*2.5. Phylogenetic Profiling of RTT and RTT-like Causing Proteins and Their Interaction Partners*

We retrieved 240 human proteins interacting with MeCP2, CDKL5, and FOXG1 from BioGRID and UniProt databases (Supplementary Table S5) [44,45]. To illuminate the interconnection of MeCP2, CDKL5, and FOXG1 binding partners as well as their evolutionary relationship, we conducted phylogenetic profiling and cluster analysis of 326 eukaryotes using the retrieved sequences and the sequences of the three proteins, MeCP2, CDKL5, and FOXG1, as queries (Figure 3, Supplementary Table S6). The results showed that the dataset was divided into four clusters, which were defined as Classes 1 to 4. There were 58 conserved proteins in chordates of Class 1, 92 in metazoans of Class 2, 17 in multicellular of Class 3, and 73 in eukaryotes of Class 4. MeCP2 and CDKL5 belonged to Class 1, whereas FOXG1 belonged to Class 2 (Figure 3). FOXG1 and MECP2 showed to have many binding partners that act as a transcription factor or gene expression regulator. In contrast, CDKL5 tend to bind to a fewer number of proteins having functions in regulating cell adhesion, ciliogenesis, and cell proliferation; however, this protein has been shown to interact with MeCP2. As RTT has been determined to occur from the altered functionality of MBD and NID of MECP2, we focused on the widely known binding partners of these domains, such as SIN3 transcription regulator family member A (SIN3A), histone deacetylase (HDAC)1, and nuclear receptor corepressor (NCOR) which play roles as co-repressor complexes. Even though FOXG1 does not directly bind to MeCP2, we found that the binding partners of MeCP2 co-repressor complex are also associated with FOXG1 binding partners that also act as co-repressor complexes such as special AT-rich sequence-binding protein (SATB)2, lysine-specific histone demethylase (KDM)1A, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily (SMARC)A member 5, A-kinase anchor protein (AKAP)8, of which are ancient proteins within Classes 3 and 4.

**Figure 3.** Phylogenetic profiling of MeCP2, CDKL5, and FOXG1 proteins and their interaction partners. The horizontal axis shows 326 eukaryotes for which whole genome sequences are available, and the vertical axis shows 240 human proteins related to RTT. Bar in a1 and a2 shows MeCP2-interactor (red), CDKL5-interactor (green), FOXG1-interactor (blue), respectively. The human orthologous proteins in each species are shown in black. The phylogenetic tree was divided into four clusters (Class 1–4); those conserved across chordates, metazoan, multicellular, and eukaryotes are shown.
