Figure 2.

ADNP Guides TFIIIC Selective Recruitment to AEs and TFIIIC-Associated Promoters

(A) Area plot ranking unique spectral counts (average of three replicates) of proteins identified in a TFIIIC-RIME experiment using GTF3C2 as bait in T47D cells grown in the presence (+S) or absence of serum (–S). Data from control immunoprecipitations using normal IgG in the presence of serum are also included (blue). Prey proteins belonging to the ChAHP complex are shown in purple.

(B) Direct interaction of recombinant ADNP and TFIIIC. For TFIIIC expression, insect cells were infected with a single baculovirus expressing GTF3C1-3XFLAG and the rest of the TFIIIC subunits (GTF3C2-6) untagged. Left panel: Coomassie staining of anti-FLAG immuno-purifications of insect cell lysates overexpressing FLAG-TFIIIC (lane 1), HA-ADNP (lane 2), or both simultaneously (lane 3). Note that GTF3C1 immunopurification via the FLAG-tag pulled down the whole TFIIIC complex (lane 1). ADNP co-purifies with TFIIIC (lane 3). GTF3C5 fractionates on top of the heavy IgG chain (IgG_H). Right panel: Coomassie staining of anti-HA immunopurification of lysates expressing FLAG-TFIIIC alone (lane 4), HA-ADNP alone (lane 5), or both together (lane 6). The presence of the TFIIIC subunits is detected in the ADNP immuno-complexes. The position of the IgG heavy and light chains (IgG_H and IgG_L, respectively) is indicated. The identity of the bands was confirmed by mass spectrometry and western blotting with HA and GTF3C2 antibodies (Figure S2A).

(C) Heatmap of ADNP-eGFP binding in K562 cells (GEO: GSE105573) spanning ±1 kb across all TFIIIC-bound AEs in T47D, ranked from high to low ADNP-eGFP enrichment. Color bar scale with increasing shades of color stands for higher enrichment (–log10 of the Poisson p value).

(D) Venn diagram with the number of TFIIIC peaks upon ADNP depletion (siADNP, yellow) compared to control cells (siCTRL, blue) in serum-starved T47D cells.

(E) Sitepro profile of TFIIIC occupancy in control (siCTRL) and ADNP-depleted cells (siADNP) in serum-starved T47D cells. The graphs are plotted over the summit of TFIIIC peaks in the siCTRL cells (p value, two-tailed unpaired t test). ADNP depletion levels are shown in Figures S2E and S2F.

(F) Genome browser view of representative TFIIIC-associated genes FEM1A and HELLS showing TFIIIC occupancy in ADNP-depleted (siADNP) and T47D control cells (siCTRL). ADNP binding from GEO: GSE105573 is represented by the blue track (eGFP-ADNP). The gene structure and the direction of transcription (arrow) are shown at the bottom.