Programmable pathway profiles reveal signaling principles of TGF-β superfamily receptors

Background:

The TGF-β superfamily is an intercellular signalling pathway whose many ligands and receptors control context-dependent processes such as germ-layer specification, tissue patterning and disease progression. This pathway consists of more than 30 ligands that signal through complexes assembled from type I and type II receptors (Massagué, 2012). It has been canonically divided into two branches, triggered by BMP and TGF-β ligands, that respectively signal through distinct receptors and SMAD effectors. These branches nevertheless share components, including ACVR-class receptors and SMAD4, and can interact antagonistically or synergistically depending on context (Schier and Talbot, 2005, Lee et al., 2007).

Given the high number of signalling components, understanding how this context-dependency is encoded through traditional single gene perturbations has been challenging. To investigate how multiple receptors interact to shape the TGF-β pathway response, the authors of this preprint developed Pathway Sculptor, a multiplexed CRISPR-interference platform that can achieve same-cell knockdown of at least 12 target genes. By applying it to perturb the expression of receptor subgroups in the TGF-β pathway, they uncovered intriguing crosstalk between the two distinct signalling branches.

Key Findings:

1. Pathway Sculptor enables potent, high-order repression of receptor profiles

To enable multiplexed gene inhibition, the authors optimised a design based on a dead Cas12a variant that can autonomously process arrays of CRISPR RNAs (crRNAs) fused to an epigenetic silencing domain. The design optimisation process included testing multiple dCas12a variants, silencing domains and various configurations of long crRNA arrays to find the configuration that best supports robust concomitant inhibition of multiple genes. To test the selectivity and efficacy of this method, they targeted a panel of surface proteins, whose expression can be readily quantified by Flow Cytometry. They demonstrated that their optimised system supports selective and efficient repression of multiple genes in multiple cell lines, including HEK cells and primary bone marrow cells.

2. Pathway Sculptor provides evidence that BMP and TGF-β branches behave as a cooperatively coupled receptor network

The authors were able to generate cell lines with inducible inhibition of curated lists of TGF-β receptor subtypes: 7 BMP receptors, 5 TGF-β receptors and 6 ACVR-class receptors. Surprisingly, the authors found that depleting BMP-associated receptors not only predictably eliminated responses to BMP ligands, but also significantly reduced activity of the TGF-β-responsive reporter to TGF-β-family ligands. Conversely, removing TGF-β-associated receptors substantially reduced activity of the BMP reporter to BMP ligands. This suggests that intra-branch signalling somehow depends on the presence of receptors of the other branch. Quantitative decomposition of the signalling suggested that the interactions between the two branches are synergistic, as the resulting signalling strength in the presence of all receptors amounted to more than the sum of its parts. In line with this potential crosstalk, BMP ligands weakly activated TGF-β receptors, although the effect of TGF-β ligands on BMP receptors was more complex. Generally, the authors demonstrate the existence of positive and negative interactions between the two branches of the TGF-β pathway.

3. ACVR and branch-specific receptors perform different information-processing functions

The authors suggested a division of labor between receptor classes in HEK293-based assays. As ACVR receptors had previously been implicated in chimeric receptor complexes that might mediate crosstalk between the signalling branches, they selectively inhibited these receptors. Removing ACVRs largely preserved the relative distinguishability of ligand-response patterns, despite reducing their amplitudes. In contrast, cells retaining only ACVRs but no BMP and TGF-β receptors showed decreased ability to respond differently to distinct ligands: Activin B became more BMP-like, and TGF-β1 no longer grouped clearly with either ligand family. The authors therefore characterise ACVRs as more “generalist”, setting signalling magnitude and inter-branch connectivity, whereas BMPRs and TGFBRs act as “specialists” that preserve ligand and branch identity.

Why we chose this preprint:

Developmental signalling pathways are incredibly complex. When working with stem cells and organoids, we oftentimes find ourselves thinking about pathways mainly in terms of ligand concentrations or exposure durations. Yet, receptors are just as important, since they mediate cell type-specific responses depending on the combination of subtypes expressed. This is why we were particularly excited by the development of a novel method to repress a customisable panel of receptor subsets within a given cell. This will enable the understanding of cooperative and competitive interactions that determine signal processing of complex signalling pathways. More broadly, we are excited by the possibilities of using Pathway Sculptor to systematically investigate multiplexed ligand-receptor interactions for better understanding cell type-specific responses to signalling cues.

Questions for the authors:

1. How universal do you expect the ACVR “generalist” and BMPR/TGFBR “specialist” division of labor to be across further cell types and differentiating tissues?
2. Beyond canonical BMP and TGF-β reporters, do you think activation of distinct SMAD subsets could lead to specific activation of more combinatorial genetic elements?
3. Could Pathway Sculptor be combined with an orthogonal multiplexed CRISPRa system to increase expression of selected receptors while repressing others, thereby reconstructing naturally occurring or user-defined receptor subsets and investigate their response to specific signalling cues?
4. Do you envision a way to use Pathway Sculptor in a library-based assay to systematically map ligand-receptor interactions in any given signalling pathway?

References:

Massagué, J. (2012). TGFβ signalling in context. Nat. Rev. Mol. Cell Biol. 13, 616–630.

Schier, A.F., and Talbot, W.S. (2005). Molecular genetics of axis formation in zebrafish. Annu. Rev. Genet. 39, 561–613.

Lee, K.B., Khivansara, V., Santos, M.M., Lamba, P., Yuen, T., Sealfon, S.C., and Bernard, D.J. (2007). Bone morphogenetic protein 2 and activin A synergistically stimulate follicle- stimulating hormone beta subunit transcription. J. Mol. Endocrinol. 38, 315–330.

Tags: crispr, signal processing, signalling, tgf beta