Comparative study of methyl-CpG-binding domain proteins

Human MBD proteins

We used a bioinformatics approach with the MBD of human MECP2 as query sequence to search for new members of the MBD protein family. Initial standard BLAST searches of the NCBI, Celera and SwissProt databases resulted only in five MBD proteins (MECP2, MBD1, MBD2, MBD3 and MBD4) which had previously been described and studied intensively. However the search of protein domain family databases (NCBI, Pfam, Smart and Prosite) revealed similarities to the MBD of MECP2 for four additional proteins, i.e. BAZ2A/TIP5, BAZ2B, CLLD8 and SETDB1 and for two cDNAs, KIAA1461 and KIAA1887. These databases use Hidden Markov Models (HMM) to detect motifs in amino acid sequences. An MBD has been described in CLLD8, SETDB1 and BAZ2A/TIP5 so far [22–24].

Nine of the eleven MBD-containing protein sequences could also be detected by screening the Sequence Similarity DataBase (SSDB) [25] at GenomeNet. The cDNAs for KIAA1461 and KIAA1887 were not found in the KEGG database that underlies the SSDB. These results are summarized in Tab 1. MBD amino acid sequences of the five previously published and the six newly described human polypeptides were aligned (Fig. 1). A sequence logo derived from the alignment of all eleven sequences is shown in Fig. 2. These analyses implicate a small number of highly conserved and apparently essential amino acids within the domain. At three positions identical amino acids are present and five positions with conservative substitutions can be found.

A phylogenetic tree of the MBD amino acid sequences of all eleven polypeptides is shown in Fig. 3. Four major MBD subsets are indicated there. The MBDs of the originally described proteins (MBD1, MBD2, MBD3, MBD4 and MECP2) are found as one group besides a second (BAZ2A/TIP5, BAZ2B) and third subset (CLLL8 and SETDB1) which are joined by a very short branch. KIAA1461 and KIAA1887 appear in a fourth branch. MBDs of the original five proteins are more similar to each other than to the novel ones, which explains why BLAST analyses with the MECP2 MBD query failed to identify the second, third or fourth class.

Domain analysis

An analysis of the amino acid sequences revealed that the MBD was the only domain shared by all eleven sequences. The MBD of MECP2, MBD1, MBD2, MBD4 and BAZ2A/TIP5 mediates binding to DNA, in case of MECP2, MBD1 and MBD2 preferentially to methylated CpG [13, 24, 26–28]. MBD4 has a special role acting as a DNA repair enzyme that reverses spontaneous CpG to TpG base exchanges, thereby maintaining methylated CpG motifs. It binds preferably to m⁵CpG/GpT mismatches [29, 30].

In case of human MBD3 and SETDB1 the MBD has been shown to mediate protein-protein interactions [23, 31]. Xenopus MBD3 is exceptional in its binding to methylated CpG which can be explained by the difference of an amino acid residue within the MBD (Lys30) important for DNA binding [31]. It remains to be determined whether the MBDs of BAZ2B, CLLD8, KIAA1461 and KIAA1887 mediate DNA binding or protein-protein interactions. Additional domains found in seven of the eleven polypeptides indicate that they are associated with chromatin and function in epigenetic mechanisms of gene regulation. Some of the proteins are already known to be involved in transcriptional repression, and the domains of the remainder strongly suggest a comparable function.

MECP2 recruits the Sin3A co-repressor complex and MBD2 the NuRD co-repressor complex, which itself contains MBD3. Both complexes contain HDACs, and MBD1 is also associated with HDAC activity although the identity of the deacetylase remains unknown [13]. Within the C-terminal part of MECP2, a histidine and proline-rich region is present which is conserved in certain neural-specific transcription factors [32].

BAZ2A/TIP5 is part of the NoRC, nucleolar remodeling complex, which represses rDNA transcription by recruiting histone methyltransferases, HDACs and DNA methyltransferases [33].

BAZ2B has a domain structure similar to BAZ2A/TIP5, both contain a DDT (DNA binding homeobox and Different Transcription factors) and a tandem PHD-bromodomain. The PHD domain is a C4HC3 zinc-finger-like motif and the bromodomain consists of 110 amino acids and is found in many chromatin-associated proteins that can interact specifically with acetylated lysine. Tandem PHD-bromodomains have been found in several transcriptional co-repressors [34]. The DDT domain is exclusively associated with nuclear domains in other proteins and was found in different transcription and chromatin remodeling factors [35]. An AT_hook motif (which allows binding to the minor groove of AT-rich DNA regions) was found in BAZ2A/TIP5 but not in BAZ2B.

SETDB1 is a H3-K9 histone methyltransferase [23], its mouse homologue, ESET, has furthermore been shown to interact with the mSin3A/B co-repressor complex [36]. The SET domain is a signature motif for lysine-specific histone methyltransferases [37, 38]. This domain is also present in CLLD8 to which no function has yet been assigned.

The predicted protein sequence of KIAA1461 harbors a PWWP motif [39] named after the conserved amino acids Pro-Trp-Trp-Pro. It was first described in the WHSC1 protein, encoded by a gene within the Wolf-Hirschhorn syndrome critical region. The PWWP domain of Dnmt3b, a DNA methyltransferase, has been recently shown to bind to DNA. A common feature of PWWP containing proteins is the presence of additional domains known to be associated with chromatin [40]. Furthermore KIAA1461 has been shown to interact with the KIAA1549 protein in a yeast-two-hybrid experiment (http://www.kazusa.or.jp/huge/ppi). This interaction partner has not been studied in detail so far, but contains a serine-rich stretch as well as a helix-turn-helix motif (PS00622, LuxR family) according to Prosite (http://www.expasy.org/prosite). Helix-turn-helix motifs can be found in many transcription regulation proteins.

In the predicted protein sequence of KIAA1887, only a proline-rich extension (http://www.ebi.ac.uk/interpro) but no protein motif as such could be found.

The co-existence of MBDs and domains involved in chromatin modification, present in many of the identified polypeptides, could also point to a connection between the latter mechanism and methylated DNA. Interestingly, a very recent study [41] has shown that Mecp2 is associated with a H3-K9 methyltransferase activity, indicating a link between DNA methylation and histone methylation.

MBD proteins in other species

Homology searches in the ENSEMBL database revealed the following murine homologues of BAZ2B, CLLD8, KIAA1461 protein and KIAA1887 protein. The mouse homologue of BAZ2B, the ENSEMBL protein ENSMUSP00000028367 (gene ENSMUSG00000026987) shows a 82.6 % amino acid sequence identity. The predicted mouse gene ENSMUSG00000021980 coding for ENSEMBL protein ENSMUSP00000022552 has a 65.2 % amino acid sequence identity to the human CLLD8. We furthermore found the predicted gene ENSMUSG00000036792 coding for the ENSEMBL protein ENSMUSP00000036847 as mouse homologue of human KIAA1461 protein with a 94.0 % amino acid sequence identity. For the KIAA1887 protein, the mouse gene sequence ENSMUSG00000025409 (ENSEMBL protein ENSMUSP00000026476) with 91.1 % sequence identity was present in the database. The latter database entry however consists of only 101 amino acids. The existence of translated proteins remains to be determined for all four mouse genes.

DNA methylation as a mechanism of gene expression regulation exists also in plants. In our database searches we detected plant MBD proteins as well. The Pfam database contains polypeptides from Arabidopsis thaliana and Triticum aestivum. BLAST analyses revealed additional proteins in Zea Mays, Hordeum vulgare and Lycopersicon esculetum. Entries for MBD containing proteins from plants over C. elegans to mouse and human are present in Pfam.

Expression patterns of MBD genes

We performed Northern blot analyses for KIAA1461 and KIAA1887. Strong signals of ~8 kb were detected for KIAA1461 in skeletal muscle, heart, pancreas, kidney and placenta. A faint band could be detected in brain, lung and liver. For KIAA1887 a strong band of ~5 kb was present in heart, kidney, liver, skeletal muscle, placenta and pancreas, weaker signals could be seen for brain and lung tissue (Fig. 4).

Taken together, MBD1, MBD2, MBD3 and MECP2 as well as SETDB1, CLLL8, BAZ2A, KIAA1461 and KIAA1887 show a broad tissue distribution. The expression of BAZ2B is more restricted according to northern blot results [46]. It is of note that CLLL8, BAZ2A, KIAA1461 and KIAA1887 show a very low expression in brain.

BLAST-based and database searches

Non-redundant GenBank, high throughput genomic sequences and expressed sequence tag (EST) databases were searched using BLASTP and TBLASTN of the NCBI web tools (http://www.ncbi.nlm.nih.gov/blast) applying default conditions. The human MECP2 chain A (MBD) was used as a query input. TBLASTX was carried out applying the web tools at the European Bioinformatics Institute (http://www.ebi.ac.uk/blast2/).

Using the MBD of human MECP2 as query, the NCBI (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein), Pfam (release 7) (http://www.sanger.ac.uk/Software/Pfam), Prosite (release 17.17) (http://www.expasy.ch/prosite/) and Smart (version 3.4) (http://smart.embl-heidelberg.de/) databases were screened for proteins with this domain. The resulting polypeptides were sorted according to their species of origin and additional identified domains within their sequence.

SSDB at GenomeNet (http://ssdb.genome.ad.jp) was searched with pfm:MBD as query motif. BLASTP and BLASTN searches were carried out against the Celera database (http://www.celera.com) with standard settings.

Homologues of KIAA1461, CLLL8 and KIAA1887 were identified in the ENSEMBL database.

Alignment and Phylogeny

Alignments and phylogenetic trees were computed using the ClustalW program at GenomeNet (http://clustalw.genome.ad.jp/) with standard settings and the ClustalX program (version 1.8) [50] for graphical representation.

The sequence logo was constructed by means of the plogo script (http://www.cbs.dtu.dk/~gorodkin/appl/plogo.html). File formats were converted with the GCG package (version 10.2) when necessary.

Sequence comparison between mouse and man was carried out using the LALIGN algorithm at EMBNET (http://www.ch.embnet.org/software/LALIGN_form.html) with default settings.

Cell lines. RNA and genomic DNA isolation, cDNA synthesis

CCRF-CEM and lymphoblastoid cells were grown according to manufacturer's instructions (DSMZ, Braunschweig, Germany and the Coriell Institute for Medical Research, Camden, Netherlands) and harvested at 95 % confluency. Genomic DNA was isolated with the QIAGEN Blood Maxi-KIT (QIAGEN, Hilden, Germany). Total RNA was isolated using the Trizol reagent (Invitrogen, Karlsruhe, Germany). A reverse transcriptase reaction was performed in the presence of 50 ng/μl oligo(dT) and 2 mM dA/C/G/TTP with 10 U/μl SSII reverse transcriptase (Invitrogen, Karlsruhe, Germany). The resulting cDNA was purified with the QIAquick PCR purification kit (QIAGEN, Hilden, Germany). cDNA (200 ng) was used in subsequent 50- μl PCR amplifications with 10 pmol gene-specific primers. Standard PCR conditions and respective annealing temperatures were used.

Northern blotting

A cDNA fragment of KIAA1461 was PCR-amplified with exon specific primers KIAA1461for (5'-CTAGACCATGGGAAAAATGT-3') / KIAA1461rev (5'-ACTTGGAGACTGCTCCTCTA-3') and human genomic DNA as template using standard conditions. For KIAA1887 a cDNA fragment was PCR-amplified with exon 6 specific primers KIAA1887for (5'-CAGACCCCCTACTGTATTTC-3') / KIAA1887rev (5'-CAAAAGGTTAAAGCTTCCAT-3') and cDNA from a lymphoblastoid cell line as template using standard conditions. A cDNA fragment of β-actin amplified with primers β-actinfor (5'-TGAACCCTAAGGCCAACCGTG-3') / β-actinrev (5'-GCTCATAGCTCTTCTCCAGGG-3') was used as a loading control. These probes were radioactively labeled with 32P-dCTP in a random prime reaction and hybridized in ExpressHyb solution (CLONTECH, Palo Alto, CA, USA) to a human multiple tissue northern blot (CLONTECH, Palo Alto, CA, USA) for 16 h at 65°C. Washing was performed in 2 × SSC / 0.1% SDS at 65°C for 10 min. Signals were detected with a PhosphorImager (Amersham Biosciences, Freiburg, Germany).