Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters

Adam M. Zahm, William S. Owens, Samuel R. Himes, Kathleen E. Rondem, Braden S. Fallon, Alexa N. Gormick, Joshua S. Bloom, Sriram Kosuri, Henry Chan, Justin G. English

Posted on: 21 June 2023

Preprint posted on 11 May 2023

Gaining ground in the development of Synthetic Mammalian Promoters

Selected by Jessica L. Teo

Categories: bioengineering, cell biology, synthetic biology

Background

Given the ability of transcription factors (TF) to bind to unique DNA sequences – broadly termed transcription response elements (TRE) – to regulate gene expression, researchers have hijacked this mechanism to modulate cellular behavior ^1,2. Cells can be engineered to express synthetic promoters carrying TREs where endogenous TFs can bind and drive the expression of user-defined genes. The creation of such cellular systems helps to provide mechanistic insights into cellular behavior and gives one the ability to control cell fate specification and cellular function for therapeutic purposes ^3,4.

Despite its broad application, the use of synthetic promoters is still in its infancy. These promoters are typically developed in-house for specific applications, which involves the time-consuming and laborious process of screening for functional promoters with desired features. These include having a large dynamic range of transcription rates with minimal basal activity, and short length to accommodate vectors with limited cargo sizes. In this new preprint, Zahm A. M. and colleagues tackled this issue by creating a library containing 6184 synthetic promoters carrying candidate TREs that are recognized by 229 human and mouse TFs. This off-the-shelf library can help to accelerate molecular tool development and encourage widespread application for research and drug discovery.

Key findings

Synthetic promoter library assembly

To generate a library to study the effects of TREs and promoter combinations in gene regulation at high throughput, the authors utilized a Massively Parallel Reporter assay (MPRA) approach coupled with DNA barcoding technology. Here, four copies of a transcription factor binding motif (TFBM) were arranged in 6 configurations and placed upstream to one to three minimal promoters. This gave rise to a synthetic promoter. The luciferase gene was added downstream to determine whether the synthetic promoters were active. Following the luciferase gene, a 24-nucleotide barcode was introduced to provide an estimate of transcription rates using Next-Generation Sequencing (NGS) (Figure 1B). The authors determined baseline transcriptional activity by expressing their synthetic promoter library in human HEK293 cells and quantifying barcoded mRNA using NGS. While the synthetic promoters showed a large dynamic range of estimated baseline transcriptional rates, this effect was dependent on the minimal promoter paired with the TRE unit – highlighting the importance of identifying optimal synthetic promoters best suited to address specific research questions.

Identification of synthetic promoters activated by endogenous cellular cues

After validating their synthetic promoter library, the authors went on to identify synthetic promoters that were activated by extrinsic cues using both human HEK293 and mouse Neuro2a cells. Amongst the ten extrinsic conditions were mitogens and factors that induced the cellular stress response. Here, the authors were able to detect changes in transcription in more than 70% of the synthetic promoters in at least one condition relative to negative controls. Importantly, the authors noted significant differences in baseline transcription rates and response to extrinsic stimulus between human HEK293 and mouse Neuro2a cells, indicating the presence of tissue- and species-specific effects on driving transcription.

Figure 1. Overview of TRE-MPRA library setup and application. Figure 1 in Zahm A. M. et al.

Identification of synthetic promoters responsive to GPCR activation

G protein-coupled receptors (GPCR) govern a broad spectrum of physiological functions through G proteins and they remain a key target in drug discovery and development ⁵. Strikingly, only a limited number of TREs have been used as readouts for GPCR signaling. This can be attributed to a lack of specificity of existing TREs as readouts for unique GPCR-G protein coupling. To tackle this issue, the authors activated GPCRs in HEK293 cells transfected with the TRE-MPRA library. Here, GPCRs were activated using two independent approaches. In the first approach, one of three human GPCRs was overexpressed in HEK293 cells before activating them using receptor agonists. In the second, cells were treated with epinephrine to activate the endogenous GPCR, ADRB2. Interestingly, distinct groups of promoters that respond selectively to G proteins G_s, G_q, and G_i were identified. Furthermore, increases in transcription rates from promoters indicate that the platform is sufficiently sensitive to detect endogenous GPCR signaling upon receptor activation.

What I like about this preprint

In this study, Zahm A. M. et al. developed an extensive massively parallel reporter assay (MPRA) library that contains 6184 synthetic promoters. This library aims to expedite the process of screening for functional promoters with desired features for specific applications at a large scale. I’m inspired by the methodical work that was carried out by the authors and excited about the utility of this toolkit that they have contributed to the scientific community.

Questions for the author

Given the sensitivity of this platform, I’m curious if the library would be able to report changes in transcription factor binding kinetics.
The use of this library to identify synthetic promoters that respond to distinct cellular stimuli is very exciting! I wonder if there are orthogonal strategies one could use to modulate groups of promoters independent of a native stimulus to test if cellular responses are recapitulated similar to when cells are treated with a native cue?

References:

Lambert, S.A., Jolma, A., Campitelli, L.F., Das, P.K., Yin, Y., Albu, M., Chen, X., Taipale, J., Hughes, T.R., and Weirauch, M.T. (2018). The Human Transcription Factors. Cell 172, 650-665. 10.1016/j.cell.2018.01.029.
Li, J.W., Zhang, X.Y., Wu, H., and Bai, Y.P. (2020). Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review. Front Bioeng Biotechnol 8, 98. 10.3389/fbioe.2020.00098.
Li, H.S., Israni, D.V., Gagnon, K.A., Gan, K.A., Raymond, M.H., Sander, J.D., Roybal, K.T., Joung, J.K., Wong, W.W., and Khalil, A.S. (2022). Multidimensional control of therapeutic human cell function with synthetic gene circuits. Science 378, 1227-1234. 10.1126/science.ade0156.
Saxena, P., Heng, B.C., Bai, P., Folcher, M., Zulewski, H., and Fussenegger, M. (2016). A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells. Nat Commun 7, 11247. 10.1038/ncomms11247.
Hauser, A.S., Chavali, S., Masuho, I., Jahn, L.J., Martemyanov, K.A., Gloriam, D.E., and Babu, M.M. (2018). Pharmacogenomics of GPCR Drug Targets. Cell 172, 41-54 e19. 10.1016/j.cell.2017.11.033.

doi: https://doi.org/10.1242/prelights.34912

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The author team shared

Given the sensitivity of this platform, I’m curious if the library would be able to report changes in transcription factor binding kinetics.

This may be difficult for several reasons, but perhaps most important is binding partner identity. We want to caution users not to directly assign the identity of the transcription factor(s) bound to the synthetic promoter sequences based on their naming nomenclature. The names of these constructs are based on the transcription factor binding motifs (TFBMs) with the highest affinity to a specific transcription factor in Jolma et al. 2013, however many of these sequences appear in the binding recognition sequences of other transcription factors. In addition multiple transcription factors may be binding to a synthetic promoter simultaneously. For these reasons we believe one could measure the kinetics of transcription activation from these reporters, but it would be challenging without significant validation of transcription factor binding identity to use these constructs to measure specific TF binding kinetics.

The use of this library to identify synthetic promoters that respond to distinct cellular stimuli is very exciting! I wonder if there are orthogonal strategies one could use to modulate groups of promoters independent of a native stimulus to test if cellular responses are recapitulated similar to when cells are treated with a native cue?

Our system relies on the endogenous transcription factors in the cell. As such activation by truly orthogonal, non-native stimuli and pathways would require synthetic transcription factors. However, one could add artificial signaling proteins such as DREADDs, optogenetic channels, photoswitchable G-proteins, or chemogenetically activated kinases to probe the direct activity of these signaling proteins in the absence of an endogenously distinct stimulus.