Fig 2: DS and FXS share specific protein-level perturbations in hPSC models(A) Western blot of FMRP using in-vitro-derived glutamatergic neurons from the indicated cell lines confirming FMR1 genotypes. A blot for the GAPDH loading control is shown below FMRP. As expected, neurons generated from the FXS patient iPSC lines and FMR1y/- line lacked FMRP expression.(B) Cytogenic analysis of G-banded metaphase cells shows expected 46, XY karyotype for the DS2U euploid control iPSC line and 47, XY, +21 karyotypes for DS1 and 2DS3 patient iPSC lines. The blue arrow indicates HSA21.(C and D) Quantification of western blots performed in triplicate for CBS (C) and NCAM2 (D), using the three control cell lines, four FXS cell lines, and two DS cell lines shown in (A). An example blot is shown beneath the quantification.(E) Quantification of western blots performed in triplicate for NCAM2 using isogenic FMR1y/+ and FMR1y/- cell lines extracted from the dataset shown in (D).(F and G) Quantification of western blots performed in triplicate for APP (F) and DYRK1A (G) using the three control cell lines, four FXS cell lines, and two DS cell lines shown in (A). An example blot is shown beneath the quantification. For western blot quantifications, error bars show SEM and significance between control and disease samples was calculated by unpaired two-tailed t test. Significance is indicated by *p = 0.05, **p = 0.005, and ***p = 0.0005 relative to controls. See also Figure S1 and Tables S1 and S2.

Fig 3: DYRK1A–DCAF7 interaction is essential for efficient expression of key myogenic genes. (A) The DYRK1A KD C2C12 cells were transfected with an empty vector (-) or the vector expressing WT or ?84–95 F-DYRK1A and then analyzed by WB for the indicated proteins. A non-specific band is denoted with an asterisk. (B–D) The cells in A were cultured in GM or DM for 3 days and the mRNAs produced from the indicated genes were detected by qRT-PCR and shown. (E) WB was used to assess the levels of endogenous DYRK1A in normal C2C12 cells and the overexpressed ?84–95 F-DYRK1A in transfected cells. (F–H) The cells in E were grown in GM and DM for 3 days and the mRNAs produced from the indicated genes were detected by qRT-PCR and shown. All error bars represent mean +/- SD from three independent measurements. The asterisks indicate different levels of statistical significance as calculated by two-tailed Student's t-test. ***P < 0.001; **P < 0.01; *P < 0.05.

Fig 4: DYRK1A and DCAF7 are required for optimal myoblast differentiation and expression of key myogenic genes. (A) The levels of the indicated proteins in 1 × 105 C2C12 cells grown in the growth medium (GM) or differentiation medium for 1-7 days (DM1-7) were examined by WB. (B, C) The mRNA levels of the indicated genes in C2C12 cells cultured in GM or DM1-7 were measured by qRT-PCR and shown. (D) Extracts of C2C12 cells grown in GM, DM5, or containing an equal mixture of the two populations (M) were subjected to IP with the anti-DCAF7 Ab or a non-specific control IgG. The precipitates were analyzed by WB for the indicated proteins. (E, F) C2C12 cells stably expressing the indicated shRNAs were cultured in GM for 5 days and subjected to immunofluorescence staining with the mouse anti-MYH2 mAb plus goat anti-mouse AF680 (green). Nuclei are counterstained with DAPI (blue). Scale bars represent 100 μm. (G, H) The percentages of MYH(+) C2C12 cells expressing the indicated shRNAs were counted and shown. (I,J). The expression of the proteins marked on the left in cells expressing the indicated shRNAs and grown in GM or DM for 5 days was detected by WB. (K, L) The MYOG promoter-driven luciferase reporter construct was co-transfected with the indicated shRNA- and/or the MYOD-expressing plasmids. After 48 hr of transfection, luciferase activities were measured in the cell extracts. All error bars represent mean ± SD from at least three independent experiments/measurements. The asterisks indicate different levels of statistical significance as calculated by two-tailed Student's t-test. ***P < 0.001; **P < 0.01; *P < 0.05.

Fig 5: DYRK1A–DCAF7 complex exerts direct transcriptional control of key myogenic genes through binding and phosphorylating Pol II at the gene loci. (A, B) ChIP-qPCR analyses were conducted to assess the occupancy of DCAF7 (A) and DYRK1A (B) at the indicated gene promoters in C2C12 cells cultured in GM or DM for 3 days. The ChIP-qPCR signals were normalized to the input DNA, divided by the values obtained with the rabbit total IgG in control reactions and shown. (C, D) C2C12 cells stably expressing the indicated shRNAs were grown in DM for 3 days and then analyzed by ChIP-qPCR to detect the occupancy of DYRK1A (C), total Pol II (D), and the Ser5- or Ser2-phosphorylated Pol II (pSer5 or pSer2; D) at the indicated gene loci. Rabbit total IgG was used as a negative control. (E) A diagram showing the positions of the primer sets used in ChIP-qPCR reactions in F-I to map the bindings across the MYH2 gene locus. TSS: transcription start site. The vertical lines/blocks represent exons. (F–I) C2C12 cells in DM for 3 days were subjected to ChIP-qPCR analysis using the primer sets in E to detect the bindings of DYRK1A (F), DCAF7 (G), total Pol II (H) and BRD4 (I) to the various positions (a to g) across the MYH2 gene locus. The ChIP-qPCR signals were normalized to the input DNA and divided by those obtained with the rabbit total IgG in control reactions. All error bars represent mean ± SD from three independent reactions. The asterisks indicate different levels of statistical significance as calculated by two-tailed Student's t-test. ***P < 0.001; **P < 0.01; *P < 0.05.