Chromodomains of CDY family members

6.1.1 Chromodomains of CDY family proteins bind to H3K9me3

CDY family proteins contain a chromodomain, a connecting hinge region, and an ECH domain (compare with 1.2.3). Chromodomains are found in several nuclear proteins and they have been connected to chromatin organization and gene regulation [108]. Proteins such as HP1, Polycomb or CHD1 (Chromain helicase DNA-binding 1) contain chromodomains, which bind to methylated histones. Small amino acid exchanges within the chromodomains lead to specificity differences as shown for HP1 and Polycomb [111]. The preferred target of HP1 chromodomain is H3K9me3 and for Polycomb it is H3K27me3, both heterochromatic PTMs. In contrast, CHD1 can recognize H3K4me3, a hallmark of euchromatic regions, with its two neighboring chromodomains.

As shown in chapter 3 and 4, chromodomains of the CDY family bind also to methylated lysine marks. From fourteen analyzed peptides, the major targets of CDY and of the CDYL1 splicing variant CDYL1b chromodomains are H3K9me2 and with a slightly lower binding constant, and therefore better binding, H3K9me3 (chapter 3/Table 1 and chapter 4/Figure 3).

CDYL2 binds to H3K9me2/me3 with a Kd comparable to the other family members but surprisingly it has an even higher affinity to H1K26me3 and testis H3K27me3.

The binding behaviour to methylated histones is also reflected in the nuclear localization of the proteins. Whereas CDY and CDYL1b clearly colocalize with the heterochromatic regions enriched in H3K9me3, the CDYL2 distribution overlaps only partially with this modification (chapter 3/Figure 3 and chapter 4/Figure 4). A large proportion of the protein is also localized in the nucleoplasm with a difuse distribution or with a dotted-like structure without overlapping with H3K9me3. This and the evolutionary observations on a DNA level that CDYL1 and 2 diverged from each other long time ago and that CDY arose from a retro-transposition event of a processed CDYL1 mRNA (1.2.2 and [103, 106, 114]), point to different and maybe very specific tasks of CDY family members within the cell nucleus. In addition, the very distinct expression patterns of the CDY proteins argue also for functional specifications within different tissues or cell types. Whereas CDYL1b mRNA is ubiquitously expressed at high levels (chapter 4/Figure 2), the CDY locus has a testis-specific gene expression [106]. In contrast the CDYL2 gene is transcribed at very low levels in all tested cell types or tissues and is upregulated in the spleen, prostate, testis and in leukocytes [114].

Taken together, the CDY family members CDY and CDYL1b but not CDYL2 are newly defined heterochromatic proteins, which might affect heterochromatin establishment and maintenance.

6.1.2 Chromodomain differences within splicing variants

Interestingly, not all CDY family members recognize histone modifications or methylated lysines. Splicing variants of the CDYL1 gene CDYL1a and also CDYL1c are not able to bind to H3K9me2/3 for different reasons. CDYL1c is lacking the whole chromodomain whereas CDYL1a harbors a non-functional chromodomain and a prolonged N-terminal part (compare with chapter 4/Figure 2A/Supplementary Figure 1). Both proteins are therefore not located at heterochromatin regions (chapter 4/Figure 4A). In contrast, CDYL1b distribution within the nucleus clearly overlaps with H3K9me3 enriched loci. This localization is dependent on Suv39h1/h2, the H3K9me3 methyltransferase. Knock out of the two isoforms lead to displacement of CDYL1b from heterochromatin (chapter 4/Figure 4).

Presented in Figure 1-9 and also in chapter 4/Figure 2D, CDYL1a has only a few amino acid exchanges within the chromdomain compared to CDYL1b. The CDYL1a chromodomain lacks the first aromatic residue of the tri-aromatic cage and a neighboring glutamate, which establishes hydrogen bonds to the peptide backbone. Mutation of these two residues to corresponding amino acids in CDY improves the binding to H3K9me3 and leads to localization to H3K9me3 mark in 30% of the analyzed cells (chapter 3/Table 3 and Figure 5).

Two proline residues are located in front of the chromodomain of CDYL1a. Additional mutations of these two proline residues within the CDYL1a chromodomain to amino acids found in CDY at this position restored the CDYL1a binding to H3K9 methylation. The localization to H3K9me3 enriched loci within the nucleus was rescued in 70% of all cases (chapter 3/Table 3 and Figure 5).

Thus, a functional chromodomain is necessary for targeting CDYL1 to the heterochromatic mark H3K9me3.

A further conclusion of these experiments is that splicing events (i.e. CDYL1a, CDYL1b, CDYL1c) of the CDYL1 mRNA can actively influence the ability of the protein to bind to H3K9me3. Interestingly, under normal conditions the mRNA of the CDYL1a splice variants is upregulated in testis and the CDYL1c mRNA occurs only at very low levels in all examined tissues and cell lines (compare with chapter 4B/Figure 2). Theoretically these splicing events could occur in developmental stages, specific cell types or in response to DNA

damage or oxidative stress to regulate if CDYL1 proteins are located at H3K9me3 dense heterochromatin or not.

6.1.3 Chromodomain binding to non-histone targets

As shown in chapter 3/Table 1 and Table 2 and chapter 4/Figure 3 CDY family proteins exept CDYL1a and CDYL1c recognize methylated lysines of non-histone proteins such as the automethylation site of the histone methyltransferase G9a. Earlier studies demonstrated a direct interaction between CDYL1b (referred as mRNA isoform 2 in the original publications) and G9a using recombinant proteins and within the CoREST complex [62, 102]. It is possible that this contact is stabilized or even established by the CDYL1b chromodomain interaction with methylated G9a on lysine 185. An argument for this hypothesis is that pulldown assays carried out with the CDYL1a splicing variant failed to demonstrate a CDYL1a/G9a interaction in vitro [132].

Because CDYL1b chromodomain in contrast to the CDYL1a chromodomain is able to bind G9aK185me3 it is likely that the G9a/CDYL1 contact is mediated by the trimethylation mark (compare also with Figure 6-2).

6.1.4 Regulation of CDY family chromodomain binding to H3K9me2/me3

It is possible that CDY family proteins are not permanently associated with heterochromatin regions. One hint is supplied by the chromatin displacement of CDYL1b during mitosis (shown in Figure 5-3). The delocalization of the protein is correlated with the appearance of the phosphorylation of histone 3 serine 10 set by the kinase Aurora B [122]. The removal of the phorphorylation mark occurs at a time coincident with CDYL1b relocalization to H3K9me3 enriched regions. Thus it is possible that the CDYL1b localization to heterochromatin is regulated by the H3K9me3S10ph mark. This hypothesis is supported by in vitro binding studies, which reveal that CDYL1b chromodomain binding to H3K9me3 is abolished if the neighboring serine is phosphorylated (chapter 4/Figure 3C). Therefore it is likely that CDY family chromodomain binding to H3K9me3 is regulated by additional histone PTMs such as phosphorylation. This regulatory possibility was already shown for the heterochromatin protein 1 (HP1) [27] and indicates that this might be a mechanism for heterochromatin binding proteins in general or chromodomain containing proteins in particular.

A second regulation mechanism for chromodomains of CDY family proteins was recently suggested [133]. CDYL1 can be methylated by the histone methyltransferase G9a in vitro.

The methylated lysine residue of CDYL1 is located C-terminal to the chromodomain but in very close proximity. Methylation of this CDYL1 lysine residue leads to a about 2-fold reduced CDYL1/H3K9me3 interaction. Thus G9a mediated methylation within CDYL1 might negatively regulate the H3K9me3 binding. But interestingly, CDYL1a instead of CDYL1b was used for the study. The CDYL1a affinity to H3K9me3 is about 200-fold less than the affinity of CDYL1b and could have had a major influence on the represented results.

A third regulation mechanism of the CDY protein chromodomain binding to H3K9me3 can be splicing events (compare with 6.1.2). The only splicing events known to date regard CDYL1. Splicing of the first three exons (exons 1, 2, 3) to the six last exons (exons 5, 6, 7, 8, 9, 10) (chapter 4/Supplementary Figure 1) lead to a non-functional chromodomain in splicing variant CDYL1a. Alternatively exon 4 is spliced to the six 3’ exons resulting in CDYL1b having a functional chromodomain. Third, only the last six exons are spliced together producing the mRNA for CDYL1c, which lacks the chromodomain. It is likely that these splicing events are regulated since CDYL1a transcript is only expressed in testis, but further investigations are needed to provide detailed mechanism for CDYL1 splicing variant expression.