Transcription factor interactions explain the context-dependent activity of CRX binding sites

Loell, Kaiser; Friedman, Ryan Z.; Myers, Connie A.; Corbo, Joseph C.; Cohen, Barak A.; White, Michael A.

doi:10.1101/2023.03.05.531194

Cited by 3 publications

(11 citation statements)

References 72 publications

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“…The contribution of the CRX motifs was calculated by comparing the difference in predicted activities of the background sequences versus the motif-containing sequences. The model predicted the known repressive effects of multiple CRX sites previously observed in both genomic and synthetic CREs (16,87).…”

Section: The Model Learned Relative Affinities and Known Contextual F...mentioning

confidence: 89%

Active learning of enhancer and silencer regulatory grammar in photoreceptors

Friedman,

Ramu,

Lichtarge

et al. 2023

Preprint

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Cis-regulatory elements (CREs) direct gene expression in health and disease, and models that can accurately predict their activities from DNA sequences are crucial for biomedicine. Deep learning represents one emerging strategy to model the regulatory grammar that relates CRE sequence to function. However, these models require training data on a scale that exceeds the number of CREs in the genome. We address this problem using active machine learning to iteratively train models on multiple rounds of synthetic DNA sequences assayed in live mammalian retinas. During each round of training the model actively selects sequence perturbations to assay, thereby efficiently generating informative training data. We iteratively trained a model that predicts the activities of sequences containing binding motifs for the photoreceptor transcription factor Cone-rod homeobox (CRX) using an order of magnitude less training data than current approaches. The model’s internal confidence estimates of its predictions are reliable guides for designing sequences with high activity. The model correctly identified critical sequence differences between active and inactive sequences with nearly identical transcription factor binding sites, and revealed order and spacing preferences for combinations of motifs. Our results establish active learning as an effective method to train accurate deep learning models ofcis-regulatory function after exhausting naturally occurring training examples in the genome.

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Section: The Model Learned Relative Affinities and Known Contextual F...mentioning

confidence: 89%

Active learning of enhancer and silencer regulatory grammar in photoreceptors

Friedman,

Ramu,

Lichtarge

et al. 2023

Preprint

Self Cite

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“…Results of prior MPRA studies show that whether CRX acts as an activator or repressor at a particular CRE depends on local motif composition (White et al 2013; White et al 2016; Hughes et al 2018; Friedman et al 2021; Loell et al 2023). We previously reported that CRX-bound silencers tend to have more copies of the CRX motif than enhancers do (White et al 2016; Friedman et al 2021), while enhancers contain more motifs for other photoreceptor TFs (Friedman et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

“…This model, trained on MPRA data from synthetic CREs with different combinations of photoreceptor TF binding sites, indicated that homotypic interactions between multiple copies of the CRX binding site were necessary to create repressive CREs. While the biophysical nature of these homotypic interactions is unknown, there is clear evidence that the presence of multiple copies of the CRX motif often leads to repression in both genomic and synthetic CREs (White et al 2016; Friedman et al 2021; Loell et al 2023).…”

Section: Discussionmentioning

confidence: 99%

“…High CRX occupancy at silencers may be promoted by homotypic interactions mediated by the transcriptional effector domain, which is necessary for repression. We recently presented a neural network model of CRX-directed cis -regulation that supports a role for homotypic interactions (Loell et al 2023). This model, trained on MPRA data from synthetic CREs with different combinations of photoreceptor TF binding sites, indicated that homotypic interactions between multiple copies of the CRX binding site were necessary to create repressive CREs.…”

Section: Discussionmentioning

confidence: 99%

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Pathogenic variants in Crx have distinct cis-regulatory effects on enhancers and silencers in photoreceptors

Shepherdson

Friedman

Zheng

et al. 2023

Preprint

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Dozens of variants in the photoreceptor-specific transcription factor (TF) CRX are linked with different human blinding diseases that vary in their severity and age of onset. How different variants in a single TF cause a range of pathological phenotypes is not understood. We deployed massively parallel reporter assays (MPRAs) to measure changes to CRX cis-regulatory function in live mouse retinas carrying knock-ins of two phenotypically distinct human disease-causing Crx variants, one in the DNA binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). We found that the effects of CRX variants on global cis-regulatory activity patterns correspond with the severity of their phenotypes. The variants affect similar sets of enhancers but to different degrees. A subset of silencers were converted to enhancers in retinas lacking a functional CRX effector domain, but were unaffected by p.R90W. Episomal MPRA activities of CRX-bound sequences showed some correspondence with chromatin environments at their original genomic loci, including an enrichment of silencers and depletion of strong enhancers among distal elements whose accessibility increases later in retinal development. Many distal silencers were de-repressed by p.E168d2, but not by p.R90W, suggesting that loss of developmentally timed silencing caused by p.E168d2 may contribute to phenotypic differences between the two variants. Our findings indicate that phenotypically distinct disease variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to mis-regulation of similar sets of enhancers, while having a qualitatively different impact on silencers.

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“…One reason for this is that different regulatory proteins can compete in their binding to shared motifs 13,14 . Another reason is that gene expression can be silenced when regulatory DNA contains multiple motifs for the same TF 15,16–18 . For example, in mice, retinal “silencers” contains clusters of motifs for the TF CRX, while retinal “enhancers” contain a mixture of CRX and other TF motifs 16 .…”

Section: Introductionmentioning

confidence: 99%

The emergence and evolution of gene expression in genome regions replete with regulatory motifs

Fuqua,

Sun,

Wagner

2024

Preprint

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Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. Prokaryotic gene expression requires a stretch of regulatory DNA called a promoter, which contains two promoter motifs called -10 and -35 boxes. However, these motifs may occur in both promoters and non-regulatory DNA in multiple copies. It is possible that these motifs influence both the evolution and emergence of regulatory DNA, but this has never been thoroughly tested. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 25 DNA sequences enriched with -10 and -35 box motifs (promoter islands), mutagenize the sequences, and measure gene expression driven by 240’000 mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, but is not correlated with the number of promoter motifs. In most mutants (∼67%), the weakening, strengthening, or emergence of a promoter is caused by mutations that modify already existing motifs. Notably, creating new motifs can either increase or decrease promoter activity. Our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de-novo genes, and phenotypic evolution.

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Transcription factor interactions explain the context-dependent activity of CRX binding sites

Cited by 3 publications

References 72 publications

Active learning of enhancer and silencer regulatory grammar in photoreceptors

Active learning of enhancer and silencer regulatory grammar in photoreceptors

Pathogenic variants in Crx have distinct cis-regulatory effects on enhancers and silencers in photoreceptors

The emergence and evolution of gene expression in genome regions replete with regulatory motifs

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