Rebalancing gene haploinsufficiency in vivo by targeting chromatin - PubMed (original) (raw)
Rebalancing gene haploinsufficiency in vivo by targeting chromatin
Filomena Gabriella Fulcoli et al. Nat Commun. 2016.
Abstract
Congenital heart disease (CHD) affects eight out of 1,000 live births and is a major social and health-care burden. A common genetic cause of CHD is the 22q11.2 deletion, which is the basis of the homonymous deletion syndrome (22q11.2DS), also known as DiGeorge syndrome. Most of its clinical spectrum is caused by haploinsufficiency of Tbx1, a gene encoding a T-box transcription factor. Here we show that Tbx1 positively regulates monomethylation of histone 3 lysine 4 (H3K4me1) through interaction with and recruitment of histone methyltransferases. Treatment of cells with tranylcypromine (TCP), an inhibitor of histone demethylases, rebalances the loss of H3K4me1 and rescues the expression of approximately one-third of the genes dysregulated by Tbx1 suppression. In Tbx1 mouse mutants, TCP treatment ameliorates substantially the cardiovascular phenotype. These data suggest that epigenetic drugs may represent a potential therapeutic strategy for rescue of gene haploinsufficiency phenotypes, including structural defects.
Figures
Figure 1. Tbx1-enriched regions co-localize with H3K4me1 enrichment.
(a) Examples of ChIP-seq data profiles as shown using the UCSC genome browser. Note the similarities between Tbx1 and H3K4me1 signals. (b) Sequence logo representing the enriched Tbx1 motif identified by de novo motif discovery. (c) Distribution of Tbx1 and H3K4me1-binding sites relative to the closest TSS. Note the sharp clustering of Tbx1 peaks within the first 800 bp downstream of the TSS. H3K4me1 peaks have a similar but broader distribution. (d) Genomic distribution of Tbx1, H3K4me1 and overlapping Tbx1/H3k4me1 (intersect) binding sites relative to genes. In all cases, the majority of sites is intragenic. Feature id: ensembl gene ID. (e,f) Map of Tbx1 (e) and H3K4me1 (f) enrichments relative to the body of RefSeq genes and to 5 kbp upstream of the TSS and 5 kbp downstream of the TES. The transcribed regions were normalized to the same length. The graph shows ChIP-seq data from two independent experiments. Note the highest enrichment downstream to the start site and a gradual increase towards the 3′-end of genes. Shaded areas around each curve indicate the standard errors. (g) H3K4me1 occupancy relative to Tbx1 summits in two biological replicates. (top) Heat-map of H3K4me1 ChIP-seq enrichment across Tbx1-binding sites. Each row represents a 4 kb window centred on summits of Tbx1 peaks and extending 2 kb upstream and 2 kb downstream. (bottom) Averaged ChIP-seq enrichment profile across a 4 kb window centred on Tbx1 sites. Thus, H3K4me1 enrichment tends to center around a Tbx1 peak. TES, transcription end sites.
Figure 2. Tbx1 dosage affects gene expression and H3K4 monomethylation.
(a) Statistically significant gene expression changes in Tbx1 K/D versus control samples. The graph shows the distribution of the number of genes versus log ratios/FC. (b) P19Cl6 cells were transfected with Tbx1 siRNAs (non-targeting siRNA as control) with and without TCP treatment. (top) Acid-extracted histones from P19Cl6 cells run on a 15% SDS–PAGE and stained with Coomassie Blue dye. (bottom) Isolated histones were subjected to SDS–PAGE and monomethylation at histone H3K4 or total H3 as control were detected with specific antibodies. (c) Examples of H3K4me1 ChIP-seq signal distribution in two loci before and after Tbx1 K/D. The shaded area indicate regions with reduced methylation. (d) Venn diagram grouping Tbx1 peaks associated with differentially expressed genes (green) and Tbx1 peaks down-methylated (purple) after Tbx1 K/D. The two groups are clearly distinct with only a small overlap. (e) The graph shows the location relative to the TSS of productive Tbx1 peaks. The peaks associated with differentially expressed genes are clearly closer to the TSS. (f) Canonical pathways by InnateDB analysis of differentially expressed (green) and differentially methylated (purple) genes associated to Tbx1 productive sites. (g) The cartoon illustrates the two types of response to Tbx1 dosage (DE and differential methylation). (h) ChIP–western blot analyses of co-immunoprecipitation experiments with endogenous proteins from P19Cl6 differentiating cells. Immunoprecipitation was carried out using an anti-Tbx1 antibody or rabbit IgG (control). Interacting proteins were eluted from the beads using three consecutive elutions (EL1, EL2 and EL3). Western blots were carried out using anti-Tbx1 (control), anti-Setd7, anti-Kmt2a/Mll1, anti-Kmt2c/Mll3 and anti-Kmt2b/Mll4 antibodies. (i) q-ChIP assay on a subset of Tbx1-binding sites in P19Cl6 cells (with and without Tbx1 K/D) using an anti-Kmt2c antibody, followed by qRT-PCR. Error bars: s.e.m.; _n_=3; two-tailed Student's _t_-test. ** P<0.01. FC, fold change; q-ChIP, quantitative ChIP.
Figure 3. TCP partially rescues gene dysregulation caused by Tbx1 K/D in cell culture.
(a) Statistically significant gene expression changes in Tbx1 K/D+TCP versus Tbx1 K/D samples. The graph shows the distribution of the number of genes versus log ratios/FC. (b) Venn diagram grouping genes significantly up- or downregulated in Tbx1 K/D versus control samples (blue) and Tbx1 K/D+TCP versus Tbx1 K/D samples. Note the large overlap of 948 genes. (c) Scatter plot depicting the DE of 608 genes ‘rescued' by TCP treatment (discordant) and of 246 genes ‘worsened' by TCP treatment (concordant). (d) Canonical pathways defined in the InnateDB with significant enrichment of genes differentially expressed after Tbx1 KD (red, top) and rescued by TCP treatment (green, bottom). Co-occurrences of rescued pathways with in vivo TCP treatment (Fig. 4n) are indicated with red asterisks. (e) qRT-PCR validation of DE of a subset of rescued genes in P19Cl6 cells (with and without K/D), and WT and Tbx1+/LacZ (HET) E9.5 mice embryos, with and without TCP treatment. Asterisks above bars indicated significant differences compared with the control (*P<0.05; **P<0.01; ***P<0.001). Error bars: s.e.m.; _n_=3; two-tailed Student's _t_-test. FC, fold change.
Figure 4. TCP partially rescues the cardiovascular phenotype of Tbx1 mutants.
(a–c′) Ink injection of E10.5 embryos. (a,a′) IIIrd, IVth and VIth PAAs in WT embryos. (b,b′) untreated Tbx1 Lacz/+ embryo with aplasia of the IVth PAA. (c–c′) normal IVth PAA in a treated Tbx1 Lacz/+ embryo. DoA, dorsal aorta. Scale bars: 100 μm (a–c'). (d–j′) Coronal sections of WT (d,d′), untreated (e,f) and treated (g–j) Tbx1 Neo2/LacZ hearts (E18.5). (e) PTA and VSD (arrowhead) in a Tbx1 Neo2/LacZ untreated embryo (ps 5). (f,f′) untreated Tbx1 Neo2/LacZ embryo with incomplete PTA and VSD (black arrowed); dashed lines: unseptated truncal region; green arrowhead: bifurcation of aorta and pulmonary trunk (ps 4). (g) incomplete PTA in a treated Tbx1 Neo2/LacZ embryo. The unseptated region (dashed lines) is restricted to the OFT valve region (ps 3). (h,h′) A treated Tbx1 Neo2/LacZ embryo with DORV and VSD (black arrowhead), the tip of the VS is under the left wall of the aorta (blue arrow) (ps 2). (i,i′) DORV in a treated Tbx1 Neo2/LacZ embryo. The tip of the VS is under the middle of the aortic valve (blue arrow), suggesting improved alignment of aorta and LV, compared with the sample in h,h′ (ps 2). (j,j′) Normal conotruncus in a treated Tbx1 Neo2/LacZ embryo (ps 1). Ao, aorta; P, pulmonary trunk. Scale bars: 100 μm (d–j′). Whole mount of samples sectioned in e–j are shown in Supplementary Fig. 5, a–f, respectively. (k) Phenotypic scores (ps) for treated and untreated Tbx1 Neo2/LacZ embryos. (l) Genomic distribution of H3K4me1 in two biological replicates of WT (blue), Tbx1 LacZ/+ untreated (red) or TCP-treated Tbx1 LacZ/+ E9.75 (25 somites) embryos (green) in genes characterized by negative correlation (discordant) or positive correlation (concordant) between expression and methylation in Tbx1 LacZ/+ versus WT comparisons. (m) Canonical pathways by InnateDB analysis of discordant (blue) and concordant (green) genes. Some of these pathways (asterisks) were also identified in cell culture experiments (Fig. 3d). PV: hypergeometric test, BH correction. DORV, double-outlet right ventricle; LV, left ventricle; RV, right ventricle; VS, ventricular septum; VSD, ventricular septal defect.
Figure 5. Cartoon showing a working model for Tbx1 interactions with chromatin and TCP rebalancing effect.
In the WT state (top row), Tbx1 promotes H3K4me1 deposition (small red dots) through interaction with histone methyltransferases (HMTs). When Tbx1 is absent or of lowered dosage (middle row), H3K4me1 enrichment is lowered because of reduced recruitment of HMTs. TCP treatment (bottom row) re-establishes H3K4me1 by inhibiting the activity of the Lsd1 demethylase. Blue cylinders indicate histone octamers.
References
- Jerome L. A. & Papaioannou V. E. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat. Genet. 27, 286–291 (2001). - PubMed
- Lindsay E. A. et al.. Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410, 97–101 (2001). - PubMed
- Merscher S. et al.. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104, 619–629 (2001). - PubMed
- Yagi H. et al.. Role of TBX1 in human del22q11.2 syndrome. Lancet 362, 1366–1373 (2003). - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases