognize free DNA, but either cannot rec­ ognize or cannot function on nucleosomal DNA. Only the TFnD must be added before the his­ tones; the other transcription factors and RNA polymerase can be added later. This suggests that binding of TFnD to the promoter creates a structure to which the other components of the transcription apparatus can bind. It is important to note that these in vitro systems use disproportionate quantities of com­ ponents, which may create unnatural situa­ tions. The major importance of these results, therefore, is not that they demonstrate the mechanism used in vivo, but that they estab­ lish the principle that transcription factors or nucle­ osomes may form stable structures that cannot be changed merely by changing the equilibrium with free components. Em Chromatin Remodeling Is an Active Process Key concepts • There are severaL chromatin remodeLing compLexes that use energy provided by hydroLysis of ATP. • The SWljSNF, RSC, and NURF compLexes aLL are very Large, and they share reLated subunits. • AremodeLing compLex does not itseLf have specificity for any particuLar target site, but must be recruited by a component of the transcription apparatus. 798 CHAPTER 30 Controlling Chromatin Structure The general process of inducing changes in chro­ matin structure is called chromatin remod­ eling. This consists of mechanisms for displacing histones that depend on the input of energy. Many protein-protein and protein-DNA con­ tacts need to be disrupted to release histones from chromatin. There is no free ride: The energy must be provided to disrupt these con­ tacts. FIGURE 30.3 illustrates the principle of a dynamic model by a factor that hydrolyzes ATP. When the histone octamer is released from DNA, other proteins (in this case transcription factors and RNA polymerase) can bind. FIGURE 30.4 summarizes the types of remod­ eling changes in chromatin that can be charac­ terized in vitro: • Histone octamers may slide along DNA, changing the relationship between the nucleic acid and protein. This alters the position of a particular sequence on the nucleosomal surface. • The spacing between histone octamers may be changed, again with the result that the positions of individual se­ quences are altered relative to protein. • The most extensive change is that an octamer(s) may be displaced entirely from DNA to generate a nucleosome­ free gap. The most common use of chromatin remod­ eling is to change the organization of nucleo­ somes at the promoter of a gene that is to be transcribed. This is required to allow the tran­ scription apparatus to gain access to the pro­ moter. Remodeling is also required, however, to enable other manipulations of chromatin, including repair reactions to damaged DNA. Remodeling most often takes the form of displacing one or more histone octamers. This can be detected by a change in the micrococcal nuclease ladder where protection against cleav­ age has been lost. It often results in the creation of a site that is hypersensitive to cleavage with DNAase I (see Section 29.18, DNAase Hyper­ sensitive Sites Change Chromatin Structure). Sometimes there are less dramatic changes, for example, involving a change in rotational posi­ tioning of a single nucleosome; this may be detected by loss of the DNAase I 10 base lad­ der. Thus changes in chromatin structure may extend from altering the positions of nucleo­ somes to removing them altogether. Chromatin remodeling is undertaken by large complexes that use ATP hydrolysis to pro­ vide the energy for remodeling. The heart of the remodeling complex is its ATPase subunit. Remodeling complexes are usually classified according to the type of ATPase subunit-those with related ATPase subunits are considered to belong to the same family (usually some other subunits are common as well). FIGURE 30.5 keeps the names straight. The two major types of com­ plex are SWIISNF and ISWI (ISWI stands for . .. --. . .. .­ - - Factors and RNA polymerase bind FIGURE 30.3 The dynamic model for transcription of chro­ matin relies upon factors that can use energy provided by hydrolysis of ATP to displace nucleosomes from specific DNA sequences. . - .. - .. . .. Nucleosomes Spacing Nucteosome slide adjusted is displaced •! •t ! Sequence Gap of changes free DNA position V Spacing becomes even FIGURE 30.4 Remodeling complexes can cause nucleosomes to slide along DNA, can displace nucleosomes from DNA, or can reor­ ganize the spacing between nucleosomes. 30.3 Chromatin Remodeling Is an Active Process 799 ... . . .. .• Type of Complex SWI/SNF ISWI Other Yeast SWI/SNF ISW1 IN080 complex RSC ISW2 SWRI Fly dSWI/SNF NURF (brahma) CHRAC ACF Human hSWI/SNF RSF NuRD hACFIWCFR IN080 complex hCHRAC SRCAP WICH Frog WICH Mi-2 CHRAC ACF fIGURE 30.5 Remodeling complexes can be classified by their ATPase subunits. imitation SWI). Yeast has two SWIISNF com­ plexes and three ISWI complexes. Complexes of both types are also found in fly and in the human being. Each type of complex may under­ take a different range of remodeling activities. SWI/SNF was the first remodeling com­ plex to be identified. Its name reflects the fact that many of its subunits are coded by genes originally identified by SWIor SNF mutations in Saccharomyces cerevisiae. Mutations in these loci are pleiotropic, and the range of defects is sim­ ilar to those shown by mutants that have lost the carboxyl-terminaldomain (CTD) tail of RNA polymerase II. These mutations also show genetic interactions with mutations in genes that code for components of chromatin, in par­ ticular SINl, which codes for a nonhistone pro­ tein, and SIN2, which codes for histone H3. The SWI and SNF genes are required for expression of a variety of individual loci (-120, or 2%, of S. cerevisiae genes are affected). Expression of these loci may require the SWI/SNF complex to remodel chromatin at their promoters. SWI/SNF acts catalytically in vitro, and there are only -150 complexes per yeast cell. All of the genes encoding the SWIISNF subunits are nonessential, which implies that yeast must also have other ways of remodeling chromatin. The RSC (remodels the structure of chromatin) com­ plex is more abundant and also is essential. It acts at - 700 target loci. SWI/SNF complexes can remodel chro­ matin in vitro without overall loss of histones or can displace histone octamers. Both types of reaction may pass through the same interme­ diate in which the structure of the target nucle­ osome is altered, leading either to reformation 800 CHAPTER 30 Controlling Chromatin Structure of a (remodeled) nucleosome on the original DNA or to displacement of the histone octamer to a different DNA molecule. The SWI/SNF com­ plex alters nucleosomal sensitivity to DNAase I at the target site, and induces changes in protein-DNA contacts that persist after it has been released from the nucleosomes. The Swi2 subunit is the ATPase that provides the energy for remodeling by SWIISNF. There are many contacts between DNA and a histone octamer; fourteen are ide).1.tified in the crystal structure. All of these contacts must be broken for an octamer to be released or for it to move to a new position. How is this achieved? Some obvious mechanisms can be excluded because we know that single-stranded DNA is not generated during remodeling (and there are no helicase activities associated with the complexes). Present thinking is that remod­ eling complexes in the SWI and ISWI classes use the hydrolysis of ATP to twist DNA on the nucleosomal surface. Indirect evidence suggests that this creates a mechanical force that allows a small region of DNA to be released from the surface and then repositioned. One important reaction catalyzed by remod­ eling complexes involves nucleosome sliding. It was first observed that the ISWI family affects nude­ osome positioning without displacing octamers. This is achieved by a sliding reaction, in which the octamer moves along DNA. Sliding is prevented if the N-terminal tail of histone H4 is removed, but we do not know exactly how the tail functions in this regard. SWI/SNF complexes have the same capacity; the reaction is prevented by the introduc­ tion of a barrier in the DNA which suggests that a sliding reaction is involved, in which the histone octamer moves more or less continuously along DNA without ever losing contact with it. One puzzle about the action of the SWI/SNF complex is its sheer size. It has eleven subunits with a combined molecular weight -2 x 106. It dwarfs RNA polymerase and the nucleosome, making it difficult to understand how all of these components could interact with DNA retained on the nudeosomal surface. A transcription complex with full activity, however, called RNA polymerase II holoenzyme, can be found that contains the RNA polymerase itself, all the TFrr factors except TBP and TFrrA and the SWI/SNF complex, which is associated with the CTD tail of the polymerase. In fact, virtually all of the SWI/SNF complex may be present in holoenzyme preparations. This sug­ gests that the remodeling of chromatin and recog­ nition of promoters is undertaken in a coordinated manner by a single complex. BIt Nucleosome Organization May Be Changed at the Promoter Key concepts • Remodeling complexes are recruited to promoters by sequence-specific activators. • The factor may be released once the remodeling complex has bound. • The MMTV promoter requires a change in rotational positioning of a nucleosome to allow an activator to bind to DNA on the nucleosome. How are remodeling complexes targeted to spe­ cific sites on chromatin? They do not them­ selves contain subunits that bind specific DNA sequences. This suggests the model shown in FIGURE 30.6, in which they are recruited by acti­ vators or (sometimes) by re