The MeCP2 protein

MeCP2-like proteins were detected in different species such as human, mouse, rat, chicken, pig, cow, rabbit and frog, but not in Drosophila (Meehan et al. 1992), which is in

2

1 3 4

232 486

MBD

619 TRD

930 STOP

3’UTR

ß-MeCP2 αααα-MeCP2

polyA 10.1-kb

polyA 5-kb polyA

1.8-kb polyA 7.2-kb NLS

763 813

ATG ATG WW

973

5’UTR

ATRX-binding RG

2

1 3 4

232 486

MBD

619 TRD

930 STOP

3’UTR

ß-MeCP2 αααα-MeCP2

polyA 10.1-kb

polyA 5-kb polyA

1.8-kb polyA 7.2-kb NLS

763 813

ATG ATG WW

973

5’UTR

2

1 3 4

232 486

MBD

619 TRD

930 STOP

3’UTR

ß-MeCP2 αααα-MeCP2

polyA 10.1-kb

polyA 5-kb polyA

1.8-kb polyA 7.2-kb NLS

763 813

ATG ATG WW

973

5’UTR

ATRX-binding RG

14 | Chapter 1

accordance with the importance of DNA methylation in all vertebrates, but not in invertebrates.

In vitro and in vivo studies showed that MeCP2 protein is a nuclear chromatin-associated protein (75-100 kDa depending on the human isoform and around 80 kDa in rodents), that binds selectively to symmetrically methylated CpG dinucleotides (at least one methylated CpG pair), through its MBD; the NLS consists of amino acids 255-271 (RKAEADPQAIPKKRGRK), and the MBD of amino acids 78-162 (Meehan et al. 1992;

Nan et al. 1993; Nan et al. 1996). MeCP2 possesses in the C-terminal region a TRD (amino acids 207-310); it was shown, in vitro, that MeCP2 represses the transcription of methylated reporter genes, but not of unmethylated ones. MeCP2 has a genome-wide binding distribution and its repression capacity is dependent on the distance of the methyl-CpG from the promoter (the higher the distance the weaker the repression capacity) and on the density of the methyl-CpGs (the higher the methylation the stronger the repression capacity) (Nan et al. 1997). Recently a group II WW binding domain (from amino acid 325 to the C-terminus of the protein) was also identified; it allows MeCP2 to specifically bind to group II WW domains of splicing factors (Buschdorf and Stratling 2004). Another potential protein functional domain could be an arginine-glycine repeat stretch (RG, aminoacids 185-190), located after the MBD, which was proposed to mediate the binding of MeCP2 to RNA (Jeffery and Nakielny 2004).

The function of a protein can be sometimes be elucidated by the identification of its interacting partners. One of the first partners of MeCP2 to be identified was the Xenopus homologue of the co-repressor Sin3A, which is associated with MeCP2 in a complex with histone deacetylase activity (HDAC1 and HDAC2) (Jones et al. 1998). MeCP2 interacts with Sin3A through its TRD, and the histone deacetylase activity of the formed complex represses the transcription of target genes (figure 1.4).

Furthermore, MeCP2 also directly binds to two other co-repressors: c-Ski and N-CoR. c-Ski binds to the TRD of MeCP2 and seems to be necessary for methyl CpG-mediated transcriptional repression (Kokura et al. 2001). The entire MBD and TRD region of MeCP2, however, is necessary for the binding of N-CoR (Kokura et al. 2001). Until now, three different co-repressor molecules have been described to be involved in MeCP2-mediated transcriptional repression. It is unlikely, however, that these three

co-Introduction | 15

repressors assemble together, but rather as individual co-repressor complexes, at the same time or sequentially.

Figure 1.4. Schematic representation of the major function of MeCP2 protein. MeCP2 binds to methylated DNA through its MBD and recruits co-repressor complexes with histone deacetylase activity by binding, through its TRD to the co-repressor Sin3A.

Several other roles have been proposed for the MeCP2 protein, which are involved in histone deacetylase-independent silencing. The chromatin structure is associated with regulation of gene expression. We already discussed the role of MeCP2 in chromatin remodelling, through binding to methylated DNA and deacetylation of histones. Besides histone deacetylation, histone methylation is another epigenetic modification involved in the organization of chromatin structure and regulation of gene expression. MeCP2 was reported to be associated with a histone methyltransferase activity, specifically involved in the methylation of lysine 9 of histone H3, strengthening a repressive chromatin state by bridging DNA methylation to histone methylation (Fuks et al. 2003). The association of MeCP2 with histone H3 methyltransferase is primarily mediated by its MBD.

It is still an open question whether MeCP2 always recruits these two activities (deacetylation and methylation of histones) simultaneously, or whether there is a functional specification depending on the event that MeCP2 is regulating, such as imprinting, X chromosome inactivation (permanent) or embryonic development and activity-dependent gene transcription (transient).

There are essentially two classes of DNA methyltransferases, the de novo DNA methyltransferases (DNMT3A and DNMT3B) which define new methylation patterns, and

MeCP2 Sin3A

HDAC2 HDAC1 MeCP2

Sin3A

HDAC2 HDAC1

Histones

Acetyl group

MeCP2 domais Methyl-CpG

16 | Chapter 1

the maintenance DNA methyltransferases (such as DNMT1). DNMT1 uses as substrate hemi-methylated DNA and copies the pattern already established during DNA replication;

it thus is responsible for the maintenance of the primitive/basal DNA methylation status. It was shown that MeCP2 associates with DNMT1, through its TRD domain, and both play a role in the maintenance of the DNA methylation pattern during DNA replication (Kimura and Shiota 2003). MeCP2 binds to the template hemi-methylated double stranded DNA and recruits DNMT1, which will add a methyl group to cytosines in the newly synthesized strands.

Experimental data suggests that MeCP2 might be involved in local or higher order chromatin reorganization. In this respect, it has been shown that MeCP2 mediates the formation of a silent chromatin loop in the distal less homeobox 5 (Dlx5)-distal less homeobox 6 (Dlx6) locus, associated with methylation of lysine 9 of histone H3 (Horike et al. 2005). Additionally, the spatial organization of the heterochromatin in the nucleus has also a role in transcriptional silencing and is involved in the maintenance of cellular differentiation (Kosak and Groudine 2004). Heterochromatin aggregates in clusters that lead to the formation of large chromocenters and the levels of MeCP2 have been correlated with this process (Brero et al. 2005). The MBD of MeCP2 is necessary for this interaction, and is independent of the pathway that involves methylation of lysine 9 of histone H3.

Proteins that interact with RNA or are components of RNA-protein complexes (RNP) commonly have a RG repeat region. The MeCP2 protein has a small stretch of RG repeats following the MBD (figure 1.3). As mentioned above, the MeCP2 was shown to be able to bind mRNA and double-stranded siRNA and form a RNP in vitro (Jeffery and Nakielny 2004). The interaction occurs through the RG domain and independently of the MBD of MeCP2. Additionally, the binding of MeCP2 to methylated DNA or siRNA occurs in a mutually exclusive manner.

What would be the advantage of MeCP2 binding to specific RNAs? This could provide specificity of transcription regulation by driving the binding of MePC2 to specific methylated chromatin regions. This feature of MeCP2 was not yet demonstrated in vivo, but if this is the case, this fact could eventually link RNA to chromatin regulation, through DNA-methylation. But, it is also possible that the MeCP2-RNP complex has a function different from its well characterized role in the regulation of gene expression. Could

Introduction | 17

MeCP2 also be involved in the post-transcriptional regulation of gene expression, controlling mRNA stability, localization and translation, which regulate many important events in development and plasticity? Identification of which specific RNA molecules are targets of the MeCP2 protein will help the scientific community to unveil the link between RNA and MeCP2.

In accordance with the previous finding, another function has been attributed to MeCP2, as a splicing regulator. MeCP2 was described to interact with Y-box binding protein 1 (YB-1), which is a nuclei acid binding protein involved in many cellular functions, including alternative splicing (Stickeler et al. 2001). MeCP2-YB1 binding requires RNA for its formation and stabilization. The proposed idea is that MeCP2 regulates alternative splicing through its WW C-terminal domain (amino acids 195-329), promoting exon inclusion (Young et al. 2005). It was hypothesized that the posttranscriptional modulation of alternative splicing could represent an epigenetic control of gene expression (Young et al. 2005). Alternative splicing allows for the existence of several transcripts from the very same gene. In this way, a dysfunction in alternative splicing may have more drastic consequences on the expression of one gene than a dysfunction in its transcription.

Very recently, another partner of MeCP2 has been identified. The alpha-thalassemia, mental retardation syndrome, X-linked (ATRX) is described to be a SWI2/SNF2 DNA helicase/ATPase, which has been shown to alter the structure of chromatin (Berube et al. 2000). Mutations in the ATRX gene are responsible for the neurological syndrome ATR-X^†. MeCP2 interacts with ATRX (both in vitro and in vivo), through a domain (ATRX-domain) that partially overlaps the MBD of MeCP2; it was described that certain human MeCP2 mutations (such as R133C, which causes a mild RTT phenotype, and A140V, present in males with X-Linked Mental Retardation) disrupt this interaction (Nan et al. 2007).

MeCP2 was initially proposed to be a “global silencer” acting at the chromatin structure level. Accumulating evidence seems to suggest that the way MeCP2 plays its role(s) might depend on the cellular and molecular context. The identification of the molecular targets of MeCP2 will help in elucidating the contribution of MeCP2 in those pathways.

† ATR-X (OMIM, #301040). Present with severe psychomotor retardation, characteristic facial features, α-thalassemia and genital abnormalities.

18 | Chapter 1