Dynamic regulation of chromatin structure

As already mentioned, epigenetics describes the alterations of gene expression due to structural changes of chromatin. There are several mechanisms which can determine the chromatin structure and the access of transcription factors to their regulatory sequence, which further on will be called epigenetic mechanisms. Their balance is interdependent and essential for normal development and cellular function. There are five categories of epigenetic mechanisms known to affect chromatin structure: DNA methylation, posttranslational modifications (PTM) of histones, several classes of non-coding RNAs, chromatin remodeling complexes which are ATP dependent protein complexes that perform nucleosomal sliding, and the exchange of histone variants which influence the regional chromatin condensation (Fig. 2.8) (Dulac 2010).

There are at least two classes of RNAs that are part of controlling epigenetic phenomena. On the one hand, there are long non-coding RNAs that can induce long-term silencing and can be inherited through cell division (Bernstein and Allis 2005). This is done by non-coding RNAs.

Figure 2.7. Chromatin structure. DNA (red) is wrapped around histone octamers (blue). A more open structure which allows access of DNA binding factors is called euchromatin. A more compact and therefore silenced structure is called heterochromatin. See also text.

Figure 2.8.

Epigenetic mechanism affecting chromatin structure.

Arrows indicate cross-talk. See text.

adapted from Dulac 2010

imprinting of the second X chromosome in female mammals together with other components of the chromatin and DNA methylation (Bernstein and Allis 2005). Small RNAs and long non-coding RNAs can recruit chromatin-modifying complexes and target it to specific chromosome regions (Moazed 2009; Tsai et al. 2010). On the other hand, there is post-transcriptional RNA interference, which might not be considered to be epigenetic by nature due to its sequence specificity. Small interfering RNAs (siRNAs) can degrade mRNA in a sequence specific manner (Hutvagner and Zamore 2002). MicroRNAs (miRNAs) bind to the 3’untranslated region of target mRNAs and down-regulate their expression (Bushati and Cohen 2007). miRNAs have been shown to play major roles in many processes. For example, specific miRNAs can promote neuronal differentiation (Stappert et al. 2013). Also, miRNA profiling has evolved as an exciting tool to study pathological and toxicological processes (Smirnova et al. 2012) as discussed below.

DNA methylation occurs at the C5 position of cytosines. DNA methyltransferases methylate cytosines in CpG islands, whereby Dnmt1 maintains the methylation state and Dnmt3a and Dnmt3b methylates de novo. Dnmt3b is only expressed in stem cells. Methylated CpG islands in the promoter of a gene are associated with repressed gene expression. The different epigenetic mechanisms are strongly interdependent. It was shown that MeCP2 binds to methylated DNA and recruits HDAC1 thereby transferring the chromatin to a silenced state (Jones et al. 1998; Nan et al. 1998; Razin 1998).

Histones can be modified by several mechanisms. The best described histone PTMs are acetylation of lysines, methylation of lysines and arginines and the phosphorylation of serine and threonine residues. But during the last decade, several additional modifications have been identified such as ADP ribosylation, ubiquitination, sumoylation, or the transfer of β-N-acetylglucosamine (Bannister and Kouzarides 2011). The different PTMs are catalyzed by the

“writer”-enzymes and removed by “erasers” (Bannister and Kouzarides 2011; Weng et al.

2012). Acetylation of lysine residues is set by histone acetyl transferases (HAT) which utilize acetyl-CoA as cofactor (Bannister and Kouzarides 2011). Several classes of histone deacetylases can remove the acetylation from the histones (see Chapter 4 and 5). Methylation of histones can occur at lysine and arginine residues, which can be mono-, di or tri-methylated or mono- or symmetrically or asymmetrically methylated, respectively. Histone lysine methyltransferases (HKMT) are very specific enzymes and all contain a so-called Set domain (Bannister and Kouzarides 2011). Removal of the methyl-groups from histones was a mystery for many years until the lysine-specific demethylase 1 (LSD1) was found which recognizes

nucleosomes if complexed with the Co-REST repressor complex (Klose and Zhang 2007).

Nowadays many histone lysine demethylases are known which, except for LSD1, all possess a catalytic jumonji domain (Mosammaparast and Shi 2010).

Not only the charges transferred to the histone tails by acetylation or phosphorylation can regulate chromatin structure, but also chromatin factors that specifically bind to the modifications. Those so-called readers or binders contain domains which recognize the modifications. Methyl-lysine-recognizing domains are PHD fingers and the Tudor “royal”

family which comprises chromodomains, Tudor- and MBT- domains, whereby numerous of those domains can bind to the same modified histone (Bannister and Kouzarides 2011).

Bromodomains can bind to acetylated lysines. Some proteins contain several binding domains allowing higher affinity. Cross-talk between the different modifications adds an extra level of complexity. This cross-talk can happen by competitive antagonism between modifications, modifications that depend on each other, disruption of protein-binding by an adjacent modification, decreased enzyme activity due to its modified substrate and cooperation between modifications to recruit specific factors (Bannister and Kouzarides 2011).

PTM of histones are known to be one of the key mechanisms for regulating proper gene expression (Waldmann and Schneider 2013). It has been debated if the structure and interactions by net charge of the aminotails of histones define the structure and function of chromatin (Zheng and Hayes 2003) or if a so-called histone code exists. The latter describes the hypothesis that distinct histone amino-terminal modifications are responsible for interaction of chromatin-associated proteins and therefore dictate transcriptionally active and silent chromatin states (Jenuwein and Allis 2001). Other groups have shown that, depending on the site at the H4 tails, acetylation can have a specific and a non-specific effect on gene expression (Dion et al. 2005). Although there has been a tendency to believe in the histone code as the governor of gene expression (Margueron et al. 2005) and although the expression is useful to define that a specific set of histone modifications is needed for a given task, it is debated if there really is a strictly predictable “code” (Kouzarides 2007). Taken together, it seems likely that both, net charge effects and specific binding to modifications, are the basis for regulating chromatin structure and gene expression (Allis et al. 2007).

There are more types of chromatin than the two well-known chromatin structures euchromatin and heterochromatin. However, until now they are not well studied. What is known is that there are histone marks that have been clearly associated with one chromatin state (Fig. 2.9).

Such histone modifications, known to be associated with the open, transcriptionally active

chromatin (euchromatin), are H3K4me3, H3K9ac and H3K36me3. Other modifications like H3K27me3 and methylated H3K9me3 correlate with transcriptionally silenced chromatin (heterochromatin). Nonetheless, the exclusiveness of histone marks to one kind of chromatin is in the most cases not as clear as H3K4 trimethylation (Bannister and Kouzarides 2011) which adds to the difficulties in predicting chromatin states from epigenetic modifications.

Chromatin structure and transcriptional regulation are especially important for differentiation processes. During differentiation, trithorax protein complexes set the marks for active chromatin (especially MLL sets HeK4me3) and polycomb protein complexes set the marks for facultative heterochromatin (especially EZH2 sets H3K27me3). In pluripotent cells, marks for open and for closed chromatin coexist in some promoters, in particular H3K4me3 and H3K27me3. This is referred to as a bivalent state in which the genes are poised for activation.

During neural differentiation of stem cells, for example, neural genes like Pax6 are de-repressed from their bivalent state to an active state by demethylation of H3K27me3 (Hirabayashi and Gotoh 2010) (Fig. 2.10).

Figure 2.9. PTM

adapted from (Hirabayashi and Gotoh 2010)

Due to this multitude of enzymes and cofactors that regulate epigenetic modifications, it seems likely that chemicals may be able to disturb this fine-tuned regulatory network in many steps (Smirnova et al. 2012). In particular, epigenetic gene regulation can be disturbed by toxicants on several levels. First, chemicals can influence the activity of the epigenetic modifiers. Second, toxicants can alter gene expression levels of the epigenetic modifiers or, third, can be in general geno-toxic and induce mutations in the genes of the epigenetic modifiers which leads to miss-regulation or - function (Waldmann and Schneider 2013).

Changes in gene expression levels of epigenetic modifiers can be tested by standard transcriptomics methods (Weng et al. 2012). However, in order to obtain information on altered activities of chromatin-modifying enzymes, methods to quantify changes of histone PTMs or DNA methylation levels are necessary. Toxicological test systems evaluating such endpoints are hard to establish, although it is well-known that certain classes of pharmaceuticals (e.g. HDAC inhibitors) do modify epigenetic marks (Collotta et al. 2013;

Smirnova et al. 2012; Stoccoro et al. 2012). Also, it is known that exposure to several environmental chemicals and other stressors can result in altered epigenetic marks. For this reason, it appears highly important to establish new test systems that evaluate epigenetic changes, and to incorporate epigenetic endpoints into already existing test systems.