CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development - PubMed (original) (raw)

CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development

Hui Yao et al. JCI Insight. 2018.

Abstract

CHD7, an ATP-dependent chromatin remodeler, is disrupted in CHARGE syndrome, an autosomal dominant disorder characterized by variably penetrant abnormalities in craniofacial, cardiac, and nervous system tissues. The inner ear is uniquely sensitive to CHD7 levels and is the most commonly affected organ in individuals with CHARGE. Interestingly, upregulation or downregulation of retinoic acid (RA) signaling during embryogenesis also leads to developmental defects similar to those in CHARGE syndrome, suggesting that CHD7 and RA may have common target genes or signaling pathways. Here, we tested three separate potential mechanisms for CHD7 and RA interaction: (a) direct binding of CHD7 with RA receptors, (b) regulation of CHD7 levels by RA, and (c) CHD7 binding and regulation of RA-related genes. We show that CHD7 directly regulates expression of Aldh1a3, the gene encoding the RA synthetic enzyme ALDH1A3 and that loss of Aldh1a3 partially rescues Chd7 mutant mouse inner ear defects. Together, these studies indicate that ALDH1A3 acts with CHD7 in a common genetic pathway to regulate inner ear development, providing insights into how CHD7 and RA regulate gene expression and morphogenesis in the developing embryo.

Keywords: Development; Embryonic development; Genetic diseases; Neuroscience.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Misregulation of retinoic acid–related genes with Chd7 deficiency.

(A and B) RNA-seq data from Chd7Gt/+ (A) and Chd7Gt/Gt (B) E10.5 inner ears, visualized by volcano plot. Chd7Gt/+ inner ears show significant downregulation of 95 transcripts and upregulation of 43 transcripts. Chd7Gt/Gt inner ears show significant downregulation of 669 transcripts and upregulation of 416 transcripts. Chd7 and retinoic acid (RA) signaling–related genes are labeled. n = 2 for each genotype. (C) RNA-seq data from E10.5 inner ears showing fold change in mRNA levels of RA-related genes. *P < 0.05. (D and E) Gene ontology term analysis of RNA-seq results describing processes (D) and functions (E) disrupted in E10.5 Chd7Gt/+ inner ears compared with Chd7+/+ inner ears. Significance was determined using DESeq (v. 1.22.1).

Figure 2. Aldh1a3 is misregulated in Chd7 mutant mouse ears.

(A) Representative qRT-PCR data showing fold change in expression of Chd7 and Aldh1a3 in Chd7Gt/+ E10.5 mouse otocysts compared with Chd7+/+ otocysts. This experiment was repeated 6 times. (B) Representative qRT-PCR data showing fold change in expression of Aldh1a3 and Chd7 in Aldh1a3+/– and Aldh1a3–/– E10.5 mouse otocysts compared with Aldh1a3+/+ otocysts. This experiment was repeated 13 times. (C) Representative qRT-PCR data showing fold change in Chd7 and Aldh1a3 expression in neural progenitors differentiated from Chd7Gt/+ and Chd7Gt/Gt mouse embryonic stem cells compared with those differentiated from Chd7+/+ mouse embryonic stem cells. This experiment was repeated twice. Significance was determined by Student’s t tests, and P values were corrected using the Bonferroni method. ***P ≤ 0.001. (D–F) In situ hybridization of sectioned littermate E10.5 inner ears shows increased Aldh1a3 expression in Chd7Gt/Gt inner ears compared with Chd7Gt/+ and Chd7+/+ inner ears (n = 6 ears per genotype). Regions of high expression in the otocyst are demarcated by solid bars. Scale bars: 100 μm.

Figure 3. Increased CHD7 affects expression of retinoic acid–related genes.

(A and B) 293T cells transfected with a vector containing human CHD7 and FLAG. CHD7 and FLAG levels were assayed by Western blot (A) and quantified (B) using ImageJ software. This experiment was repeated twice. Images were derived from duplicate samples run contemporaneously on parallel gels. (C) Expression of retinoic acid–related genes examined using qPCR of mRNA extracted from 293T cells transfected with CHD7 overexpression or empty vector. This experiment was repeated 2 times. Significance was determined by Student’s t tests. *P ≤ 0.05.

Figure 4. CHD7 binds the region upstream of the ALDH1A3 gene in human and mouse cells.

(A) ChIP-qPCR analysis in human SH-SY5Y neuroblastoma cells targeting CHD7 upstream of ALDH1A3. Four candidate sites (red squares) were selected based on publicly available CHD7 ChIP-seq data from human embryonic stem cells (GEO accession GSM1003473) and the high degree of sequence conservation among vertebrates (67). (B) Chd7 ChIP-qPCR analysis in mouse E10.5 microdissected otocysts upstream of Aldh1a3. BLAST was used to identify the homologous sequences upstream of mouse Aldh1a3; site 2′ (purple square) was selected for Chd7 ChIP-qPCR analysis. qPCR reactions were repeated two times in triplicates. Data are represented as mean ± SEM, and significance was determined by 2-tailed unpaired t tests.

Figure 5. Retinoic acid does not affect CHD7 levels in SH-SY5Y cells.

Western blotting of CHD7 and CHD5 levels in various cell types. HDAC2 is used a loading control. Each experiment was performed at least 2 times. (A) Treatment with 10 μM all-trans retinoic acid (ATRA) increased CHD5 levels and had no effect on CHD7 levels over 12 days. (B) Treatment with 1, 5, or 10 μM ATRA for 7 or 12 days mildly induced CHD5 levels and had no effect on CHD7. (C) Treatment with 10 μM 13-cis retinoic acid (13-_cis_-RA) for 6 days increased CHD5 levels but had no effect on CHD7 levels relative to treatment with DMSO only. (D) Treatment with 1, 5, or 10 μM 13-_cis_-RA for 7 or 12 days induced CHD5 levels and had no effect on CHD7 levels. (E) Treatment with ATRA (1, 5, or 10 μM) or citral (1, 5, or 10 μM) for 3 days had no effect on CHD7 levels. (F) Treatment with 10 μM DEAB for 3 days had no major effect on CHD7 levels. This experiment was repeated twice. (G) CHD7 levels were lower in neural progenitor cells derived from forebrains of E12.5 Chd7Gt/+ embryos compared with Chd7+/+ embryos. (H) Treatment with 10 μM ATRA or 10 μM citral had no effect on CHD7 levels relative to DMSO vehicle control in neural progenitors derived from forebrains of E12.5 Chd7+/+ or Chd7Gt/+ mice.

Figure 6. CHD7 does not directly bind RAR in human SH-SY5Y neuroblastoma cells, and there is minimal overlap in genomic binding sites in mouse embryonic stem cells.

Representative coimmunoprecipitation blots of CHD7 and RAR show no direct binding between CHD7 and RAR in (A) untreated (n = 2) or (B) all-trans retinoic acid–treated (ATRA-treated) (10 μM for 7 days) SH-SY5Y cells (n = 2). Images in B were derived from duplicate samples run contemporaneously on parallel gels.

Figure 7. Chd7 dosage does not affect global retinoic acid reporter activity.

(A–G) E10.5, E11.5, or E12.5 embryos containing RARE-lacZ transgene and 2 (Chd7+/+), 1 (Chd7+/–), or no (Chd7–/–) wild-type alleles for Chd7 were stained with X-gal to reflect RA activity. Chd7–/– embryos were hypoplastic and did not survive beyond E10.5; otherwise, no differences in X-gal staining pattern between wild-type and Chd7 mutant embryos were noted. Embryo counts are as follows: Chd7+/+ — E10.5, n = 4; E11.5, n = 4; E12.5, n = 5; Chd7+/– — E10.5, n = 8; E11.5, n = 5; E12.5, n = 7; Chd7–/– — E10.5, n = 1. (H) Overexpression of FLAG-HA-hCHD7 in 293T cells has no effect on RARE-luciferase reporter in the presence or absence of 1 μM all-trans retinoic acid (RA). K998R CHD7 missense mutant construct also has no effect on RARE-luciferase reporter activity. Data are shown as luciferase normalized to Renilla. (I) siRNA knockdown of hCHD7 in 293T cells does not alter RARE-luciferase reporter activity, in the presence or absence of 1 μM RA. Control siRNA against cyclophilin B enhances RA activity as expected (35). Data are shown as luciferase normalized to Renilla. Significance was determined by ordinary 1-way ANOVA tests. **P ≤ 0.001.

Figure 8. Aldh1a3 loss rescues _Chd7_-deficient inner ears.

Paint-filled inner ears from E14.5 embryos obtained by crossing Chd7Gt/+ and Aldh1a3+/– mice. (A) Shown are the cochlea (COC), endolymphatic duct (ED), posterior semicircular canal (PC), anterior semicircular canal (AC), lateral semicircular canal (LC), and saccule (SAC). Ears with loss of 1 (Chd7+/+;Aldh1a3+/–; B) or both (Chd7+/+;Aldh1a3–/–; C) copies of Aldh1a3 appear similar to wild-type ears. Ears from Chd7Gt/+;Aldh1a3+/+ (D) and Chd7Gt/+;Aldh1a3+/– (E) embryos exhibit lateral canal truncation (*) and posterior canal defects (#), whereas 42% (12 of 28) of ears from Chd7Gt/+;Aldh1a3–/– (F) mice appear normal. (A–C) Left ears; (D–F) right ears. There were no differences in left-right laterality of phenotypes. Table 1 shows the number of affected ears for each genotype, as well as n values. Scale bars: 500 μm.

Figure 9. Model depicting the influences of CHD7 and RA signaling on gene expression.

In the cytoplasm, ALDH1A3 catalyzes the oxidation of retinaldehyde to retinoic acid (RA). RA then enters the nucleus and binds RA receptor (RAR). RAR forms a heterodimer with retinoid receptor (RXR), and the complex binds RA responsive elements (RAREs) to regulate transcription of downstream genes. Shown are three potential interactions between CHD7 and RA that we investigated in this study. In the first potential mechanism, CHD7 forms a complex with RAR/RXR heterodimers that acts to activate transcription of downstream target genes. In the second potential mechanism, RAR/RXR heterodimers bind upstream of CHD7 to regulate its transcription. In the third potential mechanism, CHD7 binds upstream of ALDH1A3, repressing its transcription. Our results provide evidence against the first two mechanisms, in favor of the third.

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CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development - PubMed (original) (raw)