Histone H4


Histone H4 Acetylation and chromatin assembly

Newly synthesized histone H4 is acetylated in the cytoplasm on residue 12 by Histone acetyltransferase B (HAT B). The S. cerevisiae histone acetyltransferase acetylates the lysine at residue 12 of free histone H4 but does not modify histone H4 when packaged in chromatin. HAT B consists of two proteins, Hat1p and Hat2p. Hat1p is the catalytic subunit of the histone aetyltransferase and has an intrinsic substrate specificity that modifies lysine in the recognition sequence GXGKXG. The specificity of the enzyme in the yeast cytoplasm is restricted relative to recombinant Hat1p, suggesting that it is negatively regulated in vivo. Recombinant Hat1p can acetylate histone H2A, while cytoplasmic extracts of yeast Hat1p do not. In addition, recombinant Hat1p acetylates histone H4 on both residues Lys-5 and Lys-12 while yeast derived enzyme modifies only Lys-12. Hat2p is a second subunit of histone acetyltransferase, and is physically associated with Hat1p. Hat2p, which is required for high affinity of the acetyltransferase to histone H4, binds to histone H4 tails. Hat2p is highly related to human Rbap48, which is a subunit of the chromatin assembly factor CAF-1, and copurifies with the human histone deacetylase HD1. The Hat2p/Rbap48 family serve as escorts of enzymes involved in histone metabolism, serving to facilitate enzymatic interaction with histone H4. Curiously, there is no obvious phenotypic consequence to the deletion of either HAT1, HAT2, or both genes from yeast cells. Such a lack of effect is likely explained by an apparent redundancy of HAT activity in yeast. For example, even in the absence of HAT1 there are still HAT activities in the cytoplasm. HAT2 has an apparent homolog in yeast, MSI1, which may substitute for its role in some processes (Parthun, 1996)

Assembly of newly synthesized Histones H3 and H4 is carried out by Chromatin assembly factor 1 (CAF-1). CAF-1 is a complex of three polypeptides with apparent molecular masses of 150, 60 and 50 kDa. CAF-1 assembles nucleosomes in a replication-dependent manner. The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins homologous to Drosophila p55, a component of Drosophila CAF-1 (Tyler, 1996), and homologous to a subunit of the yeast cytoplasmic histone acetyltransferase B complex component Hat2p. Each of these proteins (p55, Hat2p, and p48) are members of the p48 family of histone escorts. Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60. p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150k, p60 and p48) and histones H3 and H4, and promotes DNA replication-dependent chromatin assembly. CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps (but remains distinct from) the lycine acetylation reported for newly synthesized H4, isolated from nascent chromatin. Lysine 8 of CAC H4 is acetylated, in addition to lysines 5 and 12. CAC H3 also displays a distinct modification pattern. 40% of the CAC H3 molecules are modified, but it is uncertain whether by phosphorylation or acetylation. Most of the p48 polypeptide present in crude nuclear fractions is not actually associated with CAC. It is thought that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in various aspects of histone metabolism (Verreault, 1996).

p48 proteins are involved as histone escorts in a multistep process of cytoplasmic histone acetylation, the assembly of histones into chromatin, and the subsequent deacetylation of incorporated histones. The p48 family component of human CAF-1 is RbAp48, so named because it was isolated in association with the Retinoblastoma protein and has a molecular weight of 48 kD. RbAp48 also associates with histone deacetylase (HD1). The two proteins show sequence homology to yeast Hat2p, a component of the yeast cytoplasmic histone acetyltransferase complex of the B type (HAT B). The p48 family members constitute a conserved subfamily of WD-repeat proteins. The fact that RbAp48 is found associated both with CAF-1 and with HD1 implies that p48 family members assume different roles at different times. In the cytoplasm p48 members participate in acetylation of newly synthesized histones. The initial histone acetylation could be required to neutralize its high positive charge, allowing it to be assembled into chromatin. In the nucleus, in association with CAF-1, p48 family members are involved in replication linked chromatin assembly. Later, in association with HD1, p48 family members are involved in histone deacetylation, a requirement for maturation of newly synthesized chromatin (Roth, 1996 and references).

Transcriptionally related nuclear histone acetylation is carried out by HAT A. The yeast transcriptional adaptor protein (Gcn5p) functions as the catalytic subunit of a HAT A activity. Gcn5p is highly similar in sequence to a ciliate HAT A complex protein. The initial histone acetylation could be required to neutralize the high positive charge of histone, allowing histone to be assembled into chromatin. Deacetylation of histones carried out by histone deacetylase, could be a prerequisite to maturation of chromatin. In any case, it is now clear that chromatin assembly and maturation involves histone acetylation and that this process begins in cytoplasm and is subsequently transferred to the nucleus (Roth, 1996 and references).

Tetrahymena Histone Acetyltransferase A (HAT A), p55, is involved in transcriptionally related histone acetylation. Tetrahymena HAT A is a homolog of yeast Gcn5p that exists as a heterotrimeric complex in yeast cells with at least two other polypeptides. Gcn5p is a regulatory molecule that facilitates the action of acidic activators in yeast such as GCN4 and Gal4-VP16. Current models suggest that the Gcn5p complex bridges enhancer-binding factors to the basal transcription machinery. Both the Tetrahymena protein and Gcn5p possess histone acetyltransferase activity and a highly conserved bromodomain. p55 preferentially acetylates histone H3. The presenceof a bromodomain in nuclear A-type histone acetyltransferases (but not in cytoplasmic B-type HATs), known to function in protein-protein interaction, suggests that HAT A is directed to chromatin through protein interaction to facilitate transcriptional activation. It is also believed that there is a functional interaction between the Histone Acetyltransferase A type of complex and components of the SWI-SNF complex in yeast (note their Drosophila homologs: ISWI and Brahma). Although the exact mechanism by which the SWI/SNF complex operates is unclear, an implication of this interaction is that one function of the SWI/SNF complex is to direct HAT A to specific sites in chromatin. These findings, combined with the recent demonstration that the SWI/SNF polypeptides are integral components of the RNA polymerase II holoenzyme in yeast, suggest a mechanism whereby HAT A is targeted to chromatin during transcriptional activation, establishing a direct link between histone acetylation and gene activation (Brownell, 1996).

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. SRC-1 possesses intrinsic histone acetyltransferase activity; it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates. (Spencer, 1997).

There are major transitions in the type and modification of chromatin-associated proteins during the early development of Xenopus laevis. Histone H4 is stored in diacetylated form in the egg only to be progressively deacetylated during normal development. If histone deacetylases are inhibited with sodium butyrate, hyperacetylated histone H4 accumulates only after the mid-blastula transition. The type of linker histone in chromatin also changes during embryogenesis, from predominantly the B4 protein at the mid-blastula transition to predominantly histone H1 (See Drosophila histone H1) at the end of gastrulation. These transitions in chromatin composition correlate with major changes in the replicative and transcriptional activity of embryonic nuclei (Dimitrov, 1993).

The distribution of acetylated isoforms of histone H4 along Chinese hamster chromosomes has been studied by immunostaining with antibodies recognizing H4 acetylated at defined lysines in its N-terminal domain. The heterochromatic long arm of the X chromosome in both female (CHO) and male (DON) cell lines is underacetylated at three out of four lysines (5, 8, and 12). In contrast, the level of acetylation at lysine 16, which is the first to be acetylated in mammals, is similar in X chromosomes and autosomes. Labeling of the cells with bromodeoxyuridine (BrdU) to mark late-replicating chromosome domains, followed by double immunostaining with antibodies to BrdU and acetylated H4, show a close, though not perfect, correlation between late replication and low levels of H4 acetylation. The results show that levels of histone acetylation are associated with the replication timing of defined domains on both the X chromosome and autosomes, but the exceptions observed suggest that this link is not absolute or essential (Belyaev, 1996).

Underacetylation of histone H4 is thought to be involved in the molecular mechanism of mammalian X chromosome inactivation, which is an important model system for large-scale genetic control in eukaryotes. However, it has not been established whether histone underacetylation plays a critical role in the multistep inactivation pathway. Differential histone H4 acetylation is found that distinguishes the X chromosomes of a female marsupial, Macropus eugenii. Histone underacetylation is the only molecular aspect of X inactivation known to be shared by marsupial and eutherian mammals. Its strong evolutionary conservation implies that, unlike DNA methylation, histone underacetylation was a feature of dosage compensation in a common mammalian ancestor, and is therefore likely to play a central role in X chromosome inactivation in all mammals (Wakefield, 1997).

Core histones isolated from normal and butyrate-treated HeLa cells have been reconstituted into nucleosome cores in order to analyze the role of histone acetylation in enhancing transcription factor binding to recognition sites in nucleosomal DNA. Moderate stimulation of nucleosome binding is observed for the basic helix-loop-helix factor USF and the Zn cluster DNA binding domain factor GAL4-AH using heterogeneously acetylated histones. However, nucleosome cores containing the most highly acetylated forms of histone H4 show the highest affinity for these two transcription factors. Acetylated histone H4 predominates relative to acetylated histone H3. Immunoprecipitation of USF-nucleosome complexes with anti-USF antibodies also demonstrated that these complexes are enriched preferentially in acetylated histone H4. USF and GAL4-AH preferentially interact with nucleosome cores containing highly acetylated histone H4. Acetylation of histone H4 thus appears to play a primary role in the structural changes that mediate enhanced binding of transcription factors to their recognition sites within nucleosomes (Vettese-Dadey, 1996).

Defined model systems consisting of physiologically spaced arrays of H3/H4 tetramer complexed with 5S rDNA have been assembled in vitro from pure components. Analytical hydrodynamic and electrophoretic studies have revealed that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. The reptation in agarose gels of H3/H4 tetramer arrays is essentially indistinguishable from naked DNA; the gel-free mobility of H3/H4 tetramer arrays relative to naked DNA is reduced by only 6% compared with 20% for nucleosomal arrays, and H3/H4 tetramer arrays are incapable of folding under ionic conditions, whereas nucleosomal arrays are extensively folded. The cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 tetramer arrays. These data establish that there is a direct physicochemical basis for competition between transcription factor binding and nucleosome assembly immediately after replication. Importantly, whether the stretches of H3/H4 tetramer arrays mature into bulk chromatin or become programmed into transcriptionally active genes will largely depend on both the local concentrations of H2A/H2B dimers and transcription factors in the vicinity of the replication fork and their respective affinities for H3/H4 tetramer⋅DNA complexes. In this regard, it seems likely that the mechanisms required to precisely regulate these parameters throughout the entire genome have evolved in conjunction with the unique nucleoprotein structure that is formed on each DNA strand immediately after replication in vivo (Tse, 1998).

The human ISWI-containing factor RSF (remodeling and spacing factor) mediates nucleosome deposition and, in the presence of ATP, generates regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation is impaired in directing transcription. Histone-tail modifications regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 increases transcription. Methylation of the histone H3 tail by Suv39H1 represses transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system is refractory to the effect imposed by acetylation and methylation (Loyola, 2001).

The reaction catalyzed by RSF appears mechanistically different from the reaction catalyzed by ACF, which requires the histone chaperone NAP-1. RSF appears to function stoichiometrically with DNA, whereas ACF functions catalytically. The assembly of regularly spaced nucleosomes, catalyzed by RSF, appears to proceed by at least two steps. The first step is nucleosome deposition, and it is likely mediated by the large subunit of RSF. This subunit (p325) is encoded by a novel gene. The amino acid sequence of p325 shows the presence of a large charged region, which might participate directly in the nucleosome assembly reaction. The small subunit of RSF, hSNF2h, does not interact directly with the histone octamer; however, RSF, like histone chaperones, interacts with the H3/H4 tetramer and the octamer. Moreover, the binding of RSF to DNA is dependent on the histone octamer. The interaction of RSF with the octamer is independent of the histone tails and does not require posttranslational modifications, since RSF interacts with the histone octamer generated with bacterially produced histones and with recombinant octamers deleted of the histone tails. Although, the nucleosome deposition step catalyzed by RSF is independent of the histone tails, the second step of the reaction, the ATP-dependent nucleosome spacing, is dependent on histone tails. Moreover, the efficiency of array formation is stimulated by p300-mediated acetylation. In agreement with previous studies showing that the Drosophila ISWI polypeptide requires the H4 tails for ATP-dependent nucleosome mobilization, the histone H4 tail is necessary for array formation. However, the histone H4 tail is not sufficient for the formation of regularly spaced nucleosome arrays, since the tails of the other histones influence array formation. p300-mediated stimulation of array formation requires acetylation of the tails of the H2A/H2B dimer. Surprisingly, however, the histone H3 tail negatively affects the p300-mediated stimulation of array formation. It is speculated that the negative effect imposed by the histone H3 tail might be overcome by other modifications, such as phosphorylation of histone H3 at Ser 10 or that an alternative HAT mediates acetylation of this tail. This remains to be elucidated (Loyola, 2001).

One of the predictions of the histone code hypothesis is the existence of functional interactions between chromatin remodeling complexes, such as SWI/SNF, and histone acetylase complexes, such as GCN5. Recent studies have elucidated the temporal sequence in which these coactivators of transcription are recruited to promoters in vivo and how their enzymatic properties contribute to gene activation. The best-characterized example in mammals is provided by the human IFN-ß gene. The gene is switched on by three transcription factors (NF-kappaB, IRFs, and ATF-2/c-Jun), and an architectural protein [HMG I(Y)], all of which bind cooperatively to the nucleosome-free enhancer DNA to form an enhanceosome. The enhanceosome targets the modification and repositioning of a nucleosome that blocks the formation of a transcriptional preinitiation complex on the IFN-ß promoter. This is accomplished by the ordered recruitment of HATs, SWI/SNF, and basal transcription factors. Initially, the GCN5 HAT-containing complex is recruited, and it acetylates the nucleosome. This is followed by the recruitment of the CBP-PolII holoenzyme complex. Next, the SWI/SNF remodeling machine arrives at the promoter via bivalent interactions with CBP and the acetylated histone N tails. SWI/SNF alters the structure of the nucleosome via an unknown mechanism, thus allowing recruitment and DNA binding of TFIID to the TATA box. The DNA bending induced upon TFIID binding to the promoter causes sliding of the SWI/SNF-modified nucleosome to a new position 36 bp downstream, thus allowing the initiation of transcription. This ordered recruitment and nucleosome sliding is consistent with the view that histone acetylation sets the stage for ATP-dependent remodeling by establishing a recognition surface for the bromodomains present in SWI/SNF-like remodeling machines. Furthermore, since histone acetylation precedes the recruitment of additional complexes bearing bromodomains, such as TFIID, it is possible that this modification also regulates recruitment of TFIID to promoters (Agalioti, 2002).

Experiments were carried out to test the histone code hypothesis. Only a small subset of lysines in histones H4 and H3 are acetylated in vivo during viral infection, and this modification is carried out by the GCN5 transcriptional coactivator complex. Reconstitution of recombinant nucleosomes bearing mutations in these lysine residues reveals a biochemical cascade for gene activation via a point-by-point interpretation of the histone code through the ordered recruitment of bromodomain transcription complexes. More specifically, acetylation of H4 lysine 8 is required for recruitment of the SWI/SNF complex, whereas acetylation of lysines 9 and 14 in histone H3 is critical for the recruitment of the general transcription factor TFIID. Thus, the information contained in the DNA address of the enhancer is extended (transferred) to the histone N termini by generating novel adhesive surfaces that participate in the recruitment of transcription complexes (Agalioti, 2002).

The precision by which the histone code is decoded is remarkable. Most likely, the code is translated via specific interactions of bromodomains with the acetylated histone N termini. The bromodomain in BRG1 associates with the H4 tail acetylated at K8, whereas the double bromodomain in TAFII250 binds to the doubly acetylated (at K9 and K14) H3 tail. The competition assays using either acetylated histone N termini peptides or bromodomain polypeptides as competitors revealed an unprecedented level of specificity for the interactions between bromodomains and acetyl-lysine histone N termini. Again, this remarkable degree of specificity may be dictated by the conformational changes forced upon these complexes by their interactions with other proteins in the complex. Thus, the point-by-point interpretation of the histone acetylation code may rely on the precise allosteric changes induced in many proteins upon their association with transcription factor complexes. For example, the initial recruitment of SWI/SNF via its association with CBP is stabilized on the promoter through the association of the BRG1 bromodomain with the H4 K8 acetylated tail. Although, BRG1's bromodomain could interact at least in principle with other acetylated lysine residues on H3, these interactions may not be of sufficient strength to ensure stable binding of the SWI/SNF complex to the promoter. Similarly, recruitment of TFIID to the SWI/SNF-modified promoter is stabilized via two types of interactions. The first with various enhanceosome components and the second with the interaction between the two bromodomains of TAFII250 and the two acetyl groups on residues K9 and K14 of histone H3. Several observations support this notion: (1) both TBP and TAFII250 are simultaneously recruited to the promoter with almost identical kinetics in vivo; (2) recruitment of TFIID in vivo occurs only when both H3 K9 and K14 are acetylated; (3) mutations in either K9 or K14 abrogate TFIID recruitment. The data show that the TAFII250 double bromodomain, when recruited to the natural IFN-ß promoter, interacts specifically with the H3 K9 and K14 acetylated residues and not with the acetylated H4 tails. However, when tested in isolation and out of the promoter/chromatin natural context, the double TAFII250 bromodomain interacts with similar affinities to both H4 and H3 acetylated tails, a result consistent with in vitro observations. It is proposed that the inordinate set of interactions occurring with purified bromodomains and acetylated histone tails is 'fixed' when present in natural promoter/chromatin contexts. In the latter case, it is possible that the competing interactions cannot take place either because the alternative target is occupied (e.g., the H4 tail is already bound by SWI/SNF) or the specific three-dimensional conformation of the transcription complex does not permit these interactions to occur (Agalioti, 2002).

A model is presented that depicts the ordered biochemical cascade decoding the DNA and histone acetylation code during activation of human IFN-ß gene transcription following Sendai virus infection. It is thought that the promoter DNA code contains all the information for the assembly of the enhanceosome in response to virus infection. The enhanceosome that assembles at the promoter element recruits the GCN5 histone acetyltransferase. Subsequently GCN5 acetylates initially H4K8 and H3K9. An unknown kinase recruited by the enhanceosome phosphorylates H3 Ser 10, a prerequisite for H3K14 acetylation by GCN5. The histone code is subsequently translated by recruiting of additional components required for transcription. The bromodomain containing transcription complexes SWI/SNF and TFIID are recruited to the promoter via bivalent interactions between the enhanceosome and specifically acetylated histone N termini, and this subsequently stimulates transcription of the IFN-ß gene (Agalioti, 2002).

The histone code hypothesis proposes that covalently modified histone tails are binding sites for specific proteins. In vitro evidence suggests that factors containing bromodomains read the code by binding acetylated histone tails. Bromodomain Factor 1 (Bdf1: possibly a homolog of Female sterile homeotic of Drosophila), a protein that associates with TFIID, binds histone H4 with preference for multiply acetylated forms. In contrast, the closely related protein Bdf2 shows no preference for acetylated forms. A deletion of BDF1 but not BDF2 is lethal when combined with a mutant allele of ESA1 (a histone H4 acetyltransferase) or with nonacetylatable histone H4 variants. Bromodomain point mutations that block Bdf1 binding to histones disrupt transcription and reduce Bdf1 association with chromatin in vivo. Therefore, bromodomains with different specificity generate further complexity of the histone code (Matangkasombut, 2003).

Structural basis for the histone chaperone activity of Asf1

Anti-silencing function 1 (Asf1; Drosophila homolog) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 Å shows how Asf1 binds the H3/H4 heterodimer, enveloping the C terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C terminus of histone H4 that forms a mini-β sheet with histone H2A in the nucleosome undergoes a major conformational change upon binding to Asf1 and adds a β strand to the Asf1 ß sheet sandwich. Interactions with both H3 and H4 are required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a 'strand-capture' mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that may be used ubiquitously by histone chaperones (English, 2006).

The ubiquitous function of Asf1 in eukaryotes is highlighted by the sequence conservation of the residues involved in the interactions between Asf1 and histones H3/H4. Budding yeast Asf1 is 56% identical to Xenopus Asf1 in the conserved core, and the Xenopus histones are 88% and 92% identical to H3 and H4 from yeast, respectively. Only the following three residues of Xenopus H3 that contact Asf1 differ in other species: C110 (Ala in yH3), Q125 (Lys in yH3), and I130 (Leu in yH3); these substitutions would appear to cause only minor and possibly compensated differences in interprotein packing. Furthermore, none of these interspecies differences occur in residues of H4 that contact Asf1. Therefore, the interactions observed in this structure will likely be applicable to Asf1-histone H3/H4 complexes from different species (English, 2006).

The Asf1 histone chaperone forms extensive contacts with both histones H3 and H4. The Asf1-H3/H4 structure reveals the details of the interface between Asf1 and a3 of H3 and has identified a new interaction between Asf1 and a2 of H3. The implications of the mutagenesis study, with regard to Asf1 and H3, are that disruption of this intricate interface has severe consequences in the context of the cellular activity. For example, mutations in the regions of Asf1 that bind to only H3 (R145E/S48R, Y112A/R145E, V94R, and S48R) or the region of H3 that binds to Asf1 (K115 and K122) weakened the interaction between Asf1 and H3 and disrupted Asf1 function in vivo and in vitro. As such, the interaction between histone H3 and Asf1 is clearly critical for its cellular functions (English, 2006).

The Asf1-H3/H4 structure shows extensive contacts between Asf1 and histone H4. This interface has two parts: (1) the globular core of Asf1 interacts with the C-terminal tail of H4 to form a strand-swapped dimer and (2) the C-terminal tail of Asf1 binds to the histone-fold region (a3) of histone H4. These interactions are also important because mutations in residues of Asf1 that contact H4 (T147, L6, V109, and V146) weaken histone binding and alter the functions of Asf1 in yeast. Similarly, mutation of histone H4 residues R92, H75, Y72, Y88, and F100 that contact Asf1 in the Asf1-H3/H4 structure reduces the chromatin assembly and/or disassembly functions of Asf1 in vivo. Clearly, interactions of Asf1 with both histones H3 and H4 are required for Asf1 function, and neither interaction is sufficient (English, 2006).

The mutations that affect the interaction between Asf1 and H3/H4 fall into two distinct functional classes; (1) those that reduce the function of Asf1 and (2) those that cause a gain-of-function phenotype. The former was expected, but the latter uncovered specific mutations that overcome the requirement for CAF-1 in transcriptional silencing. These include Asf1 S48R, V109M, Y112E, and V146L that weaken the interaction with histones H3/H4 in vivo and in vitro. Interestingly, the histone H4 H75Y mutation that had reduced Asf1-mediated chromatin-disassembly activity and Zeocin sensitivity has also been shown to bypass the requirement for CAF-1 in silencing. The same ability to bypass the requirement for CAF-1 in silencing has been demonstrated by truncations or insertion mutations in the C terminus of Asf1. Specifically, inactivation of CAF-1 leads to reduced histone deposition onto DNA, while additional mutations in the C terminus of Asf1 restores the histone deposition onto DNA. Although the C terminus of Asf1 is not present in the determined structure, it may extend toward histone H4 from its current location in the structure and may contribute further to histone binding affinity. It is possible that the Asf1 L6M, S48R, V109M, Y112E, V146L, and T147E mutations enhance transcriptional silencing by the same mechanism as the C-terminal mutations in Asf1 (English, 2006).

A human protein complex homologous to the Drosophila MSL complex is responsible for the majority of histone H4 acetylation at lysine 16

A stable, multisubunit human histone acetyltransferase complex (hMSL) contains homologs of the Drosophila dosage compensation proteins MOF, MSL1, MSL2, and MSL3. This complex shows strong specificity for histone H4 lysine 16 in chromatin in vitro, and RNA interference-mediated knockdown experiments reveal that it is responsible for the majority of H4 acetylation at lysine 16 in the cell. hMOF is a component of additional complexes, forming associations with host cell factor 1 and a protein distantly related to MSL1 (hMSL1v1). Two versions of hMSL3 were found in the hMSL complex that differ by the presence of the chromodomain. Lastly, it was found that reduction in the levels of hMSLs and acetylation of H4 at lysine 16 are correlated with reduced transcription of some genes and with a G2/M cell cycle arrest. This is of particular interest given the recent correlation of global loss of acetylation of lysine 16 in histone H4 with tumorigenesis (Smith, 2005).

Reversible histone acetylation plays an important role in regulation of chromatin structure and function. The human orthologue of Drosophila melanogaster MOF, hMOF, is a histone H4 lysine K16-specific acetyltransferase. hMOF is also required for this modification in mammalian cells. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by Western blot analysis and mass spectrometric analysis of endogenous histones. Evidence is provided that, similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 small interfering RNA-treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in the G(2) and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by a reduction in hMOF protein levels. Furthermore, hMOF-depleted cells show an increased number of phospho-ATM and gammaH2AX foci and have an impaired repair response to ionizing radiation. Taken together, these data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression (Taipale, 2005).

Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF

A stable complex containing MLL1 (Drosophila homolog, Trx) and MOF has been immunoaffinity purified from a human cell line that stably expresses an epitope-tagged WDR5 subunit. Stable interactions between MLL1 and MOF were confirmed by reciprocal immunoprecipitation, cosedimentation, and cotransfection analyses, and interaction sites were mapped to MLL1 C-terminal and MOF zinc finger domains. The purified complex has a robust MLL1-mediated histone methyltransferase activity that can effect mono-, di-, and tri-methylation of H3 K4 and a MOF-mediated histone acetyltransferase activity that is specific for H4 K16. Importantly, both activities are required for optimal transcription activation on a chromatin template in vitro and on an endogenous MLL1 target gene, Hox a9, in vivo. These results indicate an activator-based mechanism for joint MLL1 and MOF recruitment and targeted methylation and acetylation and provide a molecular explanation for the closely correlated distribution of H3 K4 methylation and H4 K16 acetylation on active genes (Dou, 2005; full text of article).

PTEN interacts with histone H1 and controls chromatin condensation

Chromatin organization and dynamics are integral to global gene transcription. Histone modification influences chromatin status and gene expression. PTEN plays multiple roles in tumor suppression, development, and metabolism. This study, performed with HeLa cells, reports on the interplay of PTEN, histone H1, and chromatin. Loss of PTEN leads to dissociation of histone H1 from chromatin and decondensation of chromatin. PTEN deletion also results in elevation of histone H4 acetylation at lysine 16, an epigenetic marker for chromatin activation. PTEN and histone H1 physically interact through their C-terminal domains. Disruption of the PTEN C terminus promotes the chromatin association of MOF acetyltransferase and induces H4K16 acetylation. Hyperacetylation of H4K16 impairs the association of PTEN with histone H1, which constitutes regulatory feedback that may reduce chromatin stability. These results demonstrate that PTEN controls chromatin condensation, thus influencing gene expression. It is proposed that PTEN regulates global gene transcription profiling through histones and chromatin remodeling (Chen, 2014: PubMed).

Histone crosstalk between H3S10ph and H4K16ac generates a histone code that mediates transcription elongation

The phosphorylation of the serine 10 at histone H3 has been shown to be important for transcriptional activation. This study reports the molecular mechanism through which H3S10ph triggers transcript elongation of the FOSL1 gene. Serum stimulation induces the PIM1 kinase to phosphorylate the preacetylated histone H3 at the FOSL1 enhancer. The adaptor protein 14-3-3 binds the phosphorylated nucleosome and recruits the histone acetyltransferase MOF, which triggers the acetylation of histone H4 at lysine 16 (H4K16ac). This histone crosstalk generates the nucleosomal recognition code composed of H3K9acS10ph/H4K16ac determining a nucleosome platform for the bromodomain protein BRD4 binding. The recruitment of the positive transcription elongation factor b (P-TEFb) via BRD4 induces the release of the promoter-proximal paused RNA polymerase II and the increase of its processivity. Thus, the single phosphorylation H3S10ph at the FOSL1 enhancer triggers a cascade of events which activate transcriptional elongation (Zippo, 2009).

Histone H4-K16 acetylation controls chromatin structure and protein interactions

Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, a native chemical ligation strategy was used to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber (Shogren-Knaak, 2006).

The structural effect of H4-K16Ac may directly contribute to regions of decondensed chromatin in eukaryotic organisms. In budding yeast, over 80% of H4 is acetylated at lysine 16, and most of the genome exists in a decondensed state. Likewise, evidence suggests that the transcriptionally enhanced X chromosome of male flies, a site of ubiquitous H4-K16Ac, is decondensed. Such decondensation of chromatin may contribute to the establishment of transcriptionally active euchromatic regions. In vitro transcription studies suggest that the adoption of higher order chromatin structure reduces gene transcription. In contrast, acetylation of H4-K16 increases gene transcription both in vitro and in vivo, and the decompaction resulting from such modification may increase the accessibility of factors that promote transcription (Shogren-Knaak, 2006).

Do other histone marks regulate chromatin folding? The phosphorylation of H3-S10 (S, serine) does not disrupt chromatin folding, and triacetylation of the H3 tail by Gcn5p does not disrupt chromatin compaction. Similarly, residues 1 to 13 of histone H4 that include three sites of acetylation are dispensable for folding of nucleosome arrays. Thus, H4-K16 is likely to be a unique acetylation site of histone tails, which function as a dual switch for higher order chromatin structure and protein-histone interactions, promoting chromatin function in a mutually reinforcing manner (Shogren-Knaak, 2006).

EGFR modulates DNA synthesis and repair through Tyr phosphorylation of histone H4

Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical for DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. This study identified a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 monomethylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair (Chou, 2014).

Acetylation of Histone H4 and double-strand break repair

Although the acetylation of histones has a well-documented regulatory role in transcription, its role in other chromosomal functions remains largely unexplored. Distinct patterns of histone H4 acetylation are essential in two separate pathways of double-strand break repair. A budding yeast strain with mutations in wild-type H4 acetylation sites shows defects in nonhomologous end joining repair and in a newly described pathway of replication-coupled repair. Both pathways require the ESA1 histone acetyl transferase (HAT), which is responsible for acetylating all H4 tail lysines, including ectopic lysines that restore repair capacity to a mutant H4 tail. Arp4, a protein that binds histone H4 tails and is part of the Esa1-containing NuA4 HAT complex, is recruited specifically to DNA double-strand breaks that are generated in vivo. The purified Esa1-Arp4 HAT complex acetylates linear nucleosomal arrays with far greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near a break site. Together, these data show that histone tail acetylation is required directly for DNA repair and suggest that a related human HAT complex may function similarly (Bird, 2002).

Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1-dependent transcription during Drosophila metamorphosis

Gene regulation by AP-1 transcription factors in response to Jun N-terminal kinase (JNK) signaling controls essential cellular processes during development and in pathological situations. The histone acetyltransferase (HAT) Chameau and the histone deacetylase DRpd3 act as antagonistic cofactors of DJun and DFos to modulate JNK-dependent transcription during pupal thorax metamorphosis and JNK-induced apoptosis in Drosophila. It has been demonstrated, in cultured cells, that DFos phosphorylation mediated by JNK signaling plays a central role in coordinating the dynamics of Chameau and DRpd3 recruitment and function at AP-1-responsive promoters. Activating the pathway stimulates the HAT function of Chameau, promoting histone H4 acetylation and target gene transcription. Conversely, in response to JNK signaling inactivation, DRpd3 is recruited and suppresses histone acetylation and transcription. This study establishes a direct link among JNK signaling, DFos phosphorylation, chromatin modification, and AP-1-dependent transcription and its importance in a developing organism (Miotto, 2006).

The functional partnership between Chm and DRpd3 in the control of JNK signaling seems specific to this HAT/HDAC pair, since mutations in other HDAC or HAT genes do not modify the chm thorax closure phenotype. Particularly illustrative is the absence of genetic interactions with mutant alleles of mof, since Mof is a MYST HAT of the same substrate specificity as Chm (Histone4 K16) and DFos interacts with Chm through the conserved MYST domain. Interestingly, a recent report has connected Chm and DRpd3 functions during Drosophila oogenesis (Aggarwal, 2004). The two proteins exhibit opposite effects on H4 acetylation and activity of replication origins, indicating that the functional antagonism of Chm and DRpd3 regulates several chromatin-dependent processes, including transcription and replication (Miotto, 2006).

Study of the mode of action of the antagonistic cofactors Chm and DRpd3 in cultured cells has provided further insights into a chromatin-based mechanism that executes a modulation of the transcriptional response to JNK signaling. The following model is proposed, based on the dynamics of cofactor recruitment and activity, chromatin modification and transcriptional status related to reversible activation of the pathway. In response to JNK signaling, Chm HAT activity sets up a histone modification pattern that is instructive for transcriptional enhancement. Consistent with this notion Chm acetylates H4, with a marked preference for K16, and facilitates H3K4 trimethylation. However, AP-1 likely also engages HATs of different substrate specificity, since H4K8 acetylation, a modification required for the recruitment of the SWI/SNF-activating complex, is directed by DFos in the absence of Chm. After JNK signaling has ceased, DRpd3 gets recruited to promoters occupied by unphosphorylated DFos and counteracts the effects of Chm by reversing histone modifications, which results in transcriptional down-regulation. Strikingly, the recruitment of DRpd3 seems not to result from the displacement of Chm from the promoter, since invariant levels of Chm are associated with the promoter in sorbitol experiments, whereas DRpd3 starts to be recruited only once the signal has been eliminated. Thus, as opposed to an exchange of a HAT coactivator complex for an HDAC corepressor complex, which occurs for instance between Pcaf/NF-kappab and DRpd3/AP-1 complexes at the attacin promoter (Kim, 2005), a complex containing both Chm and DRpd3 could then form at the target promoter whose activity changes the histone modification pattern back to a pattern less permissive to transcription. Thus, DRpd3 most likely functions during a transient phase from a transcriptionally active to silent status. Its absence from the promoter at the inactive steady state in nonstimulated cells, suggests that unphosphorylated DFos then lies in a conformational environment that prevents DRpd3 recruitment by the ZIP domain (Miotto, 2006).

Interaction of transcription factors with Histone H4

TFIID is a large multiprotein complex that initiates assembly of the transcription machinery. It is unclear how TFIID recognizes promoters in vivo when templates are nucleosome-bound. Here, it is shown that TAFII250, the largest subunit of TFIID, contains two tandem bromodomain modules that bind selectively to multiply acetylated histone H4 peptides. The 2.1 angstrom crystal structure of the double bromodomain reveals two side-by-side, four-helix bundles with a highly polarized surface charge distribution. Each bundle contains an Nepsilon-acetyllysine binding pocket at its center -- this results in a structure ideally suited for recognition of diacetylated histone H4 tails. Thus, TFIID may be targeted to specific chromatin-bound promoters and may play a role in chromatin recognition (Jacobson, 2002).

The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, nucleosome arrays containing albumin enhancer sequences were assembled and they were compacted with linker histone. HNF3 and GATA-4 but not NF-1, C/EBP, and GAL4-AH, bind their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events (Cirillo, 2002).

How might HNF3 access its binding sites in vivo? Under physiological salt conditions, nucleosome arrays exist in a dynamic equilibrium between folded and compacted states. Additionally, linker histone is rapidly exhanged on chromatin in living cells. HNF3 could exploit both of these properties to initially bind its sites in compacted chromatin. Previously published data indicate that the essential HNF3 binding sites eG and eH flank the dyad axis of the nucleosome particle to which HNF3 binds. This would place HNF3 on the bound particle in the vicinity of where X-ray crystallography data places histones H3 and H4 in the nucleosome. Histones H3 and H4 make internucleosomal contacts which have been implicated in the formation of nucleosomal arrays, and nucleosome arrays lacking the H3/H4 amino-terminal tails fail to fold into a fully compacted state or undergo mitotic chromosome condensation. It is suggested that HNF3 disrupts internucleosomal interactions promoted by H3/H4 tetramers, thereby decompacting the array locally and making it accessible to other proteins. In addition, HNF3 binding helps stabilize the position of an underlying nucleosome, which could affect the length of linker DNA on either side of the N1 particle. Small variations in linker length can have dramatic effects on the compaction of nucleosomes in chromatin. It is therefore suggested that HNF3 disrupts local chromatin structure by a combination of core histone interactions and by inducing changes in the position or orientation of nearby linker regions (Cirillo, 2002).

Histone H3 K36 methylation is mediated by a trans-histone methylation pathway involving an interaction between Set2 and histone H4

Set2-mediated H3 K36 methylation is an important histone modification on chromatin during transcription elongation. Although Set2 associates with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), the mechanism of Set2 binding to chromatin and subsequent exertion of its methyltransferase activity is relatively uncharacterized. This study identified a critical lysine residue in histone H4 that is needed for interaction with Set2 and proper H3 K36 di- and trimethylation. It was also determined that the N terminus of Set2 contains a histone H4 interaction motif that allows Set2 to bind histone H4 and nucleosomes. A Set2 mutant lacking the histone H4 interaction motif is able to bind to the phosphorylated CTD of RNAPII and associate with gene-specific loci but is defective for H3 K36 di- and trimethylation. In addition, this Set2 mutant shows increased H4 acetylation and resistance to 6-Azauracil. Overall, this study defines a new interaction between Set2 and histone H4 that mediates trans-histone regulation of H3 K36 methylation, which is needed for the preventative maintenance and integrity of the genome (Du, 2008).

Interaction of Histone H4 with TFIID

Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, it has been shown that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacts with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone is essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persists on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions (Kanno, 2004).

Given that H3 is a preferred substrate for many HATs in in vitro assays, the absence of FRET between H3 and Brd2 was striking. This could not be explained by a lack of H3 acetylation, since HeLa cells contained significant amounts of acetylated H3. This led to a test of another bromodomain protein, TAFII250, for interaction with H3. TAFII250 is a component of the basal transcription factor TFIID, and its double bromodomain module binds to an acetylated H4 peptide in vitro and acetylated H3 in a reconstituted chromatin target (Agalioti, 2002). Transfected CFP-TAFII250 associated with other components of TFIID in HeLa cells, indicating that it was incorporated into a stable TFIID complex. It was found that TAFII250 produced a significant FRET signal with H3 as well as H4 and to a lesser extent H2B but not with H2A. Because TAFII250 has a HAT activity and contains a histone recognition site outside the bromodomains (Mizzen, 1996), deletions were tested that removed the HAT region, but retained the bromodomains. The deletions produced FRET with H3 and H4 as well as did full length TAFII250. It is of note that these deletions also lacked the TBP binding surface, and TAFII250-BD is further devoid of the putative HMG-like region, indicating that DNA binding of TAFII250 is dispensable for its histone association. Thus, H3 is recognized by the bromodomains of TAFII250 but not of Brd2. Moreover, in contrast to FRET with Brd2, both mutants H4-K(5,12)G and H4-K(8,16)G partially reduced FRET with TAFII250, indicating that K5/K12 and K8/K16 of H4 both critically contribute to the interaction with TAFII250. Peptide precipitation analysis showed that TAFII250 bound H4 peptides mono-acetylated at K8, K12, or K16 as well as H3 peptides acetylated at K14. It is concluded that specific lysine residues of both H3 and H4 are recognized by the TAFII250 bromodomains in vivo and that TAFII250 has broader recognition specificity than Brd2 (Kanno, 2004).

Cell cycle dependent methylation of Histone H4 by the mammalian PR-Set protein

Distinct patterns of histone methylation during human cell cycle progression are described. Histone H4 methyltransferase activity is cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely follows the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7 (see Drosophila Pr-Set7). Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted (Rice, 2002).

To determine histone methyltransferase activity during the human cell cycle, HeLa cells were arrested by treatment with thymidine followed by mimosine. Every 2.5 h following release from the G1 arrest, synchronized cells were isolated for analysis, and the cell cycle phase was determined by fluorescence-activated cell sorting (FACS). Nuclei were isolated from the cells at these various time points for the in nucleo HMT assay and for Western blot analysis (Rice, 2002).

The in nucleo assay was used to provide a preliminary indication of alterations in nuclear methyltransferase activity during specific phases in the cell cycle. The assay was performed by incubating synchronized nuclei in the presence of a radiolabeled methyl donor (3H-S-adenosyl-methionine), followed by SDS-PAGE and autoradiography. No apparent changes in the H3 HMT activity occur during the cell cycle. In contrast, the enzymatic methylation of histone H4 in this assay is greatly increased during mid-S-phase through mitosis and returned to levels observed during G1. Little, if any, H4 HMT activity is detected outside of this time frame. It is interesting to note that histones H3 and H4 were the only nuclear proteins in this assay that were detectably methylated. There are several potential limitations to this assay including occupancy of preexisting methylation sites, complications resulting from neighboring histone modifications, and/or decreased detection of HMT activity caused by as-yet undiscovered histone demethylases. Nonetheless, this assay provides a preliminary indication that H4 methylation, as opposed to the H3 methylation, is highly regulated throughout the human cell cycle (Rice, 2002).

To further characterize the HMT activity from the in nucleo assay, integrated densitometry was used to quantitate the intensity of the radiolabeled histone bands and the intensity of the Coomassie-stained histone bands. Once these values were determined, the radiolabeled histone band was divided by the loading control Coomassie-stained histones, yielding an arbitrary number. These values were then standardized by assigning the G1 value to 1 such that the fold change at each of the time points could be determined. Although undetectable by the gels, the analysis indicates a moderate increase in H3 HMT activity that peaks during mid-S-phase (2.5-fold), declines through G2/M, and reaches a baseline at G1. In contrast, the analysis of the histone H4 bands indicates a significant increase in HMT activity during mid-S-phase to G2 (5-10 h) that abruptly declines during mitosis and the transition to G1 (12.5 h). The H4 HMT activity then reaches a baseline upon return to G1 (Rice, 2002).

These results show that the enzymatic methylation of histones H3 and H4 occurs during distinct phases in the human cell cycle under these assay conditions. Although there is a modest increase in histone H3 methyltransferase activity during S-phase, it has yet to be determined which Arg or Lys residues are being methylated during this time point. This will be difficult to dissect because many arginines and lysines in histone H3 are known to be methylated in vivo. In contrast, a dramatic increase in histone H4 methyltransferase activity is observed during mid-S-phase and G2/M in the HeLa cell cycle. Because Arg 3 and Lys 20 are the only histone H4 residues presently known to be methylated in vivo, an investigation was carried out to determine which of these two residues are being methylated during these time points in the human cell cycle (Rice, 2002).

Histones from synchronized HeLa cells were fractionated by SDS-PAGE and Western-blotted using antibodies specific for the H4 Arg 3-methyl or Lys 20-methyl modifications. Methylation of H4 Arg 3 is readily detected in HeLa cells arrested in G1 (0 h). Upon entry into S-phase (0-2.5 h), there is a decrease in this modification that increases back to the observed G1 levels during the transition from mid- to late-S-phase (5-7.5 h) and remains constant through mitosis (Rice, 2002).

One possible explanation for the observed decrease in the H4 Arg 3-methyl modification is the deposition of newly synthesized histone H4, which occurs immediately following DNA replication. Thus, the apparent decrease may be caused by a dilution due to the deposition of newly synthesized and unmethylated histone H4 proteins that ultimately become methylated during later points in the cell cycle, most likely mediated by the H4 Arg 3-specific HMT, PRMT1. In addition, the methylation of H4 Arg 3 during mid-S-phase suggests that this modification may be associated with euchromatin, or transcriptionally active regions, which are replicated early in S-phase. This would be consistent with a recent finding that H4 Arg 3 methylation plays a role in transcriptional activation of nuclear hormone receptors (Rice, 2002).

Although the increase in H4 Arg 3 methylation accounts for the observed increase in H4 HMT activity during S-phase (2.5-7.5 h), it does not explain the increased activity during G2/M. Because Lys 20 is the only other histone H4 residue known to be methylated, it was hypothesized that Lys 20 methylation is increased during this phase in the cell cycle. Blot analysis shows that H4 Lys 20 methylation also decreases during S-phase, albeit later in the cell cycle compared with H4 Arg 3 methylation. During the transition from early- to mid-S-phase (2.5-5 h), H4 Lys 20 methylation drops and remains low through late-S-phase (7.5 h) and G2 (10 h), which may be caused, again, by dilution of this modification by the deposition of newly synthesized and unmethylated histone H4 proteins. The observed decrease in H4 Lys 20 methylation in mid-S-phase suggests that this modification is associated with heterochromatin, which is known to replicate later in S-phase compared to euchromatin. This theory is supported by a recent report showing that the H4 Lys 20-methyl modification is associated with transcriptionally silent regions of the genome (Nishioka, 2002; Rice, 2002).

During mitosis (12.5 h), H4 Lys 20 methylation returns to levels similar to those observed in G1 and stay constant through the remainder of the cell cycle. Although FACS analysis shows approximately equal numbers of cells in mitosis as G1 at the 12.5-h time point, the single appearance of the mitotic-specific phosphorylation of histone H3 serine 28 indicates that H4 Lys 20 methylation occurs during mitosis rather than during the transition to G1. Furthermore, because histone H3 serine 28 is phosphorylated specifically in the early phases of mitosis and is dephosphorylated abruptly during the metaphase-to-anaphase transition, the results suggest that the methylation of H4 Lys 20 also begins early in mitosis (Rice, 2002).

To further expand the observation that H4 Lys 20 methylation decreases in S-phase and peaks at mitosis, immunofluorescence studies were performed in Drosophila embryos. A recent report shows that the H4 Lys 20-methyl modification is present in Drosophila and is essential for Drosophila development and viability (Nishioka. 2002b). In addition, the embryos provide an excellent model to study H4 Lys 20 methylation during the cell cycle since they rapidly and repeatedly shift from S-phase to mitosis -- this can be easily determined by DAPI staining. Consistent with the above findings, H4 Lys 20 methylation is clearly detected on chromosomes during both metaphase and anaphase, whereas staining during S-phase results in a faint signal even upon overexposure. It is hypothesized that the faint signal during S-phase most likely reflects a combination of dilution of the modification by histone deposition as well as the decrease in chromatin condensation, which could contribute to a dispersion of the signal resulting in a decreased ability to detect the modification. Regardless, these data confirm that H4 Lys 20 methylation is decreased during S-phase and increased specifically during mitosis (Rice, 2002).

It was recently reported that histone H4 Lys 20 methylation inhibits acetylation of H4 Lys 16, and vice versa (Nishioka, 2002b). Based on this, it was predicted that these two modifications would occur at distinct points in the cell cycle. Western blot analysis showed that H4 Lys 16 acetylation is low during G1 (0 hours) when H4 Lys 20 methylation is the highest. However, H4 Lys 16 acetylation significantly increases and peaks during mid-S-phase, the time when H4 Lys 20 methylation is the lowest. During mitosis (12.5 h), the acetylation of H4 Lys 16 dramatically decreases just as H4 Lys 20 methylation peaks. These observations show that H4 Lys 20 methylation inhibits H4 Lys 16 acetylation and vice versa (Rice, 2002).

Immunofluorescence studies were performed on mitotic HeLa cells to provide a qualitative estimate of the relative phase during mitosis in which H4 Lys 20 methylation increases. The specific phases of mitosis were determined by staining of DNA with DAPI. At prophase, the H4 Lys 20-methyl modification displays a more punctate and less intense staining pattern compared with interphase cells that have high levels of H4 Lys 20 methylation. This suggests that the observed decrease in H4 Lys 20 methylation during S-phase and G2 persists through the early stages of mitosis. It is unlikely that the observed decreased staining at prophase is a consequence of chromatin condensation since H4 Lys 20 methylation is clearly detected at other phases of mitosis when chromatin is even more condensed. In contrast to prophase, there was a visually pronounced increase in H4 Lys 20 methylation at metaphase that coincides directly with alignment of chromosomes on the metaphase plate. This suggests that the increase in H4 Lys 20 methylation occurs prior to or during metaphase. The H4 Lys 20-methyl modification persists through the rest of mitosis. Taken together, these results document the mitotic-specific enzymatic methylation of H4 Lys 20 (Rice, 2002).

A novel histone H4 Lys 20-specific methyltransferase, PR-Set7, has been identified in HeLa cells (Nishioka, 2002b). It was hypothesized that PR-Set7 expression would increase simultaneously with H4 Lys 20 methylation during cell cycle progression. Northern blot analysis in synchronized HeLa cells indicates that PR-Set7 mRNA expression is greatly increased during late S-phase and G2/M and declines during transition to G1. To determine if PR-Set7 protein expression is also increased during these times, a polyclonal PR-Set7 antibody was developed and confirmed to be specific for PR-Set7. The antibodies detect recombinant as well as endogenous PR-Set7. Consistent with the RNA expression findings, Western blot analysis shows that PR-Set7 protein is not detected during G1. The PR-Set7 protein levels elevate steadily beginning at early S-phase through G2 (10 h) and peak during mitosis (12.5 h). The increase during mitosis was confirmed by the appearance of the mitotic-specific phosphorylation of histone H3 serine 28. Moreover, Western blot analysis in G1-arrested cells confirms that the PR-Set7 protein is undetectable, whereas in mitotic-arrested cells there are significantly abundant levels of PR-Set7. Subsequent to mitosis, the PR-Set7 protein abruptly decreases and continues to decrease as more cells entered G1 (15-20 h). These findings indicate that PR-Set7 RNA and protein expression are up-regulated during cell cycle progression, consistent with the observed increase in H4 HMT activity and H4 Lys 20 methylation (Rice, 2002).

Immunofluorescence studies in HeLa cells were performed to determine the localization of PR-Set7 during different phases in the cell cycle. At metaphase and anaphase PR-Set7 is clearly associated with mitotic chromosomes. PR-Set7 is also detected at prometaphase, although the localization was relatively dispersed compared with metaphase and anaphase (Rice, 2002).

These data indicate that PR-Set7 expression is cell cycle-regulated and that the PR-Set7 protein is localized to mitotic chromosomes, coincident with the increase in H4 Lys 20 methylation. The steady increase in PR-Set7 expression during cell cycle progression is directly correlated with the observed increase in H4 HMT activity during these same times. Although these data indicate that there is sufficient enzymatically active PR-Set7 in the nucleus during S-phase and G2, the methylation of H4 Lys 20 is delayed prior to metaphase. It is presently unknown what mechanisms account for this delay; however, it is clear that PR-Set7 expression and the PR-Set7 protein must be tightly regulated by, as yet, uncharacterized mechanisms to prevent the premature methylation of H4 Lys 20. Consistent with this theory, studies performed with Xenopus egg extracts showed that the Xenopus PR-Set7 protein is phosphorylated during mitosis. Although phosphorylation of human PR-Set7 is not essential for its HMT activity in vitro, phosphorylation of the enzyme in vivo may serve to regulate its association with mitotic chromosomes (Rice, 2002).

The cell cycle-regulated methylation of H4 Lys 20 suggests that this modification is localized to specific regions in the genome and inherited in an epigenetic fashion. Using telomere position effect variegation as a model for epigenetic silencing, data in yeast suggest that this repressive chromatin state is disassembled during S-phase and reassembled by G2/M. This coincides with the decrease in H4 Lys 20 methylation during S-phase and its increase during mitosis. Once established following replication, telomeric silent chromatin is relatively stable, much like histone methylation. The similarities between these two suggest that the H4 Lys 20-methyl modification may serve as a stable epigenetic mark that aides in the establishment of discrete chromosomal regions involved in specific chromatin-mediated events. The association of PR-Set7 with mitotic chromosomes may represent a mechanism through which the H4 Lys 20-methyl mark is epigenetically transmitted. It is likely that the localization of PR-Set7 to mitotic chromosomes allows recognition of this mark on the parent chromosomes, which are then duplicated to the daughter chromosomes (Rice, 2002).

Although methylation does not affect the overall charge of the histone tail, it does increase the hydrophobicity and basicity of the lysine residue, suggesting an increased attraction for the negatively charged DNA. However, a more likely function for this modification is that it serves as a recognition motif for the binding of chromatin-associated proteins that mediate changes in higher-order chromatin structure during mitosis, similar to HP1 binding of methylated H3 Lys 9 to establish heterochromatic regions. Alternatively, methylation of H4 Lys 20 may serve to prevent the association of factors to the H4 tail, similar to H3 Lys 4 methylation, which prevents the association of the NuRD complex with the H3 tail. Based on these findings, a likely candidate would be a histone acetyltransferase that modifies H4 Lys 16 (Rice, 2002).

Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage

Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. A previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a 'histone mark' required for the recruitment of the checkpoint protein Crb2 (Sanders, 2004).

Though present in the N-terminal tail region of H4, evidence argues that lysine 20 is not exposed but 'buried' in the context of stacked nucleosomes and that the H4 tail has a critical role in higher order nucleosome packing. Based upon this and the apparent presence of the modification throughout much of the genome, a model of H4-K20 methylation function is presented. During unperturbed cell growth, the methylated H4-K20 residue is hidden or buried in the context of packed chromatin. Introduction of a DSB would then generate a region of unstacked or open chromatin exposing a preexisting methylated H4-K20 residue. Recognition of the modification by Crb2 then leads to recruitment of Crb2 to the DSB. It is further suggested that the independent association of Rad3 and subsequent phosphorylation of H2AX is further required for the stable association of Crb2 at DSBs. In the absence of H4-K20 methylation or H2AX phosphorylation, the ability of Crb2 to recognize a DSB is compromised, leading to a lack of focal enrichment and an attenuated checkpoint signal insufficient to maintain an extended cell cycle arrest after IR (Sanders, 2004).

It is currently unknown whether the proposed recognition of methyl H4-K20 by Crb2 is through direct binding, but it is predicted that the binding will be direct. Outside of its tandem BRCT domains, which may bind phosphorylated H2AX, Crb2 contains no other obvious functional domains. However, data suggest that Crb2 does contain a noncanonical tudor fold similar to that of 53BP1. It has been postulated that tudor domains may bind methylysines, since they are part of the Royal Family of chromo-like domains. This suggests that Crb2 may recognize chromatin through its tudor domain by directly binding to methyl H4-K20. Interestingly, 53BP1 contains two adjacent tudor domains and has the ability to recognize methylated H3-K79. This raises the intriguing possibility that Crb2's mammalian counterpart may recognize chromatin via interaction with other modifications in addition to methyl H4-K20 (Sanders, 2004).

Specificity. mechanism and structure of the histone methyltransferase Pr-Set7

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. The crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20) is presented. The enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. The structure of Pr-Set7 was examined along with a 10-residue peptide based on histone H4 (mono-methylated on the target lysine) and the cofactor product S-adenosylhomocysteine. Evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor; a model is developed for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. A highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and is also probably responsible for how this part of the structure ensures that Pr-Set7 is nucleosome specific (Xiao, 2005).

A family of enzymes containing a conserved domain, called the SET domain, was initially implicated in gene silencing and position effect variegation and has subsequently been shown to be responsible for methyl transfer from S-adenosylmethionine (AdoMet) to the histone lysine side-chain nitrogen (N). It seems that all but one of the methylated lysine residues present on histones is subject to modification by SET family enzymes. A key biological question asks how members of this family of enzymes achieve specificity for their particular target lysine residue. A second important issue is establishing whether a particular lysine residue is mono-, di-, or tri-methylated and whether these distinct modifications give rise to different physiological consequences (Xiao, 2005).

Mono-methylation of lysine H4-K20 has been shown to be cell-cycle regulated, and its methylation status contributes to chromosome behavior during mitosis and proper cytokinesis. Moreover, using antibodies able to discriminate between H4-K20 mono-, di-, and tri-methylation states, staining of mouse fibroblasts revealed that tri-methylated H4-K20 is associated with transcriptionally inactive pericentric heterochromatin whereas mono-methylated and di-methylated H4-K20 were found to be broadly distributed but generally associated with euchromatic regions. A number of different SET proteins have been reported as being able to methylate H4-K20 including Pr-Set7, also separately reported as Set8, Suv4-20h1 and Suv4-20h2, and NSD1. Both Suv4-20h enzymes are capable of tri-methylating H4-K20. Pr-Set7 is highly selective for H4-K20 and has a marked preference for nucleosomal substrate over either histone octamer or H4 polypeptide. Gene knockout techniques involving deletion of a homolog of the Pr-Set7 (Set8) gene in Drosophila have shown that the gene is essential for development (Nishioka, 2002). Also in Drosophila it has been shown that K20 methylation is associated with transcriptionally silent dense chromatin regions. Therefore the structural and functional characterization of Pr-Set7 was undertaken to better understand its biological role. This study reports the crystal structure of a ternary complex of the enzyme and demonstrates that it adds a single methyl group to H4-K20. A residue from the peptide substrate contributes to the active site of the enzyme and thus plays an important part in determining the enzyme's specificity, and it is suggested how the enzyme interacts with its nucleosome substrate. Finally, it is considered how the C-flanking domains of SET proteins are likely to become ordered upon the addition of cofactor and how this influences the catalytic cycle of these enzymes (Xiao, 2005).

SET8 (also known as PR-SET7) is a histone H4-Lys-20-specific methyltransferase that is implicated in cell-cycle-dependent transcriptional silencing and mitotic regulation in metazoans. This study reports the crystal structure of human SET8 (hSET8) bound to a histone H4 peptide bearing Lys-20 and the product cofactor S-adenosylhomocysteine. Histone H4 intercalates in the substrate-binding cleft as an extended parallel beta-strand. Residues preceding Lys-20 in H4 engage in an extensive array of salt bridge, hydrogen bond, and van der Waals interactions with hSET8, while the C-terminal residues bind through predominantly hydrophobic interactions. Mutational analysis of both the substrate-binding cleft and histone H4 reveals that interactions with residues in the N and C termini of the H4 peptide are critical for conferring substrate specificity. Finally, analysis of the product specificity indicates that hSET8 is a monomethylase, consistent with its role in the maintenance of Lys-20 monomethylation during cell division (Couture, 2005).

PR-Set7-dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis

The histone methyl transferase PR-Set7 mediates histone H4 Lys 20 methylation, a mark of constitutive and facultative heterochromatin. A null mutation was isolated in Drosophila PR-Set7 that suppresses position effect variegation, indicating that PR-Set7 indeed functions in silencing general gene expression. In PR-Set7 larval leg and eye discs, the number of cells is lower than normal, and the DNA content in these cells is significantly increased. These data show that PR-Set7-dependent methylation is essential for the process of mitosis. The methylation mark is highly stable and is maintained even in the absence of PR-Set7 protein (Karachentsev, 2005).

The existing l(3)neo41 mutation is caused by a P-element insertion into the 5'UTR of the PR-Set7 gene and is lethal at the late pupal stage over Df(3R)red31. Such insertions frequently result in partial loss of function of the gene. In order to obtain a complete loss of function allele, the advantage was taken of a new P-element insertion obtained from the Drosophila stock center, PR-Set7EY0466 8. This allele is lethal in homozygous pupae. By mobilizing this P-element a deletion was isolated in which the entire PR-Set7 protein coding region is missing (PR-Set720). PR-Set720 homozygotes or hemizygotes over Df(3R)red31 show a somewhat stronger phenotype: most animals die at the larval-to-pupal transition, with rare escapers surviving into early pupal stage. The presence and perdurance of PR-Set7 protein was determined in wild-type and mutant animals using anti-Drosophila PR-Set7 antibody raised in Rats against Drosophila recombinant PR-Set7 (Karachentsev, 2005).

In Western blots, a band of ~100 kDa is present in extracts of wild-type ovaries, early embryos, and throughout development, suggesting that PR-Set7 is deposited in the egg during oogenesis. In extracts from homozygous PR-Set7 animals, the ~100-kDa band is missing, while alpha-tubulin is clearly present. These results show that the antibody recognizes the PR-Set7 protein specifically. They further show that the maternally deposited PR-Set7 protein does not perdure into first instar larvae, since the band is missing in homozygous mutants (Karachentsev, 2005).

Using the antibody to stain salivary gland chromosomes, it was found that the PR-Set7 protein is associated with the chromocenter and with the chromosome arms, mostly with densely packed DNA, indicating that PR-Set7 is associated with facultative and constitutive heterochromatin as well as with euchromatin. The chromosomal staining appears specific for PR-Set7, sinces no staining is observed on PR-Set720 salivary gland chromosomes. These results show that even though the protein is missing in the mutants from first instar larval stage onward, the homozygous mutant PR-Set720 animals can survive until late larval to early pupal stages (Karachentsev, 2005).

Based on the localization of methylated H4-K20 on salivary gland chromosomes at transcriptionally inactive regions, it is proposed that this methylation functions to nucleate higher-order structures that would maintain chromatin in an inactive state. To determine whether indeed PR-Set7 functions as a silencer, its capability to suppress variegation was examined. When euchromatic genes such as the wild-type white (w+) gene in Drosophila are inserted into heterochromatic regions, the condensed chromatin structure of heterochromatin often spreads into the euchromatic region, resulting in full or partial inactivation of the genes. This results in variegated expression of the genes, easily seen in Drosophila eyes (position effect variegation, PEV) (Karachentsev, 2005).

To measure the level of expression of the w+ gene the amount of red pigment was measured in extracts of fly heads; both Df(3R)red31 and the PR-Set720 alleles function as dominant suppressors of PEV. The expression of w+ transgenes inserted into centromeric and telomeric heterochromatin of the fourth chromosome is significantly activated by the loss of function of one copy of PR-Set7. PEV of w+ insertions on chromosomes two and three were not changed. This result shows that indeed PR-Set7 functions as a repressor of gene activation and supports a previous supposition that was based only on distribution of the protein on chromosomes (Karachentsev, 2005).

The mono-, di-, and tri-methylated state of histone H4-K20 was investigated in wild-type and mutant larvae by antibody staining of salivary gland chromosomes and by Western blot. The staining of salivary gland chromosomes with the antibody specific for mono-, di-, or trimethylated histone H4-K20 shows that all three methyl marks have an indistinguishable distribution, with the exception of trimethyl H4-K20 appearing more abundant in centromeric heterochromatin. This result agrees with studies demonstrating that trimethylation of H4-K20 is preferentially a pericentromeric mark. On salivary gland chromosomes derived from homozygous PR-Set720 mutants, the level of all three forms of methylated H4-K20 was reduced. This reduction was only observed in late-stage larvae and was usually stronger on the chromosome arms than in the centromeric regions. The salivary chromosome preparations were costained with antibody recognizing the transcriptionally active form of RNA polymerase II. Both wild-type and mutant glands showed strong polymerase staining, indicating that the chromosomes are intact and that the reduced levels of the methylated forms of H4-K20 are not due to degradation of the chromosomes (Karachentsev, 2005).

Homozygous PR-Set7 larvae generally die at the larval-to-pupal transition. Although the salivary glands and the chromosomes of such mutants look relatively normal, a distinct phenotype can be observed in tissues with higher rates of cell divisions such as larval imaginal discs. Imaginal discs are epithelial sacs made up of a folded, columnar epithelium on one side and a squamous epithelium on the other side. The columnar discs cells are very small, only a few microns in size. Mutant discs are 10%–20% smaller than the wild-type discs and contain a significantly smaller number of cells. The number of cells was counted in optical sections prepared on a confocal microscope, and wild-type and mutant eye discs were compared. The amount of DNA in each cell was determined by measuring the intensity of Hoechst fluorescence. It was found that in wild-type discs there are approximately four times as many cells as in the mutant discs, and the fluorescence of nuclei indicates that there are ~3.5 times more DNA in mutant nuclei compared with wild-type. Further, hetero-allelic PR-Set7 mutants l(3)neo41/PR-Set720 rarely survive to adulthood and die soon after eclosion. In these escapers the eye is reduced in size and irregular, consistent with the phenotype observed in the eye discs of homozygous PR-Set720 larvae. These results show that PR-Set7-controlled methylation of H4-K20 is essential for normal cell cycle progression. The mutant cells seem to undergo DNA replication but fail to complete mitosis (Karachentsev, 2005).

Analysis of PR-Set7 protein in wild-type animals reveals a protein on Western blots that appears to be significantly larger than the predicted size of 76 kDa. This difference in size could, at least in part, be due to as yet uncharacterized modifications of the enzyme. Several experiments show that the antibody specifically recognizes the Pr-Set7 protein. The protein is absent in extracts from mutant animals, and it is not detectable on mutant salivary gland chromosomes. Further, Flag-tagged PR-Set7 expressed in flies is recognized as a larger protein in Western blots of extracts from transgenic lines (Karachentsev, 2005).

The Western blots also show that mutant first instar larvae are devoid of the enzyme. Nevertheless, the homozygous mutant animals live until the late third instar larval stage. PR-Set7 is expressed in oogenesis and is deposited into the egg and is still detectable in early embryos, resulting in the synthesis of monomethyl H4-K20, present in oocytes and early embryos. Zygotic transcription of PR-Set7 presumably starts during embryogenesis, and PR-Set7 is present throughout development, as expected from a gene functioning in the cell cycle. The presence of monomethylated H4-K20 in the third instar PR-Set7 mutant larvae indicates that this methylation mark of histone H4-K20 is stable over several days and several cell generations, or that an additional methylase exists that controls the methylation of H4-K20 up to late larval stages (Karachentsev, 2005).

These results agree well with observations obtained in HeLa cells. In these cells PR-Set7 expression is controlled transcriptionally and fluctuates with different stages of the cell cycle. Both mRNA and protein levels are lowest in G1 and highest during G2/M. In parallel, methylation levels of H4-K20 also cycle but the cycling is not pronounced, suggesting that the methylation is stable over more than one cell generation. The results suggest that monomethylation is a prerequisite for di- and tri-methylation to occur or be stabilized. At least two scenarios can explain this result: (1) Suv4-20 and a possible dimethylase may modify primarily a monomethylated H4-K20 substrate. (2) The arrest in the cell cycle in the PR-Set7 mutant could result in reduced levels of the di- and trimethylated forms of H4-K20. Only a partial loss-of-function allele of the newly identified Suv4-20 gene exists. Further work will be necessary to fully understand the connection between mono-, di-, and trimethylation (Karachentsev, 2005).

The results indicate that PR-Set7 functions as a suppressor of gene expression and hence as a SUVAR. This observation is consistent with the finding that monomethylated H4-K20 is strongly associated with silent chromatin of polytene chromosomes. It is not clear why the suppression is only observed with w+ transgenes inserted on the fourth chromosome. Since in this genetic interaction experiment the amount of Pr-Set7 function is reduced by only 50%, it is possible that a stronger reduction of the enzyme would also affect the expression of w+ transgenes inserted into the heterochromatin of other chromosomes. The results suggest that the heterochromatin organization on the fourth chromosome differs from that of the other autosomes (Karachentsev, 2005).

It is likely that monomethylated H4-K20 regulates the packaging of silent chromatin domains in constitutive and facultative heterochromatin. Because PR-Set7 mutants show a cell-cycle defect phenotype, it is proposed that the monomethylated H4-K20 is associated with regions containing cell-cycle genes that must remain repressed in early mitosis. Alternatively, the chromosomal regions associated with monomethylated H4-K20 may represent a flag for a cell-cycle checkpoint control or be otherwise involved in separation of chromosomes (Karachentsev, 2005).

The cell cycle-regulated expression of PR-Set7 is consistent with the observed phenotype of PR-Set7 mutants that go through S phase normally but enter into an endoreplication cycle. This phenotype is most easily seen in diploid tissues such as imaginal discs. Imaginal disc cells undergo ~12 divisions from the end of embryogenesis to the late third instar larval stage. Because PR-Set7 discs have about four times fewer cells and 3.5 times more DNA, mitosis probably is affected about two cell divisions before the final development of the discs, in mid-third instar larvae. This is about the same time when the H4-K20 methyl mark begins to disappear (Karachentsev, 2005).

When the function of PR-Set7 was investigated using a short interfering RNA (siRNA) approach in HeLa cells, no change in mitosis was observed. In the current experiments the mitosis phenotype is apparent in mutant larvae only ~10 cell divisions after the PR-Set7 enzyme is no longer detectable. The discrepancy in these results may lie in the fact that the time-frame of tissue culture experiments does not allow the detection of the mitosis phenotype. However, it was also found that cells treated with HCF-1 and PR-Set7 siRNA have a weaker multinucleate phenotype than when HCF-1 siRNA is applied alone. It was proposed that reduction in PR-Set7 and resulting changes in H4-K20 methylation have a function in mitosis. Based on the current results it is suggested that the effect of lowering PR-Set7 levels may arrest mitosis, resulting in fewer multinucleated cells than in cells treated with HCF-1 siRNA alone. The PR-Set7 phenotype may be more easily discernible in the sensitized background, when HCF-1 is reduced (Karachentsev, 2005).

In fission yeast, Schizosaccharomyces pombe, the Suv9 gene has been shown to be responsible for methylation of histone H4-K20. No role for the gene in control of gene expression could be demonstrated; rather, lack of H4-K20 methylation results in a defect in double-strand break repair and does not seem to affect mitosis. It remains to be seen whether mono-, di- or tri-methylation of histone H4-K20 functions in double-strand break repair in Drosophila (Karachentsev, 2005).

Methylation of histone H4 by arginine methyltransferase PRMT1 is essential in vivo for many subsequent histone modifications

Deposition of variant histones provides a mechanism to reset and to potentially specify chromatin states. The distribution of H3 and its variant H3.3 were determined relative to chromatin structure and elongating polymerase. H3.3 is enriched throughout active genes similar to polymerase, yet its distribution is very distinct from that of several euchromatic histone modifications, which are highly biased toward the 5' part of active genes. Upon gene induction, displacement of both H3 and H3.3 are observed, followed by selective deposition of H3.3. These results support a model in which H3.3 deposition compensates for transcription-coupled nucleosomal displacement yet does not predetermine tail modifications (Huang, 2005).

These results demonstrate an important role in vivo for PRMT1, and the H4 Arg3 methylation that it catalyzes, in broad regulation of the patterns of histone acetylation over transcriptionally active loci. Earlier reports have shown that PRMT1 methylates Arg3 on histone H4, and that methylation of H4 tails in vitro facilitates subsequent acetylation of these tails by p300. There have also been reports of synergy between histone acetyltransferases and protein arginine methyltransferases. The interdependence of these modifications has been explored further in a cell-free system with reconstituted chromatin templates carrying a p53-dependent reporter gene; it was shown that PRMT1, the methyltransferase CARM1, and p300 act cooperatively to stimulate p53-dependent transcription. Furthermore, greater stimulation was observed when PRMT1 was added before p300 rather than simultaneously with it, and H4 Arg3 methylation facilitated histone acetylation (Huang, 2005).

The chicken ß-globin locus and surrounding chromatin were used to study in vivo the distribution of H4 Arg3 methylation and its interplay with other histone modifications in the presence and absence of PRMT1. Depletion of PRMT1 results (as expected) in disruption of methylation at H4 Arg3, but also causes major decreases in histone acetylation in vivo at many of the normally enriched sites across the locus. Acetylation levels decrease not only at residues on H4, as might perhaps be expected from earlier studies, but on H3 Lys9 and Lys14 as well (Huang, 2005).

PRMT1 can be targeted to chromatin by a variety of transcription factors. PRMT1 interacts with nuclear hormone receptors, transcription factor YY1, and p53. PRMT1 also interacts with the regulatory factor USF1 at the 5'HS4 ß-globin insulator. Thus, it seems reasonable to suggest that PRMT1 depletion might affect those genes normally activated by recruitment of this enzyme to the promoter, and that the absence of PRMT1 might work indirectly through a failure subsequently to recruit histone acetyltransferases because of the absence of methylation on H4 Arg3. A closer examination of the acetylation patterns over HSA and 5'HS4 reveals that PRMT1 depletion reduces the abundance of H4 Lys5Ac and H4 Lys12Ac but has no effect whatever on H4 Lys8 acetylation at these sites. The fact that H4 deacetylation over HSA and 5'HS4 is limited to Lys5 and Lys12 can be viewed in the light of the earlier observation that diacetylated H4 Lys5 and Lys12 are appropriately spaced for preferential binding by the two tandem bromodomains of TAFII250, a major component of the TFIID complex. In this way, TFIID may be targeted to specific chromatin structures of the promoter to play its role in transcriptional regulation. TAFII250, which itself harbors intrinsic histone acetyltransferase activity, may read these acetyl modifications and contribute to the establishment of an open chromatin domain. It was reported that PRMT1 and Arg3 methylation are linked to nuclear-receptor-mediated transcriptional regulation. The finding that Arg3 methylation affects H4 Lys5/Lys12 acetylation, together with the potential for TAFII250 recruitment, suggests one possible detailed molecular mechanism of PRMT1-mediated transcriptional activation (Huang, 2005).

It is noted that the specific H4 Lys5/Lys12 acetylation pattern over HSA and 5'HS4 does not extend into the globin domain (~23-30 kb), where Lys5, Lys8, and Lys12 are all acetylated in a PRMT1-dependent manner. This difference may result from recruitment by PRMT1 of different factors for 5'HS4/HSA and for the globin genes. Consistent with this distinction, the chromatin of the 5'HS4 insulator element is constitutively acetylated in all cell lines, whereas acetylation is not found over the globin domain in nonerythroid cells such as the DT40 lymphocyte line or those from brain. In addition, despite the observed hyperacetylation of H3 and H4 throughout the majority of the 30-kb globin domain in 10-d erythrocytes, p300/CBP and PCAF are recruited only to the 5'HS4 insulator site. It is also noted that the enzymes that acetylate H4 Lys5/Lys12 are different from that responsible for acetylating H4 Lys8/Lys16. This suggests that a different or additional mechanism may control acetylation in this region (Huang, 2005).

The effect of PRMT1 depletion on H4 Arg3 methylation confirms that in these cells PRMT1 is the principal or sole enzyme responsible for this modification. The additional effects on histone H3 and H4 acetylation, taken together with results of earlier studies in vitro, strongly suggest also that H4 Arg3 methylation is necessary for lysine acetylation of both H3 and H4. However the data do not in themselves exclude other possibilities. For example, PRMT1 might regulate expression of histone acetylases, either directly through modification of histones in the neighborhood of the corresponding genes, or of other upstream genes. Alternatively, PRMT1 could methylate sites on important regulatory proteins other than histones, with downstream affects on histone acetylase activity (Huang, 2005).

In order to distinguish among these possibilities, experiments were carried out under conditions such that methylation by PRMT1 had no other targets available but the nucleosomes isolated from PRMT1-depleted cells. Purified nucleosomes were treated with PRMT1 in vitro before the addition of nuclear extract from the same cells. The nucleosomal histones are acetylated in the extract, but only if PRMT1 is present in the preincubation step. Furthermore, acetylation does not occur even in the presence of PRMT1 if the inhibitor AMI-1 is added before the enzyme in the preincubation step. Therefore, the mere presence of PRMT1 is ineffective in the absence of arginine methylation. Finally, if PRMT1 is allowed to methylate nucleosomes and the inhibitor AMI-1 is added afterward, but before addition of the nuclear extract, histone acetylation occurs when the extract is added. Under these conditions there could be no effects, direct or indirect, on the level of expression of any genes or on the activity of the corresponding proteins, thus eliminating mechanisms of this kind for the role of PRMT1 in histone acetylation and supporting a model in which local H4 Arg3 methylation is a critical requirement for subsequent acetylation of histones H3 and H4. The data also indicate that addition of PRMT1 even to wild-type 6C2 nucleosomes can stimulate histone acetylation, suggesting that many H4 Arg3 sites in these cells are normally unmethylated (Huang, 2005).

The data reveal the important role in vivo of PRMT1 and H4 Arg3 methylation in the establishment of an open chromatin domain, marked by histone acetylation and by the absence of histone methylation at residues associated with inactive chromatin, and suggest a role for H4 Arg3 methylation 'upstream' of lysine acetylation. These results shed light on the intimate connection between Arg3 methylation and other histone modifications in the regulation of chromatin states. This methylation event appears to affect multiple modifications on both histones H3 and H4, either directly or through a cascade that it initiates. The separate question of how the H4 Arg3 methylation mark is recognized by proteins that affect the acetylation reactions remains open, and should be a major subject of future studies (Huang, 2005).

ISWI chromatin remodeling complex NURF, dosage compensation and histone acetylation

The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).

A striking feature of male animals that lack either NURF301 or the catalytic subunit ISWI is the distorted, bloated morphology of the male X chromosome. This implicates NURF in the maintenance of male X chromosome morphology. In flies, X chromosome dosage compensation is achieved by up-regulating transcription from the male X chromosome. One characteristic of the male X chromosome is the specific acetylation of histone H4 at Lys 16 (H4-K16), which is believed to favor a looser chromatin structure that allows increased transcription. These patterns of acetylation are established by the male-specific expression of components of the MSL complex that are tethered on the male X chromosome and subsequently recruit the histone acetyl transferase MOF (Badenhorst, 2002).

Genetic studies demonstrate that H4-K16 acetylation antagonizes ISWI function on the X chromosome. Biochemical characterization of the ISWI-containing ACF and CHRAC complexes has revealed that they can assemble and slide nucleosomes to establish regular ordered arrays. Regular nucleosome arrays are presumed to provide better substrates for chromatin compaction and, thus, it was speculated that ACF and CHRAC might be the complexes that help compact the male X chromosome. However, NURF is the ISWI complex required for normal male X chromosome morphology. Unlike ACF or CHRAC, NURF disrupts regular, ordered arrays of nucleosomes. While it is possible that NURF is required for global aspects of higher order chromosome morphology that are needed to maintain normal male X chromosome structure, other local or transcription-based mechanisms could also account for the nurf301 and iswi phenotypes. The dosage compensation machinery is recruited to the male X chromosome at specific, high affinity sites or entry points and subsequently spreads into flanking chromatin. NURF may regulate chromatin accessibility at one or a number of these initiation sites. In the absence of NURF, entry of the dosage compensation machinery at such sites may be changed. Alternatively, NURF may control transcription of components of the sex-determination and dosage compensation pathway. Irrespective, the observed antagonistic relationship between ISWI function and H4-K16 acetylation suggests that the action of NURF on the X chromosome is correspondingly influenced by H4 lysine acetylation. This influence on NURF could be direct, as suggested by effects of acetylated H4 tail peptide on ISWI ATPase activity in vitro (Badenhorst, 2002).

Hori, T., Shang, W. H., Toyoda, A., Misu, S., Monma, N., Ikeo, K., Molina, O., Vargiu, G., Fujiyama, A., Kimura, H., Earnshaw, W. C. and Fukagawa, T. (2014). Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly. Dev Cell 29: 740-749. PubMed ID: 24960696

Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly

In vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A (see Drosophila Centromere identifer). Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. This study used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. H4K20 monomethylation (H4K20me1) was found to be enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. It is concluded that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly (Hori, 2014).

Modification of histones in gene activation and silencing

Continued Histone 4 - Evolutionary Homologs part 3/3 ! Return to part 1/3

Histone H4: Biological Overview | Regulation | Developmental Biology | References

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