Frontiers in Life Science, 2013 Vol. 7, Nos. 1–2, 63–79, http://dx.doi.org/10.1080/21553769.2013.838193 Marit Terweij and Fred van Leeuwen Division of Gene Regulation, Netherlands Cancer Institute and Netherlands Proteomics Centre, 1066CX Amsterdam, the Netherlands (Received 8 July 2013; ﬁnal version received 22 August 2013 ) Chromatin not only serves as a packaging material, but also functions as a platform for integrating signals that act upon the genome. Indeed, chromatin is a dynamic macromolecular structure that can be dramatically altered in many ways to facilitate the diﬀerent transactions at the genome. Examples of such alterations are relocalization of genomic loci within the nucleus upon transcriptional activation or induction of DNA damage, adding or removing post-translational modiﬁcations on histones or other chromatin-binding factors, or altering the basic organization of chromatin by moving or removing nucleosomes, i.e. modifying the occupancy of histone octamers. New insights into the scope and mechanisms of chromatin dynamics have recently been obtained by the development of novel techniques to visualize chromatin protein mobility and stability. Here we discuss the developments in this area, with special emphasis on histone exchange, which we deﬁne as the replacement of histone proteins without a prerequisite change in occupancy. Although histone exchange may not aﬀect chromatin organization per se, recent studies suggest that it can inﬂuence key epigenetic processes such as histone inheritance, the distribution of histone post-translational modiﬁcations, and the output of transcription factors. Importantly, errors in histone exchange in humans can contribute to malignant transformation. Keywords: histone; chromatin; epigenetics Introduction: chromatin protein dynamics Methods to measure histone protein dynamics The basic building block of chromatin is the nucleosome, There are various ways to capture the dynamics of pro- which consists of DNA wrapped around an octamer of his- teins in the cell. Here we focus on methods that have been tone proteins. The histone octamer consists of one tetramer applied to histones and chromatin-bound proteins (reviewed of histones H3–H4 and two dimers of H2A–H2B. The in Deal & Henikoﬀ 2010a). A short overview of the diﬀerent nucleosome is a robust structure. Indeed, at a bulk level techniques and their applications will be given here. Some histone proteins are stable proteins with long half-lives and methods are aimed at visualizing spatial movement of chro- relatively little mobility in the nucleus of the cell. This, matin proteins; others measure protein stability, turnover, or however, does not mean that histone proteins are static exchange. What these methods have in common is that they entities. In fact, recent studies suggest that chromatin is provide a means to distinguish between resident and newly a highly dynamic platform that coordinates many diﬀer- assembled or newly synthesized proteins. ent processes in which the genome is involved, such as transcriptional regulation, RNA processing, DNA repair FRAP and replication. These processes can involve extensive and complex post-translational modiﬁcation of histones, sub- Movement of chromatin proteins within the nucleus can nuclear genome organization, recruitment of regulatory be visualized by combining the use of ﬂuorescent fusion factors, or alterations in nucleosome occupancy. In addition proteins with live cell imaging. By ﬂuorescence recov- to these processes that can be readily visualized, more subtle ery after photobleaching (FRAP) a discrete region of the chromatin rearrangements can occur by replacing resident nucleus is subjected to laser photo bleaching. The time it histones for newly synthesized ones. This process of histone takes to regain ﬂuorescence in that region is a measure for exchange or turnover has until recently remain unnoticed the replacement of resident proteins by proteins from non- but may nevertheless have an important impact on the bleached regions or newly synthesized proteins, thereby epigenome and its function. Here we discuss technologies informing on the residence time of the labeled protein in that have been developed to measure histone dynamics and chromatin. FRAP has been used to study a wide range of what they have taught us on the mechanisms and functions proteins including histones, transcription factors, and chro- this novel layer of epigenetic regulation. matin remodeling enzymes. These studies have shown that Corresponding author. Email: firstname.lastname@example.org © 2013 Taylor & Francis 64 M. Terweij and F. van Leeuwen the residence time of regulatory proteins is in the order of culture (SILAC) in combination with MS, global dynamic seconds and that of linker histone H1 is in the order of min- changes in modiﬁcations can be monitored due to the mass utes. The residence time of bulk canonical histones is in the diﬀerences of old and new proteins. With this method, order of hours, with H2A and H2B showing more exchange and consistent with previous studies, histone acetylation than H3 and H4 (Kimura & Cook 2001; Phair et al. 2004; and deacetylation have been found to be highly dynamic Catez et al. 2006). Imaging approaches to measure protein processes. Histone methylation generally is a more sta- dynamics or interactions using conditional time-controlled ble PTM. For example, in SILAC studies on HeLa cells, ﬂuorescent proteins have recently been reviewed elsewhere mono-methylation occurred on most lysines in H3 (K4, K9, (Wu et al. 2011; Toyama & Hetzer 2013). Although very K27, and K36) shortly after incorporation into the chro- powerful and applicable to single cells, imaging based matin, whereas di- and tri-methylation accumulated more methods do not provide information about the dynamics slowly during the cell cycle. Methylation of H3K79 even of chromatin proteins at speciﬁc genomic regions. accumulates across multiple cell generation (Sweet et al. 2010). MS-based technologies are very powerful. They can not only be used to measure levels and dynamics of Conditional ﬂuorescent labeling: SNAP-tag, modiﬁcations occurring on proteins but also of the pro- FlAsh-ReAsh, and TimeSTAMP teins themselves. SILAC-MS analysis of histone proteins The SNAP-tag is a modiﬁed variant of O6-alkylguanine- in HeLa cells has shown long half-lives of canonical his- DNA alkyltransferase. The normal function of this alkyl- tones and somewhat shorter half-lives for histone H1, which transferase is in DNA repair (Jansen et al. 2007). The is generally in agreement with the FRAP studies described SNAP-tag polypeptide can bind covalently to its substrate above (Zee et al. 2010). O6-benzylguanine. Derivatives of O6-benzylguanine are cell-permeable and can be ﬂuorescent or non-ﬂuorescent, Pulse-chase labeling combined with aﬃnity puriﬁcation: allowing both labeling and quenching approaches. By alter- CATCH-IT and QuaNCAT nating the substrates, pulse-chase assays can be performed to follow newly synthesized proteins (Figure 1A). Using Recently, two new methods were developed that do not this method, distinct mechanisms and timing of deposi- need the use of inducible gene copies or fusion proteins. tion have been described for canonical histone H3 and the CATCH-IT (covalent attachment of tags to capture histones variant histone H3.3; in HeLa cells histone variant H3.1 and identify turnover) has been used to capture histone is incorporated during S phase while histone variant H3.3 exchange genome-wide. In this method, cells are treated is incorporated throughout the cell cycle (Ray-Gallet et al. with a methionine analog L-azidohomoalanine (AHA), 2011). The HaloTag, a protein fusion tag that covalently which is incorporated into all newly synthesized proteins binds to a series of speciﬁc ligands, may oﬀer simi- in the cell and which can subsequently be linked to a lar approaches (Kovalenko et al. 2011). The FlAsh and biotin tag. Newly synthesized proteins can be puriﬁed using ReAsh technique employs a small tetracysteine tag that streptavidin beads and histones and the DNA bound to can bind to biarsenical ﬂuorescent tags (Adams & Tsien these new histones can be extracted under stringent wash- 2008). These biarsenical tags can permeate the cell mem- ing conditions (Deal & Henikoﬀ 2010b) (Figure 1B). With brane and are not ﬂuorescent until they are bound to the CATCH-IT all proteins in the cell will be labeled, which tetracysteine tag. Although this method may not be as sen- makes this method at the moment especially suited for sitive as conventional Green Fluorescent Protein- (GFP) studying the dynamics of (H3–H4) tetramers, since these fusion protein labeling, an advantage is that there is no remain associated with the DNA under conditions that lead need to wash away unbound dye. Moreover, the tag is to dissociation of most other proteins (Deal & Henikoﬀ relatively small when compared to GFP, avoiding prob- 2010a). A recent variation to this method is QuaNCAT. This lems caused by bulky tags. The TimeSTAMP method method combines site selective labeling with AHA (BON- combines epitope tagging with protease cleavage (Lin & CAT; bio-orthoganol non-canonical amino acid tagging) Tsien 2010). The protein of interest is tagged with a small to enrich for newly synthesized proteins with quantitative epitope tag that contains a speciﬁc protease cleavage site. SILAC-based mass spectrometry to provide a way for quan- The speciﬁc protease cleaves oﬀ the epitope tag by default. titative comparison of bulk protein dynamics between two Upon inhibition of the protease the tag remains stable, conditions (Howden et al. 2013). allowing detection of the newly synthesized protein, for example by immunoblotting or immunocytochemistry. Internalization of ectopic exogenous histones The cells of the slime mold Physarum polycephalum grow Detection of protein dynamics by mass spectrometry in a natural synchronous manner and are capable of inter- Mass spectrometry (MS) has been used extensively to nalizing exogenous proteins. Taking advantage of these identify post-translational modiﬁcations (PTMs) on his- unique properties, incorporation of tagged histones has been tones. Using stable isotope labeling by amino acids in cell determined. In this system, exogenous H2A–H2B dimers Frontiers in Life Science 65 Figure 1. Methods to measure histone dynamics. (A) SNAP-tag can be used to follow newly synthesized proteins in the cell. The SNAP-tag binds covalently to labels. The labels can be either ﬂuorescent or non-ﬂuorescent, to allow pulse-chase and quench-pulse-chase assays, by alternating the types of labels. TMR (tetramethylrhodamine) conjugated to SNAP substrate is used as ﬂuorescent label to detect exchange; BG-block (O -benzylguanine) is used to block binding of TMR. (B) CATCH-IT (covalent attachment of tags to capture histones and identify turnover) can be used to measure dynamics of native histones across the genome. Newly synthesized proteins (depicted with a lighter color and dashed outline) are labeled with a methionine analog. After isolating nuclei, the label can be biotinylated and puriﬁed via streptavidin. Stringent washing strips oﬀ all proteins except H3–H4 from the DNA, which can subsequently be analyzed by microarray or deep sequencing. Green and yellow triangles depict H3–H4; blue and purple arches depict H2A–H2B. (C) In the inducible ectopic expression approach the protein of interest is expressed from the endogenous, constitutive gene and from a second ectopic gene that is under control of an inducible promoter. (D) RITE (recombination-induced tag exchange) can be used to measure protein dynamics by distinguishing between old and new protein expressed from the same gene. The ﬁrst epitope tag is ﬂanked by LoxP recombination sites and has a stop codon; the second epitope tag is located downstream of the second LoxP site. Before inducing Cre recombinase, the protein will be tagged with the ﬁrst epitope tag; after induction the ﬁrst tag is removed and the newly synthesized proteins will have the second tag. localize to the nucleus in the G2 phase of the cell cycle, polymerase II, whereby H2A–H2B exchange is higher in regardless of transcriptional activity, however, assembly coding regions than in promoter regions and higher in into the chromatin is dependent of transcription by RNA active genes than in inactive genes. H3–H4 tetramers show 66 M. Terweij and F. van Leeuwen very little replication-independent incorporation. The fate be less suitable for proteins that have a very high turnover. of resident (untagged) histones cannot be determined by this However, RITE is a ﬂexible method that can be applied to assay (Thiriet & Hayes 2005, 2006; Ejlassi-Lassallette et al. any protein of interest and combined with various down- 2011). stream approaches. For example, RITE using ﬂuorescent tag-switching has recently been employed to visualize the inheritance of organelles and macromolecular complexes in Inducible expression of epitope-tagged histones replicating yeast cells (Hotz et al. 2012; Menendez-Benito In budding yeast, several methods have been developed by et al. 2012). which newly synthesized and old chromatin proteins can be distinguished by genetically encoded diﬀerentially labeled Histone assembly, deposition, and exchange chromatin proteins. A frequently used approach involves the conditional expression of an ectopically expressed ver- Since the discovery of the nucleosome, a lot of research sion of the protein of interest in the background of the has been done to unravel its composition and complex- endogenous untagged or diﬀerently tagged protein (Cheng ity, revealing that alongside the four canonical histones and & Gartenberg 2000; Schermer et al. 2005; Linger & Tyler their PTMs, histone variants exist that have speciﬁc func- 2006; Jamai et al. 2007; Ruﬁange et al. 2007). This method, tions and PTMs. Whereas canonical histones are deposited which typically involves an inducible promoter such as the into the chromatin mainly during DNA replication, variants galactose-inducible GAL1 promoter, has been particularly can replace the canonical forms by replication-independent successful in combination with chromatin immuno precip- mechanisms. In this section, we will give an overview of itation (ChIP) to map the incorporation of new chromatin the histone deposition pathways, histone exchange, and proteins across the genome (Figure 1C). A drawback of the canonical histone replacement by histone variants. use of ectopically expressed histones is that the endoge- nously expressed copy represents a mixture of old and new Delivering histones to chromatin proteins because of its ongoing synthesis. As a consequence, Following the production of histones in the cytoplasm, they the endogenously expressed and induced histones will even- undergo a multi-step maturation process before they are tually reach a steady state. However, promoter shut-down translocated to the nucleus and incorporated into the chro- strategies applied to the endogenous copy can overcome matin. Several protein complexes work together to deliver this limitation (Katan-Khaykovich & Struhl 2011). Another the histones to the chromatin and to facilitate nucleosome point of consideration is that the introduction of an extra assembly, whereby H3–H4 and H2A–H2B are controlled copy can lead to overexpression, which in turn can lead to by distinct pathways (Figure 2). altered dynamics (Au et al. 2008). Immediately after translation, histone H3 in mammalian cells is bound by HSC70, a heat shock chaperone that assists Recombination-induced tag exchange in the proper folding of a number of proteins. It has been Another genetically encoded method that has been devel- suggested that this protein is the ﬁrst to bind to H3 after oped in budding yeast is RITE (recombination-induced tag translation because H3 bound to HSC70 is monomethy- exchange) (Verzijlbergen et al. 2010). RITE is a genetic lated at lysine 9 (K9me1), a mark that is found exclusively pulse-chase assay that makes use of the site-speciﬁc Cre- on newly synthesized H3 in human cells (Loyola et al. recombinase to switch from an old tag to a new tag. DNA 2006; Campos et al. 2010). Also, there is a lack of cassettes containing an epitope tag between two LoxP chromatin-related modiﬁcations on H3 bound to HSC70 recombination sites and an orphan epitope tag downstream and H4 is not found in this complex (Campos et al. 2010). of the second LoxP recombination site can be targeted Newly synthesized histone H4 is found in another complex downstream of the gene of interest (Figure 1D). The LoxP containing HSP70 and HSP90. In these early complexes, sites can be induced to recombine by transient activation of histone H3 and H4 are poly(ADP-ribosylated) (Alvarez Cre recombinase by adding the human hormone β -estradiol et al. 2011). It has been hypothesized that the poly(ADP- to the media. The recombination between the LoxP sites will ribosylation) mark is removed from H3 and H4 when result in a swap from the ‘old’ tag to the orphan (‘new’) tag these proteins are brought together in a complex contain- in the coding sequence, leading to an epitope tag switch at ing HSP90 and tNASP. In this complex H3K9me1 is still the protein level (Verzijlbergen et al. 2010). RITE has sev- high and H4 in this complex is not yet acetylated (Campos eral strengths. The gene of interest is expressed from the et al. 2010). The H3–H4 dimer is subsequently handed down endogenous promoter, the switch is permanent and does not to another complex containing sNASP and the HAT1 his- involve complex promoter-induction/shut-down strategies, tone acetyltransferase complex. In this complex histone and old and new proteins can be monitored simultaneously. H4 is acetylated on K5 and K12 (Campos et al. 2010). RITE has been used to map the exchange and inheri- Next, the acetylated H3–H4 dimer is transferred to the tance of histone proteins in the yeast genome. Since the nuclear import complex. It has been suggested that there recombination process can take several hours, RITE may are two diﬀerent import complexes in human cells. Each Frontiers in Life Science 67 Figure 2. Transport of new histones. Overview of factors involved in shuttling of histone proteins from the cytoplasm to the nucleus. complex associates with H3–H4 dimers, but with a diﬀerent et al. 2011). The negative crosstalk observed between pattern of histone modiﬁcations, and a diﬀerent destination H3K14ac and H3K9me1 supports the hypothesis that in the chromatin. One import complex contains importin- these two sets have diﬀerent destinations (Alvarez et al. 4 and ASF1a. This complex binds to dimers that have 2011). In budding yeast H3–H4 dimers are bound by the H3K14 acetylation as well as H3K9me1 and H4K5K12ac histone chaperone Hif1, which binds to the HAT1 com- (Alvarez et al. 2011). The second import complex con- plex (and forms the NuB4 complex) that acetylates H4. The tains importin-4 and ASF1b. This complex binds H3–H4 H3–H4 dimer is then handed down to the histone chaperone dimers containing H3K9me1 and H4K5K12ac (Alvarez Asf1. Histone acetyltransferase Rtt109 can bind to Asf1 and et al. 2011). It has been suggested that these diﬀerent sets acetylates H3K56, which is a hallmark of newly synthesized of H3–H4 dimers determine the modiﬁcation pattern the histone H3 (Verreault et al. 1996; Winkler et al. 2012). H3– dimers will eventually have in the chromatin. H3K9me1 H4 dimers are imported into the nucleus by Kap123, a mem- is necessary to establish H3K9me3 in heterochromatin, ber of the karyopherin/importin family (Mosammaparast whereas H3K14ac is found in active chromatin (Alvarez et al. 2002; Avvakumov et al. 2011). 68 M. Terweij and F. van Leeuwen Figure 3. Dynamics of histones during replication. Overview of functions proposed for histone chaperones in dealing with histones during DNA replication. In the nucleus, Asf1 hands H3–H4 dimers to factors that changes that alter the binding aﬃnity of the H3–H4 dimers: assemble them into tetrasomes or nucleosomes (Figure 3). binding of Asf1–H3–H4 to the CAF-1 complex, containing These factors are the CAF-1 chromatin assembly complex RbAP48, causes a structural change in the conformation and Rtt106 (Li et al. 2008; Clemente-Ruiz et al. 2011; of the H3–H4 dimer, which leads to a decreased aﬃnity Fazly et al. 2012). The transit of H3–H4 dimers is promoted of Asf1 for H3–H4 (Zhang et al. 2013). Interestingly, like by H3K56 acetylation, since this PTM increases the bind- Asf1, the CAF-1 complex only binds H3–H4 dimers, and ing aﬃnity of CAF-1 and Rtt106 with H3–H4. CAF-1 is not (H3–H4) tetramers, possibly due to a destabilized H3– implicated in replication-coupled H3–H4 deposition since H3 interface (Zhang et al. 2013). However, CAF-1 can bind it binds to the replication-coupled H3.1 variant, but not the two H3–H4 dimers to form and deposit an H3–H4 tetramer replication-independent H3.3 variant in metazoans (Tagami onto the DNA (Liu & Churchill 2012; Winkler et al. 2012). et al. 2004). Also, CAF-1 interacts with PCNA (proliferat- It has been suggested that CAF-1 and DNA compete for ing cell nuclear antigen) (Shibahara & Stillman 1999; Green binding to the H3–H4 tetramer and that eﬃcient deposition et al. 2005; Su et al. 2012; Winkler et al. 2012). The transfer of tetramers may require the action of ATP-dependent chro- of H3–H4 dimers from Asf1 to CAF-1 involves structural matin remodelers or post-translational modiﬁcations. For Frontiers in Life Science 69 example, acetylation of H4K5 and K12 by HAT1 facilitates that the diﬀerentiated cell has to establish its epigenome the dissociation of H3–H4 tetramers from CAF-1. H3K56ac de novo. also plays a role in this part of the assembly line. Binding The replication fork is ﬂanked by short stretches of 250– of H3–H4 tetramers to DNA is reduced by acetylation of 300 bp of naked DNA (Sogo et al. 1986; Gasser et al. 1996), H3K56, leading to unstable tetrasomes. However, once the indicating that DNA replication and chromatin assembly are H2A–H2B dimers are loaded, H3K56ac has no eﬀect on tightly coordinated. The coupling between DNA replication nucleosome stability (Andrews et al. 2010; Watanabe et al. and assembly of new histones is most likely facilitated by 2013). various interactions between replication proteins and chro- Histone H2A and H2B follow a diﬀerent transporta- matin assembly factors (Li et al. 2012; Burgess & Zhang tion route. H2A–H2B dimers are imported into the nucleus 2013). Interestingly, those interactions may also play a role by Kap114 in association with Nap1 (Mosammaparast in the reassembly of old histones following passage of the et al. 2002). Kap114 is a karyopherin that binds to the replication fork, as has been suggested for Asf1 (Groth NLS of H2A. In the nucleus, RAN-GTP binds to Kap114 et al. 2007). An Asf1–MCM complex has been described and releases its cargo upon GTP hydrolysis. Nap1 sub- that binds to H3–H4 dimers harboring chromatin-associated sequently delivers H2A–H2B to chromatin for deposition histone modiﬁcations during replication stress, suggesting (Mosammaparast et al. 2002). Nap1 also binds to H3–H4. that Asf1 can deliver histones that were previously present However due to the higher aﬃnity of the H3–H4 for DNA, in chromatin of the parental DNA (Groth et al. 2007). The Nap1 readily deposits H3–H4 onto the DNA (Andrews histone chaperone NASP provides another mechanism to et al. 2010). Similarly, Nap1 eﬃciently deposits H2A–H2B coordinate histone synthesis and supply with DNA repli- onto DNA containing H3–H4 tetramers and disfavors non- cation. NASP controls the pool of soluble histones H3–H4 nucleosomal deposition of H2A–H2B on DNA (Andrews by regulating the activity of heat shock proteins Hsc70 and et al. 2010). Hsp90, which direct H3–H4 for degradation by autophagy The FACT (facilitate chromatin transcription) com- (Cook et al. 2011). Thereby, NASP prevents the accumula- plex also plays a role in H2A–H2B deposition (Formosa tion of free histones and the possible negative consequences et al. 2001; Owen-Hughes & Gkikopoulos 2012). FACT thereof during DNA replication stress or malfunctioning has been shown to be a chaperone for H2A–H2B and of Asf1. In budding yeast the checkpoint kinase Rad53 is can facilitate nucleosome assembly but also binds to H3– involved in avoiding the buildup of soluble histones during H4 (Belotserkovskaya et al. 2003, 2004). It is unclear misregulation of histone expression or replication stress. whether the displacement of H2A–H2B by FACT is direct Rad53 phosphorylates excess histones (H3 and H4), which or indirect, but it is clearly involved in altering chromatin targets them for degradation by the proteasome (Gunjan & structure during DNA replication (Wittmeyer & Formosa Verreault 2003; Singh et al. 2009, 2010). 1997). Interestingly, human FACT interacts with the mini Early studies suggested that parental (H3–H4) chromosome maintenance complex (MCM) (Tan et al. tetramers are segregated as tetramers and not as dimers 2006) and yeast FACT interacts with replication protein A during DNA replication (Leﬀak et al. 1977; Jackson & (RPA) (VanDemark et al. 2006), supporting genetic studies Chalkley 1981; Annunziato 2005), suggesting that epige- that link FACT to DNA replication (Okuhara et al. 1999; netic information encoded on nucleosomes is not inherited Schlesinger & Formosa 2000; Gambus et al. 2006; Tan et al. equally by the two daughter cells. The identiﬁcation of his- 2006) (Figure 3). tone chaperones bound to H3–H4 dimers (Asf1, HIR com- plex) led to the suggestion that histone H3–H4 tetramers might segregate as dimers and mix with newly synthe- Replication-coupled histone assembly sized H3–H4 proteins (Tagami et al. 2004; Ray-Gallet et al. Production and delivery of new histones peaks during S- 2011). Splitting of nucleosomes or tetramers could pro- phase, when not only DNA but also its packaging material vide a mechanism to transmit epigenetic information to is duplicated. Early studies on chromatin duplication sug- both daughter strands for PTMs that are present on each of gest that the existing, parental, nucleosomes are randomly the two copies of the respective modiﬁed histone protein. distributed over the two daughter strands (Sogo et al. 1986). Recent studies suggest that tetramer splitting can indeed The gaps need to be ﬁlled with newly synthesized histones to occur, but is restricted to a small subset of nucleosomes and maintain the same nucleosome occupancy as the parent cell is not associated with DNA replication. In mammalian cells (Annunziato 2005; Alabert & Groth 2012). Interestingly, tetramers of canonical H3–H4 do not split, whereas a small random distribution of existing histones may not apply to all fraction of H3.3–H4 tetramers are present as mixed (old cell types. A recent study showed that preexisting canonical + new) tetramers through replication-independent nucle- histone H3 is preferentially retained in male germline stem osome assembly (Xu et al. 2010; Huang et al. 2013). In cells during asymmetric division, whereas the replicated budding yeast tetramer splitting only occurs at highly active DNA in the diﬀerentiating daughter cell was assembled in genes undergoing high levels of replication-independent newly synthesized histones (Tran et al. 2012). This suggests histone exchange (Katan-Khaykovich & Struhl 2011). that the stem cell maintains the epigenetic information and These ﬁndings are in line with biochemical studies showing 70 M. Terweij and F. van Leeuwen that chromatin assembly factors CAF-1 and Rtt106 deposit Replication-independent deposition of histone H3 in H3–H4 tetramers (Su et al. 2012; Winkler et al. 2012). yeast Unlike higher eukaryotes, yeast only has a canonical H3 and lacks H3 variants, with the exception of the centromere- Replication-independent histone assembly: exchange speciﬁc H3 variant Cse4. However, the one histone H3 Although DNA replication in S-phase represents the major protein is involved in replication-dependent deposition as pathway of histone deposition, histones can also be dis- well as replication-independent exchange. Several studies assembled and reassembled by replication-independent in yeast showed that histone H3 is a dynamic entity in chro- mechanisms, leading to histone turnover or exchange. matin and that resident histone H3 molecules can be evicted Indeed, histone synthesis peaks during S-phase, but canon- and replaced by new histones in-trans upon activation and ical histones are also substantially expressed outside S- subsequent inactivation of inducible genes (Boeger et al. phase. Moreover, variants of the canonical histones are 2003; Rando & Winston 2012; Reinke & Hörz 2003; typically expressed and assembled throughout the cell Schermer et al. 2005). This model system has uncovered cycle. Variant histones do not generally use the assembly roles for Asf1, Spt6, Spt16 (FACT), the HIR complex, routes of canonical histones. Instead, each variant has ded- Rtt106, H2B ubiquitination, Chd1, and the proteasome in icated chaperones, providing opportunities for diﬀerential reassembling chromatin in the wake of RNA polymerase regulation of the diﬀerent assembly processes. Importantly, (Fleming et al. 2008; Imbeault et al. 2008; Ivanovska et al. replication-independent histone exchange and deposition of 2011; Jamai et al. 2009; Kim et al. 2007; Lee et al. 2012; histone variants can inﬂuence DNA accessibility and stabil- Ransom et al. 2009; Schwabish & Struhl 2006). Using ity of histone PTMs and has been linked to critical cellular inducible copies of tagged histones (as described above) the processes such as gene regulation, development, oncogenic process of histone exchange in yeast has subsequently been transformation, and nuclear reprogramming. delineated in ﬁne detail. It was ﬁrst shown in ﬁssion yeast that replication-independent exchange occurs preferentially Replication-independent deposition of histone H3.3 in euchromatic regions (Choi et al. 2005). These ﬁndings are In metazoans, deposition of canonical H3 is restricted to in agreement with studies in budding yeast, which showed S-phase. In contrast, histone H3 variant histone H3.3 is high levels of transcription-independent histone exchange expressed throughout the cell cycle and incorporated by in gene promoter regions, as well as transcription-dependent replication-independent mechanisms (Ahmad & Henikoﬀ exchange of H3 in coding regions and boundary-associated 2002). In Drosophila, H3.3 is assembled into chromatin regions (Dion et al. 2007; Jamai et al. 2007; Ruﬁange et al. in promoters, transcribed regions, and regulatory regions 2007). While there is a relationship between replication- (Mito et al. 2005; Teves et al. 2012). A similar proﬁle independent H3 exchange and RNA polymerase density in has been described in Arabidopsis (Stroud et al. 2012). coding regions, genes with the same transcription rate can These patterns are consistent with the idea that deposi- still have diﬀerent amounts of H3 exchange. The amount tion of H3.3 is linked to disruption of chromatin by the of exchange that is not explained by transcription rate act of transcription. In mammals, H3.3 is deposited mainly has been referred to as relative exchange (Gat-Viks & in promoter regions of active and silent genes (Chow et al. Vingron 2009), a feature that describes gene-speciﬁc rather than transcription-related eﬀects. A critical gene-speciﬁc 2005) and is also found in regulatory regions (Ray-Gallet feature is the type of promoter that drives the expression et al. 2011) and telomeric and centromeric regions (Wong of a coding sequence: histone exchange in coding regions et al. 2009; Goldberg et al. 2010). Three dedicated H3.3 is higher in regulated or inducible genes (also called stress chaperones have been described: HIRA, Daxx/ATRX and genes) than in constitutive genes (also called growth genes DEK (Campos et al. 2010; Drané et al. 2010; Elsaesser or housekeeping genes) (Dion et al. 2007; Jamai et al. 2007; & Allis 2010; Sawatsubashi et al. 2010; Tagami et al. Radman-Livaja et al. 2011 Ruﬁange et al. 2007). Whereas 2004). HIRA is responsible for depositing H3.3 in euchro- growth genes are typically regulated by TFIID (Transcrip- matic regions. It has been proposed to act by a gap- tion Factor RNA polymerase II D), have no canonical TATA ﬁlling mechanism at regions where canonical H3 is absent. Daxx/ATRX mediates H3.3 deposition in heterochromatic boxes, and contain promoters with highly organized nucleo- regions while DEK may target H3.3 to speciﬁc regulatory somes ﬂanking a nucleosome depleted region, stress genes regions (Campos et al. 2010; Drané et al. 2010; Elsaesser & are typically dependent on SAGA, have canonical TATA Allis 2010; Goldberg et al. 2010; Sawatsubashi et al. 2010; boxes, and show a non-stereotypical nucleosome organiza- Wong et al. 2009). H3.3 deposition by HIRA is promoted tion around the transcription start site (Rando & Winston by phosphorylation of histone H4S47, which is catalyzed 2012). It has been suggested that the diﬀerences in his- by the kinase PAK2. This phosphorylation event acts by tone exchange may be related to the bursty nature of increasing the binding aﬃnity of HIRA1 for H3.3–H4 transcription of stress genes (Dion et al. 2007; Jamai and reducing the binding of CAF-I with H3–H4 (Kang et al. 2007; Radman-Livaja et al. 2011; Ruﬁange et al. et al. 2011). 2007). Frontiers in Life Science 71 Figure 4. Histone traﬃc during transcription. Overview of functions proposed for histone chaperones and remodeling factors in dealing with histones during transcription and the crosstalk with histone PTMs. In yeast, histone exchange has also been studied by using Several recent studies in yeast have already uncovered the RITE genetic pulse-chase assay (Verzijlbergen et al. some of the mechanisms and functions of histone exchange 2010). Using this assay, H3 exchange has been detected in (Figure 4). In yeast, newly synthesized H3 is acetylated at cells arrested in G1, G2/M, and starvation conditions. Upon K56 by Asf1/Rtt109, and regions of the genome with high exit from starvation, yeast cells show extensive replication- H3 exchange show enrichment for this modiﬁcation. Dele- independent histone exchange across the genome, suggest- tion of Rtt109 or Asf1 or mutation of H3K56 reduce histone ing large scale epigenetic remodeling. A major determinant exchange rates in coding sequences (Kaplan et al. 2008; of H3 exchange is transcription. Active genes display a Ruﬁange et al. 2007; Schwabish & Struhl 2006; Smolle higher replication-independent exchange in their promoter et al. 2012; Venkatesh et al. 2012). The chromatin assembly and coding regions than inactive genes (Verzijlbergen et al. factor Rtt106 associates with coding regions, binds H3K56 2010). The RITE assay does not only allow for the following acetylated histones, and also promotes histone exchange of newly synthesized histones, but also for the following of in transcribed regions (Imbeault et al. 2008). H3K56ac the old histones. Radman-Livaja et al. (2011) investigated has intimate connections to another PTM on histone H3. the retention of old, ancestral histones over many cell gener- Methylation of histone H3K36 by Set2 represses histone ations in a genome-wide manner. In replicating yeast cells, exchange by disfavoring the interactions of histone H3 H3 is preferentially retained at the 5 end of genes of long with chaperones Asf1, Spt16 (FACT) and Spt6 (Venkatesh lowly transcribed genes. A model that has been proposed et al. 2012). In a set2 mutant, where no H3K36 methyla- to explain the ancestral H3 patterns contains three compo- tion is present, replication-independent histone exchange, nents: (1) histone exchange, which is higher in promoter H3K56ac (a hallmark of new H3) and H4ac are all increased regions than in ORFs; (2) lateral movement of the histones over ORFs. Disrupting histone exchange by deleting Asf1 towards the 5 end, called ‘passback’ (possibly due to the or Rtt109 in the set2 background decreases this accumu- passing of the transcription machinery); and (3) spreading lation of acetylation, thereby establishing histone exchange of the histones via dissociation and re-association during as a way to load pre-acetylated histones onto ORFs in replication (Radman-Livaja et al. 2011). the absence of Set2-mediated H3K36me (Venkatesh et al. 72 M. Terweij and F. van Leeuwen 2012). Interestingly, H3K36me also recruits and/or acti- et al. 2009). This raises the hypothesis that old H3 might vates the Rpd3S histone deacetylase complex to the 3 be reassembled into nucleosomes by FACT. In human end of coding regions. Therefore, Set2/H3K36me keeps cells Spt16 is recruited by H3K36 methylation by Set2D acetylation in transcribed regions low by two mechanisms: (Carvalho et al. 2013). Upon transcriptional activation it prevents the assembly of new, pre-acetylated histones Set2D methylates H3K36, which recruits FACT and leads to and it recruits an active deacetylase complex to reset a decrease in H2B occupancy. Thus, Set2D and FACT seem co-transcriptional acetylation events. This cleaning up of to work together to reassemble nucleosome after RNA poly- chromatin in the wake of transcription is required to pre- merase has passed, thereby preventing transcription from vent the ﬁring from cryptic promoters in coding regions. cryptic promoters within coding regions (Carvalho et al. H3K36me has more functions in histone exchange because 2013). it also recruits the Isw1b chromatin remodeler to coding regions, which together with the remodeler Chd1 pre- Replication-independent deposition of histone H4 and vents histone exchange (Radman-livaja et al. 2012; Smolle H2B et al. 2012), especially at long lowly transcribed genes. By Using variants of the inducible expression strategy, exten- combining RITE with a barcode-screen called Epi-ID, sev- sive replication-independent histone exchange has been eral other histone exchange factors have been identiﬁed. observed for histone H4 and H2B in budding yeast (Jamai Whereas Gis1 and Nhp10 are negative regulators, Hat1 et al. 2007; Linger & Tyler 2006). A comparison of the and its partners Hat2 and Hif1, which together form the behavior of H3 and H2B suggests that the rate of exchange NuB4 complex, are positive regulators of histone exchange of H2B is higher than that of H3 and occurs equally in (Verzijlbergen et al. 2011). Whether the NuB4 complex coding regions and promoters (Jamai et al. 2007). This aﬀects histone exchange via acetylation of new histones on ﬁnding is in agreement with the biochemical properties of H4K5K12 remains to be established. the nucleosome, in which the H3–H4 tetramer forms a sta- The HIR complex in yeast has been suggested to ble complex and from which the H2A–H2B dimers readily reassemble H3 in cis (Kim et al. 2007) (Figure 4). The HIR dissociate under conditions of high ionic strength or after complex is a conserved key player in histone exchange. passage of RNA polymerase (Hansen 2002; Kulaeva et al. In higher eukaryotes the HIRA complex deposits H3.3 2010). Recent in vitro transcription studies have implicated (Pchelintsev et al. 2013; Rai & Adams 2012; Tagami et al. the H2A–H2B chaperone Nap1 and the nucleosome remod- 2004). In yeast, it was initially identiﬁed as a histone chap- eling complex RSC (remodels structure of chromatin) in erone complex (containing Hir1-3 and Hpc2) involved in nucleosome dynamics during transcription. RSC displaces regulation of histone gene expression, but is also involved in a whole nucleosome and facilitates elongation by RNA replication-independent nucleosome assembly (Green et al. polymerase. Nap1 assists RSC in promoting transcription 2005; Kim et al. 2007; Lopes da Rosa et al. 2011; Silva through a nucleosome. Its positive eﬀect on transcription et al. 2012). Interestingly, also the replication-coupled coincides with the capturing an H2A–H2B dimer, allowing nucleosome assembly complex CAF-1 has been shown to RSC-dependent transcription to proceed through a hexas- aﬀect histone exchange (Lopes da Rosa et al. 2011). ome and partially retaining chromatin architecture (Kuryan FACT plays a complex role in nucleosome dynamics et al. 2012). during transcription. In yeast, the FACT complex contains two proteins, Spt16 and Pob3, aided by a third protein that is involved in DNA binding and contains a HMG-box- Exchange of other histone types like structure, Nhp6 (Formosa et al. 2001). In mammalian Although most research has been directed at histone H3 cells, FACT consists of only two proteins, Spt16 and exchange in yeast or at the replacement of histone H3.1 for SSRP1, where the last protein contains an HMG box for the replication-independent variant H3.3 in multicellular DNA binding (Belotserkovskaya & Reinberg 2004). FACT organisms, histone H2A also undergoes exchange and can travels along with RNA polymerase during transcription be replaced by a range of variant histones. Here we brieﬂy (Orphanides et al. 1999). It was ﬁrst shown that FACT summarize the ﬁndings on exchange of histone H2A. can displace one H2A–H2B dimer from the nucleosome, thereby creating a hexasome (Belotserkovskaya et al. 2003). Replacement of H2A by H2A.Z RNA polymerase can transcribe through this hexasomal Histone H2A.Z is the major variant of H2A. This variant structure (Kulaeva et al. 2010), however Kulaeva et al. is common for all eukaryotes, and the high sequence con- (2010) show that multiple closely spaced RNA polymerases servation suggests that it arose early in evolution (Kusch & will evict the hexasome from the DNA. It has also been Workman 2007; Skene & Henikoﬀ 2013). H2A.Z has been shown that FACT can act in the opposite way, reassembling linked to a wide range of functions, including transcrip- the nucleosome in the wake of RNA polymerase (Fleming tional activation, repression, and chromosome segregation. et al. 2008; Formosa 2011). In histone exchange studies H2A.Z is localized at promoters, where it is believed to no increase in H2B exchange has been observed, while H3 maintain a dynamic or accessible chromatin structure. In exchange increased in the Spt16 deletion mutant (Jamai Frontiers in Life Science 73 Figure 5. Pathways for the replacement of H2A and H2A.Z. Role of chaperones and remodelers in the nuclear import, deposition, and eviction of H2A.Z. addition, H2A.Z might have an important role at the bound- dimers (as well as H2A–H2B dimers) and shuttles them into aries between heterochromatin and euchromatin (Kusch the nucleus. In the nucleus, Nap1 hands the H2A.Z–H2B & Workman 2007; Venkatasubrahmanyam et al. 2007). dimer to the SWR1 (SWI/SNF Related) complex (SWR- Structural studies have shown that nucleosomes containing C). Chz1 has been shown to bind to H2A.Z in the nucleus, H2A.Z have an extended acidic patch on the nucleo- and is also able to deliver H2A.Z to the SWR-C (Straube some core surface. Incorporation of H2A.Z is believed to et al. 2010). The deposition of H2A.Z has been proposed to make the nucleosome more unstable; however, results from be replication-independent (Svotelis et al. 2010). A recent in vitro studies are contradictory (Kusch & Workman 2007; study in mouse trophoblast stem cells is in agreement with Watanabe et al. 2013). It has been proposed that nucleo- this idea. This study revealed changes in the composition somes containing both variants H3.3 and H2A.Z are very and abundance of H2A.Z-containing nucleosomes during unstable and may in fact be lost during conventional chro- the cell cycle (Nekrasov et al. 2012) and suggests that matin extraction methods (Jin et al. 2009). Methods that new H2A.Z in promoter regions is not assembled during preserve H3.3–H2A.Z nucleosomes show that double vari- S-phase but after completion of mitosis (Nekrasov et al. ant nucleosomes are predominantly located in the NDR of 2012). SWR-C incorporates H2A.Z in a stepwise man- TSSs of active promoters, enhancers, and insulator regions. ner, replacing one H2A–H2B dimer at a time (Luk et al. Jin et al. suggest that the region around the TSS is most of 2010). This can lead to the existence of heterotypic nucleo- the time occupied, either by transcription factors, or by dou- somes containing one H2A and one H2A.Z (Luk et al. 2010; ble variant nucleosomes, and only at the transition between Nekrasov et al. 2012). Heterotypic nucleosomes might be those two a ‘nucleosome-free’ region will appear (Jin et al. more unstable than homotypic nucleosomes (H2A.Z only 2009). or H2A only), as crystal structure analyses also suggest H2A.Z is incorporated by dedicated complexes (Henikoﬀ 2009; Suto et al. 2000). The activity of the SWR- (Figure 5). Two chaperones have been identiﬁed for H2A.Z, C in the genome is increased when the tails of H2A or H4 are Nap1, which is also a chaperone for H2A–H2B, and Chz1, acetylated (Altaf et al. 2010; Ishibashi et al. 2009; Shia et al. which seems to be a chaperone exclusively for H2A.Z– 2006). Acetylation of H2A and H4 in yeast is performed H2B (Billon & Côté 2012; Luk et al. 2010; Straube et al. by the NuA4 complex (Altaf et al. 2010; Keogh et al. 2010). In the cytosol, Nap1 associates with H2A.Z–H2B 2006); in the absence of a functional NuA4 complex, 74 M. Terweij and F. van Leeuwen nucleosomal H2A.Z decreases in yeast. In yeast, the SWR- repair. Yeast does not encode H2A.X but uses canonical C is the only known H2A.Z assembly factor. In mammalian H2A instead. When a DSB occurs, H2A.X is quickly phos- cells, H2A.Z assembly is carried out by two complexes: phorylated (γ -H2A.X). After repair, γ -H2A.X is replaced TIP60/P400 and SCRAP (Snf2-related CBP activator pro- by non-phosphorylated H2A.X (Kusch & Workman 2007). tein). The TIP60/P400 complex has an intrinsic acetylation No speciﬁc deposition factors have been described for ability for the tails of H2A and H4, and this is necessary for H2A.X to date but FACT has been found to bind to H2A.X incorporation of H2A.Z. The SCRAP complex, however, (Skene & Henikoﬀ 2013). A quickly evolving variant of is able to incorporate H2A.Z without the need for H2A and H2A was discovered about 10 years ago (Chadwick & H4 acetylation (Altaf et al. 2010). The SWR-C incorpo- Willard 2002). This variant, called H2A.Bbd (Barr body rates H2A.Z in the promoter regions of most genes in yeast. deﬁcient), only exists in mammals and not invertebrates. It has been proposed that SWR-C incorporates H2A.Z by H2A.Bbd is enriched in active gene bodies, and excluded default, resulting in H2A.Z incorporation around promoter from the inactive X chromosome, suggesting a role in sites and into gene bodies and that H2A.Z is removed from transcription. It has also been suggested that H2A.Bbd is non-promoter regions by the INO80 complex (INO80-C) involved in mRNA processing, although it is unclear if this (Papamichos-Chronakis et al. 2011). INO80-C, which is involvement is direct or indirect (Tolstorukov et al. 2012). closely related to SWR-C, is involved with the exchange of It has been shown that nucleosomes containing H2A.Bbd H2A.Z by canonical H2A, thereby opposing the action of are less stable than nucleosomes containing canonical H2A SWR-C. The speciﬁcity of H2A.Z localization also involves (Gautier et al. 2004). This could be because nucleosomes its acetylation. Acetylation of H2A.Z in yeast occurs on containing this shorter variant of H2A wrap only about the N-terminal tail and is carried out by NuA4 and SAGA 120 bp, leaving more DNA accessible for the binding of histone acetyltransferase complexes and reversed by Hda1 transcription factors. In agreement with the ﬁnding that (Babiarz et al. 2006; Keogh et al. 2006; Millar 2006; Lin H2A.Bbd-containing nucleosomes are less stable, it was et al. 2008; Ishibashi et al. 2009; Mehta et al. 2010). H2A.Z found, using FRAP, that exchange of H2A.Bbd is faster than is acetylated in promoter regions and INO80-C prefer- H2A exchange (Gautier et al. 2004). Mouse H2A variant entially removes unacetylated H2A.Z form the DNA. In H2A.Lap1 may also act to destabilize nucleosome orga- the absence of INO80-C, unacetylated H2A.Z aberrantly nization (Soboleva et al. 2012). Another variant of H2A, accumulates, which has a negative eﬀect on DNA damage macroH2A is mainly found on the inactive X chromosome repair and replication fork stability (Papamichos-Chronakis and near the promoters of inactive genes, suggesting that et al. 2011). Finally, the chromatin-remodeler Fun30 con- this variant is involved in gene repression (Gamble & Kraus tributes in H2A.Z positioning; deletion of Fun30 leads to a 2010). Exchange mechanisms of this variant are currently redistribution of H2A.Z from promoters to coding regions unknown. (Durand-Dubief et al. 2012). Interestingly, the speciﬁcity of SWR-C for H2A.Z is under control of multiple levels of trans-histone crosstalk. Outlook Whereas in unmodiﬁed nucleosomes SWR-C is activated It is becoming clear that the nucleosome is a very dynamic by DNA-bound H2A, in nucleosomes containing H3K56ac, structure that is much more than a structural packaging SWR-C is also activated by H2A.Z and exchanges the vari- mechanism for eukaryotic DNA. Histone proteins within ant for H2A (Watanabe et al. 2013). This ﬁnding and pre- the nucleosome are being modiﬁed by numerous PTMs on vious ﬁndings strongly link H2A.Z to exchange of histone numerous residues and canonical histones can be replaced H3 in yeast: (1) exchange of histone H3 leads to incorpora- by variants. These variations on the nucleosome can inﬂu- tion of H3 pre-acetylated on K56; (2) H3K56ac promotes ence chromatin structure and function in many ways. turnover of H3 in promoter nucleosomes; (3) H3K56ac Indeed, histone variants have been linked to a diverse set reverses the substrate-speciﬁcity of SWR-C, leading to of functions (Skene & Henikoﬀ 2013). However, important removal of H2A.Z; and (4) H2A.Z enhances turnover of his- functions are also emerging for the act of histone exchange tone H3 in promoter-proximal nucleosomes. A model has itself. been proposed in which an H3K56ac nucleosome may be Replication-independent histone exchange provides an subject to multiple rounds of SWR-C-catalyzed exchange important mechanism to maintain epigenome integrity in of H2A and H2A.Z, and this rapid H2A–H2B/H2A.Z– non-replicating cells (Ray-Gallet et al. 2011; Skene & H2B dimer exchange might promote H3–H4 exchange Henikoﬀ 2013). It allows the cell to replace histones that (Watanabe et al. 2013). are evicted by transcription or repair and thereby repair disrupted chromatin structures. This may explain why histone H3.3 accumulates in terminally diﬀerentiated non- Other H2A variants replicating rat neurons (Piña & Suau 1987). However, H2A.X is a variant of H2A that is present throughout the histone exchange may not go without consequences since genome and involved in DNA double strand break (DSB) it leads to loss of existing PTMs and deposition of histones Frontiers in Life Science 75 containing PTMs associated with newly synthesized his- by applying the inducible expression strategy to Rap1 has tones. In fact, histone exchange may provide the cell with shown that long Rap1 residence correlates much better with a mechanism to remove damaged histones or histones con- transcriptional activation than steady-state levels of Rap1 taining certain PTMs. Several recent studies support the binding (Lickwar et al. 2012). Fast Rap1 turnover is linked idea that histone dynamics and histone PTMs can inﬂuence with low transcriptional output. Moreover, long Rap1 res- each other. One example is methylation of H3K79 by Dot1. idence time correlates with slow H3 exchange (Lickwar No H3K79 demethylases have been identiﬁed, suggesting et al. 2012). This ﬁnding suggests that nucleosomes and that removal of this PTM requires dilution of modiﬁed transcription factors dynamically compete for DNA binding histones by replication-dependent or -independent mech- and opens up the possibility that histone exchange inﬂu- anisms. Indeed, H3K79me3 levels increase when yeast ences the transcriptional output of a transcription factor cells grow slower and aged histones have higher aver- binding event. Systematic measuring of the dynamics of age levels of H3K79me3 than young histones (De Vos chromatin proteins and regulators and the identiﬁcation of et al. 2011). When examined in the epigenome, H3K79me3 histone exchange factors and mechanisms will be critical to positively correlates with inheritance of ancestral histones determine in future studies how chromatin protein dynam- in replicating cells and negatively correlates with histone ics and histone exchange inﬂuence the epigenetic landscape, exchange and relative exchange levels (Radman-Livaja gene regulation, and epigenetic inheritance. et al. 2011). Together, these ﬁndings strongly indicate that Although the mechanisms and functions of histone histone exchange and inheritance ﬁne-tune the H3K79me exchange are not yet fully understood, recent studies indi- landscape laid down by Dot1. The observed slow buildup cate that histone H3.3 deposition is involved in key cellular of H3K79me3 over successive generations also challenge processes such as epigenetic reprogramming and cancer models that propose that histone PTMs are rapidly copied development (Filipescu et al. 2013). A large fraction of following DNA replication to maintain epigenetic states of human pancreatic neuroendocrine tumors (PanNETs) has the parental cell. been found to harbor mutations that inactivate ATRX Rapid exchange of histones in promoter regions is and Daxx. Based on the telomere disfunctioning observed incompatible with the idea that cells transmit epigenetic in these tumor cells, it has been proposed that loss of information by passing on histones with speciﬁc PTMs. ATRX/Daxx activity and deposition of H3.3 at telomeres An alternative model proposes that histone exchange might leads to telomere destabilization (Heaphy et al. 2011). In perpetuate active or silent gene expression states by increas- human pediatric glioblastomas, driver mutations in ATRX- ing or decreasing accessibility to sequence-speciﬁc binding DAXX have been found as well as mutations in the H3.3 proteins (Deal et al. 2010). Histones in coding regions gene itself (Schwartzentruber et al. 2012; Sturm et al. 2012). are more stable than promoter histones and may there- Interestingly, some of the glioblastoma driver mutations in fore provide better opportunities for epigenetic memory H3.3 reduce global H3 methylation levels in cells by inhi- mechanisms. However, tracking ancestral histone H3 in bition of SET-domain enzymes, suggesting that the mutant replicating yeast cells suggests that histone H3 does not H3.3 proteins act in a dominant manner to also inﬂuence associate with the exact same locus as where it came from canonical H3 (Lewis et al. 2013). Finally, replication- prior to DNA replication (Radman-Livaja et al. 2011). This independent deposition of H3.3 by HIRA has recently been argues against inheritance of chromatin state at a single found to be important for transcriptional reprogramming of nucleosome resolution, but rather suggests that inheritance nuclei transplanted to Xenopus oocytes. During reprogram- can occur in multi-nucleosome domains (Radman-Livaja ming, H3.3 deposition occurs in regulatory regions, among et al. 2011). The fact that less exchange is observed in yeast which that of Oct4, which may destabilize chromatin and coding regions does not mean that the chromatin in cod- facilitate a shift to diﬀerent epigenetic states (Jullien et al. ing regions is less disrupted. It cannot be excluded that 2012). Together, these studies demonstrate that histone histones are evicted and recycled, a process that remains exchange is a novel layer of epigenetic control with key undetected with most assays that measure histone dynam- roles in important cellular processes. ics. 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Frontiers in Life Science
– Taylor & Francis
Published: Jun 1, 2013
Keywords: histone; chromatin; epigenetics