Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription

Figure 1.. Developmentally delayed child identified with c.781–1G > A mutation in TAF8 gene. (A) Photographs of patient at 3 years of age (top) and 4 years of age (bottom). (B) Brain MRI acquired at 2 years and 7 months of age shows (sagittal on top and two transversal levels on bottom) mildly prominent extra-axial spaces, diffuse thinning of white matter with delayed myelinization, borderline enlarged lateral ventricles, short corpus callosum with narrow posterior body and absent splenium, normal brainstem, borderline small cerebellar vermis, and mildly small posterior fossa size. (C) (Top) Patient and control genomic sequences at the intron 7–exon 8 boundary of interest. Asterisk highlights the G > A splice site mutation (in red). Capital letters show exon 8 coding nucleotides. (Bottom) Schematic representation of the TAF8 gene (not to scale) with the position of the mutation indicated by the red asterisk. (D) Sequencing chromatogram highlighting the G nucleotide missing from the beginning of exon 8 in the patient cDNA. (E) RT-qPCR performed with primer annealing to the boundaries between exons 2 and 3, showing abundance of TAF8 mRNA in control and patient cells treated with and without…

Figure 2.. The TAF8: c.781–1G > A mutant protein lacks an NLS and is degraded by the proteasome. (A) Amino acid sequence of WT TAF8 protein aligned to the in silico predicated amino acid sequence generated by the TAF8: c.781-1G > A mutation, which possesses a unique carboxy-terminus (depicted in red), and lacks an NLS (depicted in grey highlight in the WT sequence). Histone fold domain in the WT sequence is highlighted in blue. (B) Two different protein WCE (WCE1 and WCE2) preparations (see Materials and Methods) were analyzed by western blot. Blots were probed with two different polyclonal anti-TAF8 antibodies (affinity purified pAb 3478 and crude pAb 3477 serum) targeting the entire TAF8 protein, as indicated. (C) Patient and control fibroblasts were treated with MG132 for 12 or 18 h, WCEs were prepared and probed with an anti-TAF8 antibody (affinity purified pAb 3477). The presence of a smaller protein, likely corresponding to the mutant TAF8 protein was detected in the presence of MG132.

Figure 3.. Exogenously expressed TAF8 mutant protein is unstable. (A) WCEs from insect Sf9 cells were prepared with either no infection, or with infection of a recombinant baculovirus encoding the TAF8: c.781-1G > A mutant protein fused to an FLAG tag and western blot assays were carried out using purified anti-TAF8 pAb 3478. (B) Western blot analyses of WCEs generated from Sf9 cells either with no infection, infection with recombinant baculovirus encoding FLAG tagged WT TAF8, or FLAG-tagged mutant TAF8 (FL-TAF8 MUT) protein, probed either with anti-FLAG antibody (upper blot), or affinity purified anti-TAF8 pAb 3478 (middle blot). Each lane represents an independent infection. (C) RT-qPCR performed with primers annealing to the boundary between exons 2 and 3 of TAF8 mRNA normalized to Sf9 housekeeping genes 28S, GAPDH and L35, performed on cDNA generated from Sf9 cells with either no infection, or infected with recombinant baculovirus encoding WT TAF8 protein or TAF8: c.781-1G > A mutant protein. All values were normalized to the average of the WT TAF8 infection. Error bars represent SEM of three technical replicates. See Supplementary Material, Table S1 for primer sequences. (D) Schematic…

Figure 4.. mESCs induced to delete Taf8 undergo cell death potentially as a result of transcriptional defects. (A) Representative images of Taf8lox/lox or Taf8lox/lox-Rosa26-CreERT2 mESCs after 8 days of culture with or without 0.5 µM 4-hydroxy tamoxifen treatment (4-OH). See also Supplementary Material, Figure S2A. (B) Curve showing the cumulative number of viable mESCs over the course of the treatment, cell counts were plotted taking into account the dilution factor at each passage. (C) Cell death was measured by trypan blue exclusion over the course of the treatment (as indicated). (D) Western blot analyses to detect TAF8 protein with the affinity purified 3478 pAb during several days (d) of 4-hydroxy tamoxifen (4-OH) treatment. Equal loading was measured by using an anti-γ-tubulin antibody. MW markers are indicated in kDa. (E) RT-qPCR of randomly selected, highly expressed genes, measuring newly synthesized mRNA relative to Pol I transcribed pre-rRNA 45S. The list of the primers is indicated in Supplementary Material, Table S1. For all experiments three biological replicates were carried out per genotype. Error bars represent SEM.

Figure 5.. TFIID assembly is defective in fibroblasts harboring the TAF8: c.781–1G > A mutation. (A–C) Coimmunoprecipitation performed on WCEs prepared from control, or TAF8: c.781-1G > A patient fibroblasts, using either anti-TAF7 and anti-GST (negative control) antibodies (A) or anti-TBP and anti-GST (B) or anti-TAF10 and anti-GST (C). Input extracts or immunopurified complexes were separated by SDS-PAGE and western blot analyses were carried out by probing the blots with antibodies against the indicated TFIID subunits. The anti-GST antibody heavy (IgG H) and light chains (IgG L) are indicated. Ponceau-stained membranes in A–C show equal loading. In A–C the MW markers are indicated in kDa. (D) Mass spectroscopy performed on proteins co-IP-ed with anti-TAF10 antibody, normalized to TAF10 bait protein. Relative NSAFbait was calculated as described previously (52). Error bars represent SEM of two technical replicates. See Supplementary Material, Table S2 for raw mass spectrometry peptide counts.

Figure 6.. Genome wide RNA polymerase II occupancy and transcription is not significantly effected in fibroblasts harboring the TAF8: c.781–1G > A mutation. (A) UCSC genome browser snapshots of randomly selected representative genes showing Pol II occupancy in control fibroblasts (HFF-1, purple) and TAF8: c.781-1G > A patient fibroblasts (red). Arrows at TSSs show the direction of transcription. (B) Heatmap of anti-Pol II ChIP-seq generated by K-means clustering in a window showing TSS to transcription termination site (TTS) of all genes in control and patient fibroblasts. (C) Metagene plots of all expressed Pol II transcribed genes, generated from spike-in normalized anti-Pol II ChIP-seq, on control fibroblasts and TAF8 mutant fibroblasts. Average Pol II profiles were calculated for every expressed refseq gene from –5 kb region upstream of every TSS, including the gene bodies (GBs), until +5 kb downstream from the TTS. The Y-axis shows mean tag/50 bp densities. (D and E) On the basis of the clustering of Pol II occupancy in the gene bodies (Supplementary Material, Fig. S3B) Cluster 1 (D), containing 580 genes (Supplementary Material, Table S3), and Cluster 2 (E), containing 1102 genes…

Figure 7.. Schematic representation of the TFIID composition within WT, Taf8 KO and patient cells. In WT cells holo-TFIID, containing TBP and 13 TAFs, including WT TAF8, is abundant in cells and drives faithful transcription. In Taf8 knockout cells, such as the mESCs described in Figure 4 of this study, loss of TAF8 results in impaired TFIID and no transcription. In contrast, in the patient fibroblasts either only partial TAF assemblies form, or the tiny (undetectable) amount of mutant TAF8 protein integrates into a TFIID-like complex. Either, or both of these complexes could mediate Pol II transcription initiation in patinet fibroblasts.