A yeast display nanobody library contains members that silence a reporter gene in human cells.

(A) Schematic of library enrichment from yeast display and assay in human cells. Top, yeast display: a large naive nanobody library is genetically encoded in yeast, and each yeast cell expresses a single nanobody displayed on its surface. FLAG-tagged chromatin regulator complexes (CR) are overexpressed in human cells, which are then lysed and mixed with nanobody-displaying yeast.The library is enriched through multiple rounds of magnetic pulldown, and the final library is sequenced. DNA is extracted from yeast and directly cloned as fusions to a doxycycline (dox)-inducible rTetR DNA-binding domain. Bottom, HT-recruit: next, human cells encoding a reporter gene with a Tet-responsive element, fluorescent marker, and surface expression marker are infected at low MOI with rTetR-nanobody lentivirus. Cells are binned into silenced (reporter gene off, surface marker not expressed) and non-silenced (reporter gene on) pools by magnetic separation against the surface marker, and nanobody library member frequencies in each pool are computed. (B) Distributions of library member frequencies in the naive yeast library and after each round of selection. X-axis, frequency per 1000 valid sequenced library members, y-axis, count, vertical dashed line, frequency of 1 per 1000 fragments. (C) Flow cytometry distribution of yeast cells bound to FLAG-tagged DNMT3A (stained with FITC-conjugated anti-FLAG antibody) in round 3 (green line) compared to naive (black line). (D) Log2(OFF:ON) (positive = more silenced) enrichment scores plotted for each replicate of the high-throughput nanobody recruitment screen (R1 and R2) in HEK-293A cells on day 5 of dox-mediated recruitment. Stars indicate nanobodies selected for validation. Hits are defined using an enrichment score threshold of 2. (E) Representative flow distributions for selected nanobodies on day 6 of dox recruitment. Grey curves, no dox added; green curves, dox added. (F) Timecourse of dox-mediated gene silencing and epigenetic memory of selected nanobodies, plotted as the normalized fraction of silenced cells (Citrine Off).

Yeast display selects nanobodies that activate silenced genes.

(A) Schematic of silenced reporter and activation screen. Human DNMT3A was fused to rTetR and transfected transiently into HEK-293A cells harboring the TRE-surface marker-citrine reporter while dox was added. Silenced (citrine OFF) cells were sorted and a stable population was maintained. Yeast display was performed using full length human TET1, mouse TET2 and human TET3 to curate a library of nanobodies against TET1/2/3 complexes, which were fused to rTetR, delivered via lentivirus to the silenced cells, and separated into re-activated (bound) and still-silenced (unbound) pools. (B) Flow cytometry measurements of Citrine expression wild-type HEK-293A cells (gray), HEK-293A cells expressing the surface marker-Citrine reporter (blue), and HEK-293A cells expressing the reporter after transient transfection and recruitment of rTetR-DNMT3A (purple). The silenced population (purple curve) was sorted and maintained for screening use. (C) Flow cytometry measurements of the naive library (post-negative selection), round 1-TET enriched, and round 2-TET enriched nanobody-expressing yeast libraries. X-axis, Alexa-647 fluorescence (indicating nanobody expression on the yeast surface); y-axis, Alexa-488 fluorescence from anti-FLAG antibody (indicating yeast binding to FLAG-tagged TETs). (D) Distributions of library member frequencies in the naive library (top) and round 2 yeast display selection against the TETs. X-axis, frequency per 1000 valid sequenced library members; y-axis, count; vertical dashed line, frequency of 1 per 1000 fragments. (E) Log2(ON:OFF) (positive = more activated) scores per replicate of the TET nanobody recruitment screen in HEK-293 cells; nanobodies chosen for follow-up denoted as stars. (F) Flow cytometry Citrine measurements for selected nanobodies and VP64 on day 10 of dox-mediated recruitment. VP64 was chosen as an activating positive control. Grey curves = no dox added, purple curves = dox added. (G) Timecourse of dox-mediated reporter activation starting from day 3 of dox addition, plotted as the fraction of cells that have re-activated (citrine ON). Lines show the average of two biological replicates, dots are individual replicates, and shading shows standard deviation. (H) Measurements of the fraction of cells activated (Citrine ON) upon recruitment of rTetR alone (negative control), VP64 (positive control), TET1-CD (the catalytic domain of TET1 enzyme fused to rTetR), and activating nanobodies #A3 and #A4 to epigenetically silenced promoter RSV. Grey, no dox; purple, dox added. Bars indicate the average of two biological replicates, and error bars show standard deviation.

Details of DNMT3A yeast selection and screen, related to Figure 1.

(A) Individual flow cytometry measurements of selected nanobodies fused to rTetR, as in Fig. 1E, compared to their scores in the high-throughput recruitment assay. X-axis, HT-recruit screen score; y-axis, fraction cells with Citrine OFF as measured by flow cytometry. Error bars indicate standard deviation, and dots represent the average of two biological replicates. (B) Sequences of each of the CDRs (variable regions) of selected hit or non-hit nanobodies, as in Fig. 1D, compared to the consensus sequence as computed by the MEME server. Conserved residues in each CDR are highlighted in yellow.

Details of TET yeast display and recruitment screen, related to Figure 2.

(A) Flow cytometry measurements of DNMT3A-silenced and sorted HEK-293A cells after 5 days recruitment of rTetR only (gray) or rTetR-TET1(CD). (B) Flow cytometry measurements of a negative control, glucose-exposed yeast library. X-axis, Alexa-647 fluorescence (indicating nanobody expression on yeast surface); y-axis, Alexa-488 fluorescence (indicating yeast binding to FLAG-tagged TETs. (C) Flow cytometry Citrine measurements for rTetR only negative control on day 10 of dox recruitment to the epigenetically silenced pEF reporter. Grey curve = no dox added, purple curves = dox added. (D) Measurements of the fraction of cells activated (Citrine ON) upon recruitment of rTetR alone (negative control), VP64 (positive control), TET1-CD (the catalytic domain of TET1 enzyme fused to rTetR), and activating nanobodies #A3 and #A4 to the minimal promoter minCMV. Grey, no dox; purple, dox added. Bars indicate the average of two biological replicates and error bars show standard deviation.