Expansion and mechanistic insights into de novo DEAF1 variants in DEAF1-associated neurodevelopmental disorders

Abstract

De novo deleterious and heritable biallelic mutations in the DNA binding domain (DBD) of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of disorders termed DEAF1-associated neurodevelopmental disorders (DAND). RNA-sequencing using hippocampal RNA from mice with conditional deletion of Deaf1 in the central nervous system indicate that loss of Deaf1 activity results in the altered expression of genes involved in neuronal function, dendritic spine maintenance, development, and activity, with reduced dendritic spines in hippocampal regions. Since DEAF1 is not a dosage-sensitive gene, we assessed the dominant negative activity of previously identified de novo variants and a heritable recessive DEAF1 variant on selected DEAF1-regulated genes in 2 different cell models. While no altered gene expression was observed in cells over-expressing the recessive heritable variant, the gene expression profiles of cells over-expressing de novo variants resulted in similar gene expression changes as observed in CRISPR-Cas9-mediated DEAF1-deleted cells. Altered expression of DEAF1-regulated genes was rescued by exogenous expression of WT-DEAF1 but not by de novo variants in cells lacking endogenous DEAF1. De novo heterozygous variants within the DBD of DEAF1 were identified in 10 individuals with a phenotypic spectrum including autism spectrum disorder, developmental delays, sleep disturbance, high pain tolerance, and mild dysmorphic features. Functional assays demonstrate these variants alter DEAF1 transcriptional activity. Taken together, this study expands the clinical phenotypic spectrum of individuals with DAND, furthers our understanding of potential roles of DEAF1 on neuronal function, and demonstrates dominant negative activity of identified de novo variants.

Figure 1

Transcriptomic changes in the hippocampus of NKO mice highlight Deaf1 role in neuronal function. (A) Heat map and (B) volcano plot of the 347 identified differentially expressed genes (red color indicates decreased expression and blue indicates increased expression) by RNA-sequencing using hippocampal RNA from 4 control and 4 NKO mice. Biological function enrichment analysis of (B) all 347 genes, (C) genes with increased expression and (D) genes with decreased expression.

Figure 2

DEAF1 regulation of UBE2M. (A) Location and sequence of a putative DEAF1 consensus sequence within the UBE2M proximal promoter. Both mouse and human sequences are shown. (B) EMSA using WT or p.Q264P DEAF1 proteins with control 6spcon or Ube2m dsDNA probes. (C) ChIP was performed using hippocampal tissue from control or NKO mice with pre-immune serum (PI) or anti-DEAF1 antibodies. Quantitative PCR was performed using primers designed to amplify a known DEAF1 binding region within the Deaf1 promoter or the Ube2m proximal promoter. Each bar represents mean +/− SEM fold enrichment relative to control PI (N = 3). ^**P < 0.01 using one-way analysis of variance followed by Dunnett’s multiple comparisons test (versus control PI). (D) Western blots were performed on hippocampal tissue from control or NKO mice using anti-UBE2M and anti-ACTB antibodies. Each bar represents mean +/− SEM relative to ACTB expression for each animal (N = 3). ^*P,0.05 using Student’s t-test.

Figure 3

CRISPR/Cas9-mediated deletion of DEAF1 in HEK293T cells. (A) RT-qPCR was performed using RNA isolated from 293 T Control and 293 T DEAF1-KO cells to determine changes in the expression of the indicated DEAF1-regulated genes. (B) Western blots were performed on proteins isolated from 293 T Control and 293 T DEAF1-KO cells using anti-UBE2M and ACTB antibodies. Each bar represents mean +/− SEM relative to control expression (N = 3). ^***P < 0.001 using Student’s t-test. (C) ChIP was performed on 293 T control and 293 T DEAF1-KO (CRISPR/Cas9) using pre-immune serum or anti-DEAF1 antibodies. Regions of genomic DNA were amplified using the primers to DEAF1 promoter (positive control), DEAF1 Exon 6 (negative control) or UBE2M promoter. PCR products were separated on agarose gels.

Figure 4

Alterations in dendritic spine number in hippocampal regions of NKO mice. (A) Representative image of Golgi-Cox staining (Control) used to visualize neuronal dendritic spines in hippocampal slices of Control, Het and NKO mice. (B) Quantification of spine number per μm in apical and basal CA1, apical and basal CA3, and proximal and distal DG regions. Each bar represents mean +/− SEM (N = 6 per animal genotype). ^*P,0.05, ^**P < 0.01 using one-way analysis of variance followed by Bonferroni’s multiple comparisons test.

Figure 5

Dominant negative activity of de novo DEAF1 variants. (A) Immunofluorescent images of 293 T stable cells expressing GFP, WT-DEAF1, or the indicated DEAF1 heritable (p.R226W) or de novo variants (p.P174_G222del, p.Q264P) using anti-FLAG antibodies. (B) Western blots were performed on proteins isolated from 293 t stable cells using anti-FLAG (DEAF1) and anti-tubulin antibodies. (C) RT-qPCR was performed using RNA isolated from HEK293T Control and HEK293T stable cells to determine changes in the expression of the indicated DEAF1-regulated genes. Each bar represents mean +/− SEM (N = 4). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to DEAF1 using one-way analysis of variance followed by Dunnett’s multiple comparisons test.

Figure 6

Effects of DEAF1 WT and p.Q264P on DEAF1-regulated gene expression in cells lacking DEAF1. (A) HEK293T DEAF1-KO (CRISPR/Cas9) cells were transduced with AAV particles that express GFP, DEAF1, or DEAF1-p.Q264P. RT-qPCR was performed to determine changes in the expression of the indicated DEAF1-regulated genes. Each bar represents mean +/− SEM (N = 4). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to GFP-transduced Control using one-way analysis of variance followed by Fisher’s least square comparisons test. #P < 0.01 compared to GFP-transduced DEAF1-KO using one-way analysis of variance followed by Fisher’s least square comparisons test. (B) Models of WT-DEAF1, DEAF1 KO and dominant negative actions of identified DEAF1 variants on DEAF-target gene regulation.

Figure 7

DEAF1 variants and study subjects with de novo autosomal dominant DEAF1-associated neurodevelopmental disorder (DAND). (A) DEAF1 (Transcript NM_021008.4 and protein NP_066288.2) protein structure including the DNA binding domain (aa173–306), zinc finger region (ZnF), nuclear localization signal (NLS), nuclear export signal (NES) and MYND domain. Also shown are locations of the identified de novo heterozygous DEAF1 variants in this study. (B) Analysis of conservation of DEAF1 (Consurf) with 14 mammalian homologs, including human DEAF1, using 6 amino acid sliding window. De novo DEAF1 variants cluster within the highly conserved DBD. (C-H) Nonspecific minor facial dysmorphisms are observed, including thick lower lip vermilion, tented upper lip vermilion and pointed chin. (C) Study participant 3 at 8 years of age, heterozygous for the c.712A > C (p.Thr238Pro) variant; (D) study participant 4 at 6 years of age, heterozygous for the c.748A > G (p.Lys250Glu) variant; (E) study participant 5 at 14 years of age, heterozygous for the c.754 T > C (p.Trp252Arg) variant; (F) study participant 6 at 11 years of age, heterozygous for the c.767 T > G (p.Ile256Ser) variant; (G) study participant 7 at 11 years of age, heterozygous for the c.880G > A (p.Val294Leu) variant; (H) study participant 9 at 20 years of age, heterozygous for the c.332A > C (p.Asp111Ala) and deletion of exons 3–6 (GRCh37, g.683978_690053del, p.Ser130_Leu290del).

Figure 8

Effects of clinically-identified DEAF1 variants on DEAF1 transcriptional repression and activation activity. (A) and (C) HEK293T cells were transfected with DEAF1 promoter luciferase reporter plasmid with pcDNA3, WT-DEAF1 or the indicated DEAF1 variant. DEAF1 repression activity was determined relative to pcDNA3 (set to 100%). (B) and (D) HEK293T cells were transfected with Eif4g3 promoter luciferase reporter plasmid with pcDNA3, WT-DEAF1, or the indicated DEAF1 variant. DEAF1 activation activity was determined relative to pcDNA3 (set to 100%). Each bar represents mean +/− SEM (N = 5–7 de novo variants with N = 13 WT for repression studies and N = 5 de novo variants with N = 11 WT for activation studies). ^*P,0.05, ^**P < 0.01 using one-way analysis of variance followed Dunnett’s multiple comparisons test (WT versus each mutant). (E) EMSA using WT (DEAF1) or the indicated FLAG-tagged recombinant proteins isolated from transfected HEK293T cells. DEAF1 consensus dsDNA probes containing 6 bp (6spcon) or 11 bp (N52–69) nucleotides between the CG dinucleotides were examined.