Figure 6. Tiling screen discovers compact repressor domains within nuclear proteins.

A. Tiling library covering 238 nuclear proteins (15,737 elements). These tiles were fused to rTetR and tested with HT-recruit as in Figure 1A.

B. Genes ranked by maximum repressor strength. Dots=tiles. Hit threshold is log2(OFF:ON) ≥ 2 S.D. above the mean of the negative controls. Genes with a hit (gradient) and genes no hit (grey) are divided by vertical line..

C. Tiling CTCF. Protein annotations from UniProt. Horizontal bars show the tile span and vertical error bars show the S.E. from 2 biological replicates. The strongest hit tile is highlighted with a vertical gradient and annotated as a repressor (orange). D. Tiling BAZ2A (also known as TIP5).

E. Individual lentiviral rTetR(SE-G72P)-tile fusions were delivered to reporter cells, cells were treated with 100 ng/ml dox for 5 days, and then dox was removed. Cells were analyzed by flow cytometry, the fraction of cells with citrine reporter OFF was determined and the data fit with the gene silencing model (Methods) (N=2 biological replicates). KRAB repressor domains are positive controls. Tiling data corresponding to the validations shown on the right (blue) is in Figure S6.

F. Tiling MGA. Two repressor domains are found outside the previously annotated regions and labeled as Repressor 1 and 2 (dark red, purple). The minimized repressor sequences at the overlap of hit tiles are highlighted with narrow red vertical gradients.

G. The maximal strength repressor tiles from two peaks in MGA were individually validated as in (E).

H. MGA repressor 1 was minimized by selecting the region shared between all hit tiles in the peak (red shade between vertical lines). Below, the sequence conservation ConSurf score is shown (orange line) with the confidence interval (the 25th and 75th percentiles of the inferred evolutionary rate distribution, grey). Asterisks=residues Consurf predicts are functional (Methods).

I. The MGA effectors were minimized to 10 and 30 AA sub-tiles, as shown in (H), cloned as lentiviral rTetR(SE-G72P)-tile fusions, and delivered to reporter cells. Cells were treated with 100 or 1000 ng/ml dox for 5 days and the percentages of cells with the reporter silenced were measured by flow cytometry (N=2 biological replicates).