Fig 2: Weak EM density for some regions of the Drosophila IntS4-IntS9-IntS11 complex (ICM).a Cryo-EM density of the IntS4-IntS9-IntS11 complex low-pass filtered to 7 Å resolution, and viewed after 90° (b) and 180° (c) rotation around the vertical axis. Weak EM density for CTD2 of IntS9 and IntS11 and residues beyond 570 of IntS4 (CTD) becomes more visible at this resolution, but the quality of the density was not sufficient to allow an atomic model to be built for IntS4 CTD. Panels a–c created with Chimera46.

Fig 3: WDR73 interacts with Integrator complex components INTS9 and INTS11. (A) Schematic representation of WDR73 protein with its six predicted WD40 repeats (grey hexagons) and the position of GAMOS-associated mutations described in this article (asterisk). (B) GFP-IP from immortalised podocytes stably expressing either GFP or GFP-WDR73 shows interaction of GFP-WDR73 with endogenous INTS9 and INTS11. (Ci,ii) GFP-IP from HEK293T cells transiently transfected with either GFP-INTS11 (i) or GFP-INTS9 (ii) shows interaction of both tagged proteins with endogenous WDR73. (D) Immunoprecipitation of INTS11 from podocytes stably expressing either GFP or GFP-WDR73 shows that endogenous INTS11 may associate with GFP-WDR73. (E) Immunoprecipitation of endogenous INTS9 from control siRNA treated or WDR73-suppressed podocytes shows that WDR73 suppression has no effect on the ability of INTS9 and INTS11 to associate. Please note WDR73 and actin bands are cropped for clarity, as WDR73 lane shows only contamination from IgG in IP lanes. (F) GFP-IP from HEK293T cells transiently transfected with 2 µg DNA encoding GFP-WDR73, or 10 µg DNA encoding either GFP only or GFP-WDR73 p.256Profs*18 shows that the latter construct is unable to associate with either INTS9 or INTS11. (G) GFP-IP from HEK293T cells transiently transfected with the indicated constructs shows that WDR73 is able to associate with GFP-INTS11 wild type, GFP-INTS11 (1-450) and (1-493). All experiments shown are representative of at least three independent biological repeats.

Fig 4: Reduced globin gene expression and increased LCR HS2 and HS3 eRNA levels in differentiating CD34+ cells deficient for INTs11. (A) RNA sequence reads in the human LCR HS2 and HS3 genomic region. Data were obtained from CD34+ blood progenitor cells from the ENCODE database (ENCSR000CUA,(45). (B) Western blot analysis of INTs11 and β-actin in differentiating CD34+ cells treated with INTs11 siRNA (si-INTs11) or nontargeting control siRNA (si-control) for 72 h. (C) RT-qPCR expression analysis of INTs11, β-globin, γ-globin, α-globin, GATA1 and USF1 genes in differentiating CD34+ cells treated with si-INTs11 or si-control for 72 h. (D) RT-qPCR expression analysis of LCR HS2 and HS3 5′ and 3′ eRNAs in differentiating CD34+ cells treated with si-INTs11 or si-control for 72 hours. The diagram on the left illustrates position of primers used to amply different regions flanking LCR HS2 and HS3. Error bars reflect SEM from three independent experiments (***P < 0.001; **P < 0.01; *P < 0.05). Expression in (C) and (D) was normalized to that of β-actin (Actin).

Fig 5: Depletion of Mediator subunits does not significantly affect MAPK responsiveness. (A) Heat map representing the fold induction of 106 EGF-induced genes in HeLa cells following treatment with shCTRL or shRNA against INTS11, MED1, or MED12. All experiments were performed in at least two biological repeats. Each lane represents the fold ratio of gene expression changes before and after 20 min of EGF stimulation. The heat map is ranked from the highest to the lowest fold induction of EGF-responsive genes. (B,C) EGF-induced gene expression at EGR1, FOSB, and NR4A1 loci did not change by the presence of shRNA control or shRNA against MED1 (B) or MED12 (C), as revealed by ChromRNA-seq. (D) Dot plots represent significant impairments of EGF-induced activation by INTS11 knockdown but not by knockdown of MED1 or MED12. Average expression levels of 106 EGF-induced genes or control genes were measured by fold induction after EGF treatment. (***) P < 0.001 for all comparisons, t-test. (E) Knockdown of MED1 or MED12 does not affect EGF-induced NR4A1 gene activation. The gene transcription level was measured before and after 5, 10, and 20 min of EGF induction using quantitative RT–PCR (qRT–PCR). Shown in the figure is the average from three independent experiments. Error bars represent SEM. (F) Heat map representing the fold induction of 60 serum-induced genes after treatment with shCTRL or shRNA against INTS11 or MED12. Each lane represents the fold ratio of gene expression changes before and after 20 min of serum stimulation. All genes were induced by at least twofold. FPKM > 1; q-value < 0.05. The heat map is ranked from the highest to the lowest fold induction of serum-responsive genes. (G) Serum-induced gene expression at FOSB and NR4A1 loci was reduced by the presence of shRNA against INTS11 or MED12. (H, top) Dot plots showing that loss of INTS11 or MED12 abrogates the serum-induced transcriptional activation on serum-responsive genes. (Bottom) There is no significant change in control genes. The serum-responsive genes are listed in Supplemental Table S5. (***) P < 0.001, t-test.