Histone posttranslational modifications and cross talk

Posttranslational modifications (PTM) of histones and other proteins are recognized as important regulators of protein function and stability, protein-protein interactions or sub-cellular localizations (Yang 2005). Histone proteins are subjected to a multitude of different

PTMs, also referred to as histone ‘hall marks’, including lysine acetylation, methylation, ubiquitinylation and SUMOylation, arginine methylation, serine and threonine phosphorylation, glutamate ADP-ribosylation and proline isomerization (Gelato and Fischle 2008). In addition, methylated lysines can exist in a mono-, di-, or tri-methyl state, whereas targeted arginine residues can be modified into symmetric or asymmetric di-methylated or a mono-methylated state.

Conventionally, chromatin can be categorized into two main classes, euchromatin and heterochromatin. The first one is characterized by a low condensation state and a more nuclease-sensitive configuration, making it poised for gene expression, although not necessarily transcriptionally active. In contrast, heterochromatic structures comprise highly condensed regions that are in general gene-poor and form mainly on repetitive sequences, such as satellite centromeric and pericentromeric repeats as well as telomers (Baxter et al.

2002; Grewal and Elgin 2002). These structures replicate late in S-phase and are accompanied by H3K9me3 and H4K20me3, whereas H3K9ac and H3K16ac and methylated H3K4 are often found within euchromatic sites.

The inactive X-chromosome contains several hallmark histone modifications such as H3K9me2, H3K27me3, H4K20me, H3K4 demethylation, general deacetylation, as well as the histone variant macroH2A and high level of DNA methylation (Brinkman et al. 2006).

Many covalent modifications alter the electrostatic charge of histones, thereby changing the structural properties of the histones or the chromatin environment. Some histone tail modifications serve as target sites for protein recognition modules. (A.1.2.1, A.1.2.2).

The temporal overlap of various PTMs on histone N-terminal tails discloses the possibility of combinatorial effects. Accordingly, the proposed ‘histone code’ postulates that a certain set of histone modifications dictates the recruitment of particular transacting factors to accomplish specific functions (Strahl and Allis 2000; Jenuwein and Allis 2001; Turner 2007).

Several experimental data demonstrate indeed combinatorial effects of histone modifications, however the evidence for a ‘universal code’ still lacks. It rather seems that the interdependency of multiple histone PTMs refer to a ‘cross-talk’ promoting or antagonizing one another (Fischle 2008). Importantly, at present it is not clear which PTMs effectively lead to the establishment of a chromatin element, or whether an element with certain architectural features or protein composition enhance the addition or removal of certain marks (Khorasanizadeh 2004; Gelato and Fischle 2008).

In general, the inter-relationship between posttranslational histone modifications, special histone variants, chromatin remodeling (A.1.4), DNA methylation (A.3) and the RNAi machinery seem to be important for the establishment or maintenance of certain chromatin states (Narlikar et al. 2002; Robertson 2002; Hake et al. 2004; Vos et al. 2006).

A.1.2.1 Histone acetylation

Histone acetylation is set by histone acetyltransferases (HATs), which catalyze the transfer of acetyl groups from acetyl-CoA to the ε-amino terminal groups of specific lysine residues on all four core histones. This reaction is reversed by specific factors, the histone deacetylases (HDACs), which remove acetyl groups from lysines. Alterations of the histone

acetylation state appear to play an important role in chromatin assembly and gene regulation. Increased histone acetylation often correlates with transcriptional activity, whereas decreased acetylation correlates with a transcriptionally repressed state (Fischle et al. 2003a) (Gelato and Fischle 2008). The bromodomain, found in chromatin-associated proteins and HATs, functions as the sole protein module known to bind acetyl-lysine motifs (Mujtaba et al. 2007).

The first HAT identified was isolated from macronuclei from Tetrahymena (Brownell et al.

1996), and showed strong homology to Gcn5, a transcriptional co-activator in S. cerevisiae.

Gcn5 as the catalytic subunit of the ‘SAGA’ transcriptional co-activator complex (Grant et al.

1997) clearly linked histone acetylation to gene regulation. In the past years, many HATs have been identified, often in multi-protein complexes and with different histone tail specificities (Glozak et al. 2005).

Histone acetylation is believed to primarily neutralize the positive charge of histones, thus decreasing their affinity for the DNA and altering nucleosome-nucleosome interactions (Vaquero et al. 2003). In fact, eviction of linker histone and H4K16 acetylation resulted in decompaction of the 30nm fiber in vitro (Robinson et al. 2008). The resulting permissive structure facilitates binding of proteins such as those of the transcriptional machinery (Khorasanizadeh 2004). In addition, acetylated tails can directly recruit components of chromatin associated factors via the bromodomain (Mujtaba et al. 2007), including the TBP associated factor TAF_II250 and the human SWI/SNF chromatin remodeling enzyme BrgI (A.1.4).

In general, histone acetylation plays an important role in nuclear processes like chromatin assembly, DNA repair and apoptosis, VDJ recombination and dosage compensation in Drosophila ((Iizuka and Smith 2003) and references therein).

Histone deacetylases, the enzymes that remove the acetyl groups, are generally suggested to play an important role in gene inactivation. Indeed, the first identified histone deacetylase (HDAC1), was shown to be a homolog of the yeast Rpd3p transcriptional regulator (Taunton et al. 1996). Several classes of HDACs were defined, according to their expression pattern, homology and their sensitivity against specific inhibitors. Very often, HDACs are found within large multi-subunit complexes, components of which serve to target enzymes to genes, leading to transcriptional repression. In agreement, many transcriptional repressors were found to be associated with histone deacetylases, and their activity was necessary for gene silencing (Vaquero et al. 2003). In particular, class I HDACs form complexes with the transcriptional co-repressor Sin3 (David et al. 2008), the ATP-dependent remodeling complex NuRD (Zhang et al. 1999), DNA methyltransferases Dnmt1, Dnmt3a/b (Fuks et al.

2000; Rountree et al. 2000; Fuks et al. 2001; Geiman et al. 2004b) and the histone methyltransferase Suvar39H1 (Czermin et al. 2001). Hence, HDACs are involved in multiple functions such as transcriptional and epigenetic silencing, development, cell differentiation, X-chromosome inactivation in mammalian females, and position effect variegation in Drosophila.

A.1.2.2 Histone methylation

Histone methylation occurs on both lysine and arginine residues on several histone tails, although it is best described for histone H3 and H4 (Lachner and Jenuwein 2002; Fischle 2008). Histone methyltransferases (HMT) catalyze the transfer of up to three methyl-groups form S-adenosyl-methtionine (SAM) to the ε-amino group of a single lysine residue. The protein arginine methyltransferase (PRMT) generates both mono- or di-methylated arginine residues, either symmetrically or asymmetrically by transferring methyl-groups to the guanidine-group.

Of the many known lysines residues methylated, six have been well characterized to date:

five on H3 (K4, K9, K27, K36, K79) and one on H4 (K20) (Lachner and Jenuwein 2002).

Although histone methylation has been largely associated with transcriptional repression and epigenetic regulation, it is also involved in transcriptional activation, dependent on the interplay with other histone modifications (Turner 2002). Methylation of K4, K36 and K79 of histone H3 are examples of transcriptional activation (Beisel et al. 2002; Fischle et al. 2003a;

Santos-Rosa et al. 2003), whereas H3K9me3 and H3K27me3 are characteristic marks for silenced regions (Bannister et al. 2001; Lachner and Jenuwein 2002). In addition, H3K79me and H4K20me have been implicated in the process of DNA repair.

Probably one of the best-studied modifications, namely H3K9me2/3, functions as a ‘docking site’ for HP1 (heterochromatin protein 1), which is characteristic of inactive heterochromatic regions (Bannister et al. 2001). Similar, H3K27me3 is bound by the Polycomb group protein (PRC1 complex) (Cao et al. 2005), involved in maintaining the silenced state of homeotic genes during development and of the X-chromosome. Both proteins bind to the methylated lysines through a specific recognition module, the chromodomains (Fischle et al. 2003b). The PHD domain (Plant Homeodomain) is another prominent protein fold, found to specifically recognize H3K4me2/3 (Bienz 2005), thus reading part of the histone code (Jenuwein and Allis 2001; Wysocka et al. 2006). PHD fingers tend to be found in nuclear proteins that have a role in chromatin regulation and are involved in both gene activation and repression (Mellor 2006). In addition other protein domains such the Tudor domain (H3K79me, H4K20me), WD40-Repeat domain (H3K4me) are capable of specific interactions with methylated lysine residues (Martin and Zhang 2005).

In relation, Suvar3-9 and E(z) of PRC2 were the first SET domain (Suvar3-9, Enhancer-of-zeste, Trithorax domain) containing histone methyltransferases (HMT) described (Rea et al.

2000), specifically methylating H3K9 and H3K27 respectively. Subsequently, by homology search to the SET domain and functional assays other HMTs were identified (Vaquero et al.

2003).

Until recently, it was unclear whether histone lysine demethylation would take place in the cell, primarily through the observation, that methyl groups seemed to be very stable on heterochromatic regions. The discovery of the LSD1 protein, the first demethylase specific for methylated H3K4, dramatically changed the view on the dynamics of histone methylation (Shi et al. 2004). LSD1 is present in different repressor complexes and its substrate specificity was modulated from H3K4me to H3K9me when binding to the androgen receptor (Shi et al. 2005; Metzger et al. 2006), thus exerting a function in gene activation. Recently,

five new demethylases were identified that possess the JmjC-domain which is different from the LSD1 protein. Interestingly, these demethylases were found to demethylate specific methyl states (Culhane and Cole 2007; Swigut and Wysocka 2007). Of Importance are UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome) and JMJD3 (jumonji domain containing 3) that were found to specifically remove di- and tri-methyl marks on H3K27 in vivo (Hong et al. 2007; Lee et al. 2007b). Furthermore, UTX occupies promoters of Hox gene clusters and associates with MLL2/3 during retinoic acid signaling, resulting in H3K27 demethylation and H3K4 methylation respectively (Lee et al. 2007b).

A.1.2.3 Other histone modifications

All histones including H1 have been shown to be substrates for phosphorylation in vivo. In particular phosphorylation of H1 and H3 (S10, S28) have been associated with chromosome condensation and segregation (Wei et al. 1999; Vaquero et al. 2003). H3S10 phosphorylation appears early in G2 of the cell cycle, first in pericentromeric heterochromatin and then spreading, by metaphase to the rest of the chromosome (Hendzel et al. 1997). A

‘methyl-phospho binary switch’ has been proposed in that H3S10ph leads to ejection of HP1 from H3K9me3 (Fischle et al. 2005). Importantly, H3K10ph is regulated by Aurora-B a member of the Aurora/AIK kinase family that participates in mitotic regulation.

ADP-ribosylation implies the transfer of ADP-ribose molecules to either glutamic acids in a poly-glutamate stretch or single arginine residues with NAD⁺ as the source for ADP-ribose.

Thus, ADP-ribosylation is linked to the metabolic state of the cell. Although H1 and H2B are the most highly modified, all histones seem to be ADP-ribosylated (Golderer and Gröbner 1991). Interestingly, preferentially hyper-acetylated histones, especially H4, are found to be ADP-ribosylated. Due to its fast turn-over in the cells, it has been proposed to play a role in adaptation of the cell to environmental changes (Pieper et al. 1999).