Expanding the MECP2 network using comparative genomics reveals potential therapeutic targets for Rett syndrome
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Israel
- Hermelin Brain Tumor Center, Henry Ford Hospital, United States
- The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Israel
- Edmond and Lily Safra Children's Hospital, Chaim Sheba Medical Center, Israel
Figures
Figure 1 with 1 supplement
The phylogenetic profile of MECP2.
(A) The Normalized Phylogenetic Profile (NPP) of MECP2 in eukaryotes along with the top 5000 co-evolved proteins. Each row represents one human protein, ordered by Pearson correlation to MECP2, with the top 200 proteins labeled as E200. Darker color indicates higher normalized conservation of the protein in each species. Organisms are grouped by phylogenetic clade, and clustered within each clade. (B) The NPP of MECP2 in 51 mammals along with the 5000 top co-evolved proteins, ordered by Pearson correlation to MECP within mammals. The top 200 proteins are labeled as M200. (C) Gene overlap of the top 200 most co-evolved genes in eukaryotes and mammals, listing the 10 gene names listed in both.
Figure 1—figure supplement 1
Scaled phylogenetic profiles of all human genes by similarity to MECP2.
(A) NPP profiles of human genes in 1028 eukaryote species, ordered by the Pearson correlation to the profile of MECP2. (B) Phylogenetic profiles of human genes in 51 mammals, scaled to highlight the conservation patterns within the clade. (C) GeneAnalytics enrichment for the 390 union set of E200 and M200. Within each organ, individual anatomical compartment scores are displayed in dots. (D) Overlap between the 390 union set of E200 and M200 with all STRING MECP2 coexpression, experimental and textmining categories. (E) Overlap between the 200 genes most correlated to MECP2 in mammals (M200) and eukaryotes (E200) with the 390 genes most correlated to MECP2 through STRING.
Figure 2 with 2 supplements
The MECP2 druggable genes network.
(A) The 390 genes co-evolved with MECP2 in either eukaryotes or mammals contain 33 genes that are targets of known drugs/compounds. These genes are shown in a STRING interaction graph, with known gene-gene interactions shown as colored edges, and those with very strong co-evolution (Pearson correlation > 0.7) shown as gray edges. The two proteins co-evolved in both eukaryotes and mammals (EPOR and IRAK1) are colored yellow and the four other genes found at chr19p13.2 are colored purple. (B) Genomic location of MECP2 interacting genes along chromosome X in humans and mice. (C) Karyotype band locations of MECP2 and the 390 co-evolved genes. * p<0.05, **p<0.01, *** p<0.001 (D) Genomic location of MECP2 interacting genes along chr19p13.2 in humans and chromosome nine in mice. (E) Intra-chromosomal Hi-C contact heatmap for the chr19p13.2 locus in GM12878 cells, adapted from the 3D Genome Browser (Wang et al., 2018). Genes in the MECP2 network are shown, with other genes hidden for clarity. TADs called by 3D Genome Browser shown in blue/yellow track, with a super-TAD containing MECP2 genes outlined in red.
Figure 2—figure supplement 1
Synteny in the Chr19p13.2 locus.
(A) NCBI Genome Data Viewer view of the genomic locations of DNMT1, ICAM1, ICAM3, KEAP1, and EPOR (top panel, genes highlighted). The gene orthologs are shown in Mus musculus chromosome 9 (bottom panel). (B) Inference of the synteny in ancestral species from Genomicus. Each row represents the same genomic locus at an ancestral state. Each gene is represented by a different color, and selected genes are annotated below. EPOR, ICAM1, ICAM3, KEAP1, and DNMT1 maintain their relative positions as far as the mammalian ancestral genome.
Figure 2—figure supplement 2
Synteny between MECP2 and IRAK1.
(A) NCBI Genome Data Viewer view of the genomic locations of MECP2 and IRAK1 (top panel). The gene orthologs are shown in Mus musculus chromosome X (bottom panel). (B) Inference of the synteny in ancestral species from Genomicus.
Figure 3 with 2 supplements
Effects of MECP2 knockdown on neural cell phenotypes.
Human microglia (A,B), NSCs (C), astrocytes (D,E), and neurons (E) were silenced for MECP2 using lentivirus vector. The relative expression of M1 and M2 markers was analyzed in microglia cells using RT-PCR (A) and degree of phagocytosis using the pHrodo assay (B). NSCs were transduced with lentivirus vectors expressing the differentiation reporters GFAP and MAP2 and were differentiated as described in the methods. Ten days later, luciferase activity was determined (C). Astrocytes silenced for MECP2 were analyzed for the expression of EAAT2 using western blot analysis (D) and the expression of BDNF mRNA in both MECP2 silenced astrocytes and neurons was determined using RT-PCR (E). The results are a representative experiment of three separate tests analyzed in quadruplet. **p<0.01, ***p<0.001,****p<0.0001.
Figure 3—figure supplement 1
Silencing of MECP2 in neural cells.
Neurons, astrocytes, and microglia cells were transduced with lentivirus vectors expressing a control or MECP2 shRNAs. Following 3 days, the expression of MECP2 and actin were determined using western blot analysis. The results are representative of three experiments with similar results.
Figure 3—figure supplement 2
Cytotoxic effects of tested compounds.
Human microglia (A), astrocytes (B), neurons (C), and neuronal stem cells (D) were treated with different concentrations of DMF (1, 10, and 50 µM), EPO (10, 30, and 50 ng/ml), and pacrinitib (1 and 10 µM). Following 3 days of treatment, the cells were analyzed for cell death using LDH assay. The results are the means ± SD of six samples for each compound.
Figure 4
Effects of tested compounds on the polarization of microglial cells.
Human microglia cells were silenced for MECP2 using lentivirus vectors expressing MECP2 shRNA. Control cells were transduced with lentivirus vectors expressing a control shRNA. After 5 days, the expression of MECP2 was determined by western blot analysis (Figure 3—figure supplement 1). Control and silenced cells were treated with EPO 10 ng/ml (A), DMF 10 µM (B) and pacritinib 10 µM (C), and the expression of M1 and M2-associated markers were determined after 72 hr using RT-PCR. MECP2 silenced microglia cells treated with EPO, DMF, or pacritinib were also analyzed for phagocytosis using the pHrodo assay (D). MECP2-silenced microglia cells were transduced with lentivirus vectors expressing the NF-kB reporter followed by treatment with EPO, DMF, and pacritinib for 24 hr. Luciferase activity was determined (E). The results demonstrate the means ± SD of a representative experiment of three separate tests analyzed in quadruplet. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.
Figure 5
Effects of tested compounds on the differentiation of NSCs and EAAT2 expression in astrocytes.
Human NSCs (A) and astrocytes (B) were silenced for MECP2 using lentivirus vectors expressing MECP2 shRNA. Control cells were transduced with lentivirus vectors expressing a control shRNA. After 5 days, the expression of MECP2 was determined by western blot analysis (Figure 3—figure suppplement 1). (A) Control and silenced NSCs were allowed to differentiate for 10 days and the expression of differentiation markers GFAP and βIII tubulin were determined using RT-PCR. (B) Silenced astrocytes treated with the different compounds or with medium were analyzed for the expression of EAAT2 using RT-PCR. The results are of a representative experiment of three separate tests analyzed in quadruplet. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.
Figure 6
BDNF expression in neural cells.
MECP2 expression was silenced in human neurons (A) and astrocytes (B, C) and BDNF expression was analyzed in cells treated with a medium or the specific tested compounds using both RT-PCR (A) and ELISA (B, C). The combinatorial effect of EPO and DMF was assessed on BDNF expression in astrocytes (C). *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.
Figure 7
A model of MECP2 network genes converging on NF-κB signaling.
(A) We propose that reduced MECP2 levels could lead to increase in ICAM1, ICAM3, IRAK1, and KEAP1 levels, and a decrease of EPOR levels within the tissue-protective receptor. (B) IRAK-1 plays a role in NF-κB activation through IKK activation, which leads to IκB phosphorylation and subsequent degradation, NF-κB nuclear translocation and the expression of its pro-inflammatory target genes. KEAP1 binds NFE2L2 and prevents its nuclear localization. NFE2L2 target genes inhibits NF-κB signaling through a non-transcriptional mechanism involving degradation of IκBα. EPO binds EPOR and enhances the activation of Akt, resulting in inhibition of GSK-3β and inhibition of NF-κB nuclear transport.
Author response image 1
Normalized phylogenetic profiles of MECP2 and HDAC proteins.
Author response image 2
Overlap between the E200 and M200 intersection set (PP) and all STRING MECP2 interactions.
Additional files
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Supplementary file 1
Genes most co-evolved with MECP2.
- https://cdn.elifesciences.org/articles/67085/elife-67085-supp1-v1.csv
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Supplementary file 2
STRING GO-term enrichment of the E200 set.
- https://cdn.elifesciences.org/articles/67085/elife-67085-supp2-v1.txt
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Supplementary file 3
RT-PCR primer sequences.
- https://cdn.elifesciences.org/articles/67085/elife-67085-supp3-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67085/elife-67085-transrepform-v1.bpb.docx
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Expanding the MECP2 network using comparative genomics reveals potential therapeutic targets for Rett syndrome
eLife 10:e67085.
https://doi.org/10.7554/eLife.67085
