Targeted degradation of transcription factors by TRAFTACs: TRAnscription Factor TArgeting Chimeras

Abstract

Many diseases, including cancer, stem from aberrant activation or overexpression of oncoproteins that are associated with multiple signaling pathways. Although proteins with catalytic activity can be successfully drugged, the majority of other protein families, such as transcription factors, remain intractable due to their lack of ligandable sites. In this study, we report the development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) as a generalizable strategy for targeted transcription factor degradation. We show that TRAFTACs, which consist of a chimeric oligonucleotide that simultaneously binds to the transcription factor of interest (TOI) and to HaloTag-fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. Application of TRAFTACs to two oncogenic TOIs, NF-κB and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins. Thus, TRAFTAC technology is potentially a generalizable strategy to induce degradation of other transcription factors both in vitro and in vivo.

Keywords: E3 ligase; HaloTag; PROTACs; brachyury; dCas9; degradation; proteasome; transcription factors; undruggable; zebrafish.

Figure 1.. Schematic Representation of the TRAnscription Factor TArgeting Chimeras (TRAFTACs).

Heterobifunctional dsDNA/CRISPR-RNA chimera (TRAFTAC) recruits E3 ligase complex through dCas9-HT7 in the presence of haloPROTAC. Heterobifunctional TRAFTAC binds to dCas9-HT7 via its RNA moiety while dsDNA portion of the chimera binds to the transcription factor of interest (TOI). Addition of haloPROTAC recruits VHL E3 ligase complex to the vicinity of TOI. TRAFTAC-mediated proximity induced ubiquitination directs TOI for proteasomal degradation.

Figure 2.. Binding Experiments for NFκB-TRAFTAC, CT-dCas9HT7 and p50.

A) A diagram illustrating the architecture of NF-κB-TRAFTAC and the NF-κB binding kappaB sequence. B) Schematic of bacterial and mammalian expression CT-dCas9HT7 constructs. After bacterial expression, protein was purified by His-tag affinity column purification. While the bacterial expression vector has a His-tag sequence, it was replaced by HA-tag for mammalian expression. C) Different concentrations of purified CT-dCas9HT7 were incubated with NFκB-TRAFTAC and the protein:oligo complexes separated on an agarose gel. Data represent the ability of dCas9HT7 to bind to the modified-double stranded chimeric NFκB-TRAFTAC. D) Immunofluorescence data illustrating in cellulo TRAFTAC engagement with fusion dCas9HT7. Cells were transfected with fluorescein labeled TRAFTAC for 12 h and cells fixed, permeabilized and probed with anti-HA antibody, followed by secondary antibody conjugated to Alexa Fluor 568. Images were captured using a confocal microscope. Scale bar: 25 μm. E) Immunoprecipitation of p50 and ribonucleocomplex (dCas9HT7:NFκB-TRAFTAC). Stable cells lysates with dCas9HT7 were treated with NFκB-TRAFTAC and control-TRAFTAC. After 1 h, dCas9HT7 was immunoprecipitated using HA antibody conjugated beads and eluted samples were probed with antibodies as shown.

Figure 3.. NF-κB Degradation by TRAFTACs.

A) HP14 induces p50 degradation in the presence of TNF alpha. Briefly, cells were transfected with NFκB-TRAFTAC and after 16 h, cells were treated with increasing HP14 concentrations. After 1h, cells were treated with or without TNF-alpha for indicated times. B) TRAFTACs induce p50 degradation within 9 h of HP14/TNF-alpha treatment. C) Chemical structures of HP14/TNF-alpha and the inactive epimer control (HP17). D) VHL and TRAFTAC-dependent p50 degradation. Cells were transfected with either with NFκB-TRAFTAC or control-TRAFTAC followed by the treatment of HP14/TNF-alpha or the HP17/TNF-alpha for 12 h. Cell lysates were probed for p50 and GAPDH. E) TRAFTAC induced p50 degradation is dependent on dCas9HT7. Stable cells overexpressing dCas9HT7 and parental cells were transfected with chimeric oligo and the experiment performed as mentioned above. F) NFκB-TRAFTAC mediated p50 degradation elicit an effect on downstream gene expression. (Not significant (N.S); * P < 0.03; ** p<0.002; *** p<0.0002; **** p<0.0001; n: Independent biological replicates; n=2)

Figure 4.. Positioning of HT7 in dCas9 Governs the HT7 Fusion Protein Degradation by haloPROTACs.

A) HP14 induces degradation of CT-dCas9HT7 fusion protein. The cell line stably overexpressing CT-dCas9HT7 was treated with HP14 (20 μM) and HP17 (20 μM) and lysed at indicated time points. Lysates were probed for HT7 and GAPDH. B) The haloPROTAC HP3 (possessing a shorter linker) did not induce p50 degradation, in contrast to HP14. C) Cells stably expressing NT-dCas9HT7 were treated with HP14 and lysed at indicated time points. D) NFκB-TRAFTAC/HP14/TNF-alpha treated NT-dCas9HT cells were lysed and probed for p50 and tubulin levels.

Figure 5.. TRAFTACs Targeting Brachyury Induces its Degradation.

A) Schematic representation of the brachyury-GFP construct and the brachyury binding DNA sequence. B) Cell lysates that overexpress brachyury-GFP were incubated with the brachyury-TRAFTAC and the inactive control-TRAFTAC before HA immunoprecipitation. Eluted samples were probed against brachyury and HT7 levels. C) Ternary complex formation assay for the dCas9HT7:chimeric oligo and brachyury. Purified proteins were incubated with chimeric oligos and protein:oligo complexes were separated using DNA agarose gel to analyze gel shifts as depicted in the figure. D) Cells stably expressing brachyury-GFP were transfected with brachyury-TRAFTAC or control-TRAFTAC followed by HP14 treatment. Cells were then lysed and analyzed for brachyury, tubulin, c-Myc and HT7 levels. (Not significant (N.S); * P < 0.03; ** p<0.002; n: Independent biological replicates; n=2) E) Time dependent degradation of brachyury-GFP. Cells stably expressing brachyury-GFP were transfected with brachyury-TRAFTAC and treated with HP14 and HP17. Cells were lysed at indicated time points, subjected to western blotting and probed for brachyury, tubulin and HT7. F) Stable cells overexpressing brachyury-GFP were treated with HP14 and MLN4924 for 9 and 16 h. Cells were lysed and probed for brachyury, HT7 and tubulin. G) The brachyury-GFP fluorescence signal was captured after HP14 and MLN4924 co-treatment. Scale bar: 75 μm.

Figure 6.. Endogenous Brachyury Degradation by TRAFTACs.

A) Co-immunoprecipitation of brachyury in the presence and absence of brachyury-TRAFTAC or control-TRAFTAC oligos. Cell lysates were subjected to HA immunoprecipitation and eluted samples were probed for brachyury and HT7. B) Hela cells transiently transfected with NT-dCas9HT7 followed by a second transfection with brachyury-TRAFTAC. Then HP14 and HP17 were treated and cell lysates were prepared after 15 h and probed for brachyury, c-Myc and tubulin. C) Hela cells stably overexpressing NT-dCas9HT7 were transfected with either with brachyury-TRAFTAC or allscrambled-TRAFTAC followed by HP14 or HP17 treatment. Cell lysates were then probed for brachyury and tubulin levels. (Not significant (N.S); * P< 0.03; ** p<0.002; *** p<0.0002; **** p<0.0001; n: Independent biological replicates; n=2)

Figure 7.. Brachyruy-TRAFTAC Induces tail Formation Defects in Zebrafish.

A) Schematic illustration of the experimental work flow. B) Buffer (mock, a), dCas9HT7:brachyury-TRAFTAC:HP14 (b), dCas9HT7:allscrmb-TRAFTAC:HP14 (c), dCas9HT7:brachyury-TRAFTAC:HP17 (d) and dCas9HT7:brachyury-TRAFTAC (e) were introduced to zebrafish via microinjection and embryos were analyzed at 30- and 60-hours post fertilization (hpf). Embryos injected with dCas9HT7:brachyury-TRAFTAC:HP14 displayed significant, severe tail deformation compared to the embryos injected with mock and other controls RbNCs. Scale bar: 250 μm. C) Percentages of number of normal and defective embryos in each group treated with different RbNCs (A-E). (n: Independent biological replicates; n=4, *** p<0.0002; **** p<0.0001 in Two-Way ANOVA; Test: Tukey; confidence level: 95%, number of embryos per group = 30–50) D) A heat map showing the percentages of severely and mildly defective embryos in each study group. Percentages were calculated compare to the total number of embryos within the corresponding group. (n: Independent biological replicates; n=4, *** p<0.0002; **** p<0.0001, 30–50 embryos per group)