A Role for the Chromatin-Remodeling Factor BAZ1A in Neurodevelopment

Abstract

Chromatin-remodeling factors are required for a wide range of cellular and biological processes including development and cognition, mainly by regulating gene expression. As these functions would predict, deregulation of chromatin-remodeling factors causes various disease syndromes, including neurodevelopmental disorders. Recent reports have linked mutations in several genes coding for chromatin-remodeling factors to intellectual disability (ID). Here, we used exome sequencing and identified a nonsynonymous de novo mutation in BAZ1A (NM_182648.2:c.4043T > G, p.Phe1348Cys), encoding the ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1), in a patient with unexplained ID. ACF1 has been previously reported to bind to the promoter of the vitamin D receptor (VDR)-regulated genes and suppress their expression. Our results show that the patient displays decreased binding of ACF1 to the promoter of the VDR-regulated gene CYP24A1. Using RNA sequencing, we find that the mutation affects the expression of genes involved in several pathways including vitamin D metabolism, Wnt signaling and synaptic formation. RNA sequencing of BAZ1A knockdown cells and Baz1a knockout mice revealed that BAZ1A carry out distinctive functions in different tissues. We also demonstrate that BAZ1A depletion influence the expression of genes important for nervous system development and function. Our data point to an important role for BAZ1A in neurodevelopment, and highlight a possible link for BAZ1A to ID.

Keywords: ACF1; BAZ1A; CYP24A1; Wnt signaling; epilepsy; intellectual disability; neurodevelopment; vitamin D metabolism.

Figure 1

A schematic representation of the human ACF1 protein indicating known interaction domains. ACF1 domains include a WAC motif necessary for the binding of ACF1 to the DNA and involved in ACF‐mediated chromatin assembly) [Fyodorov and Kadonaga, 2002], and a DTT motif (DNA binding homeobox and transcription factors). It further contains three conserved motifs, two BAZ and a WAKZ (responsible for the binding of ACF1 to ISWI, the other subunit of the ACF complex) [Jones et al., 2000; Eberharter and Becker, 2004], a PHD finger (plant homeodomain) and bromodomain (Brds) motifs. The mutation (p.Phe1348Cys) is located in the linker region between the PHD and Brd domains. The evolutionary conservation is shown for the mutation and the surrounding amino acids.

Figure 2

Differential expression of CYP24A1. A: The left graph shows levels of BAZ1A expression in the trio. To the right are the results of targeted qrtPCR validation of the CYP24A1 expression in the affected child and the healthy parents. B: The left graph shows overexpression of the BAZ1A in the transfected Saos‐2 cells compared with untransfected cells. The right graph shows the qrtPCR validation of the CYP24A1 expression in Saos‐2 cells transfected with either wt‐BAZ1A or mut‐BAZ1A. Saos‐2 cells transfected with the wt‐BAZ1A is considered as control. All expression values were normalized to the level of B‐actin in each respective sample. Expression levels represent mean values of the biological and technical replicates and error bars are ±SD. C: Simplified schematic model showing the role of ACF1 in the regulation of expression of CYP24A1. In the absence of vitamin D3, ACF1 and SNF2H (the other member of the ACF complex) interact with N‐CoR and suppress the expression of CYP24A1 through binding to the VDR. D: ACF‐1 protein is localized to the nucleus of Saos‐2 cells. To the left, protein expression analysis was performed by western blot of nuclear versus cytoplasmic protein lysates using specific antibodies against ACF‐1 and total histone H3. Histone H3 was used as a positive control for the nuclear fraction purification. To the right, ChIP analysis of anti‐ACF1 precipitated DNA measured by qrtPCR from fresh blood monocytes of the trio family. Chromatin precipitated from each sample is reported as a percentage of input. Error bars are ±SD of the biological and technical replicates. Asterisks indicate significant statistical difference (P ≤ 0.05) of CYP24A1 enrichment in anti‐ACf1‐precipitated DNA between the ID patient and the parents. Student's t‐test was used to calculate P values.

Figure 3

The log2 fold change of the genes in the GO categories Wnt pathway (n = 123), canonical Wnt pathway (n = 45), and Wnt target genes (n = 44). Genes having no reads aligned in the RNA sequencing experiments were not counted.

Figure 4

The relative expression of SYNGAP1 and SMARCA4 in the trio. A: qrtPCR validation of the differential expression of SYNGAP1 between the patient and the parents (left). To the right are the expression levels of SYNGAP1 in a control trio family, indicating no age‐related differential expression. B: qrtPCR validation of the differential expression of SMARCA4 between the patient and the parents (left). To the right are the expression levels of SMARCA4 in a control trio family showed no age‐related differential expression. All qrtPCR results were normalized to the level of B‐actin in each respective sample. Expression levels represent mean values of the biological and technical replicates and error bars are ±SD.

Figure 5

Validation of BAZ1A knockdown in Saos‐2 cells and Baz1a knockout in mouse. A: Left, expression levels of BAZ1A in Saos‐2 cells transfected with BAZ1A siRNA and Saos‐2 with control siRNA. All expression values were normalized to the level of B‐actin in each respective sample. Expression levels represent mean values of the biological and technical replicates and error bars are ±SD. Right, protein expression of ACF1 in Saos‐2 cells transfected with BAZ1A siRNA, Saos‐2 with control siRNA and untransfected cells. B: Bar plot showing the fraction of Baz1a expression in each mouse tissue as compared with the tissue with the highest Baz1a expression (heart).

Figure 6

Venn diagram showing the number of DEGs in mouse heart (green), mouse frontal brain (brown), patient blood (yellow) and Saos‐2 cells (blue), and the overlap of the DEGs between the different tissues.