Comprehensive Analysis of a cis-Regulatory Region Reveals Pleiotropy in Enhancer Function

Noon, Ella Preger-Ben; Sabarís, Gonzalo; Ortiz, Daniela P.; Sager, Jonathan; Liebowitz, Anna; Stern, David L.; Frankel, Nicolás

doi:10.1016/j.celrep.2018.02.073

Cited by 85 publications

(82 citation statements)

References 35 publications

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“…Therefore, we tested the capacity of a DNA sequence containing the full svb cis -regulatory region to rescue the described molecular and developmental defects of the Df(X)svb 108 allele. For this purpose, we used a transgenic fly line, where a bacterial artificial chromosome (BAC) carrying the complete cis -regulatory region of svb (Preger-Ben Noon et al, 2018a) was integrated into chromosome 2. To exclude svb mRNA from effecting the rescue, this svbBAC construct drives a dsRed reporter gene instead of another copy of svb .…”

Section: Resultsmentioning

confidence: 99%

“…All fly strains used have been previously described: DG3-lacZ (Tsai et al, 2017); ubx1 (Crocker et al, 2015a); HS::ubx-1 : (Crocker et al, 2015a); Df(X)svb 108 (Frankel et al, 2010); svbBAC-dsRed (Preger-Ben Noon et al, 2018a); diBAC-gfp is CH322-35A16 EGFP tagged in VK37, covering D (Venken et al, 2009). Unless otherwise noted, they are generated from w 1118 stock, which is referred to as wild-type.…”

Section: Methodsmentioning

confidence: 99%

“…The fly line containing diBAC-gfp (CH322-35A16 EGFP) was a gift from Schulze, Karen Lynn & Bellen, Hugo J. The Df(X)svb 108 flies were a gift from Stern DL and the svb BAC flies were a gift from Preger Ben-Noon E and Frankel N (Preger-Ben Noon et al, 2018b). We thank Rafael Galupa and Nicolas Frankel for suggestions and discussions.…”

Section: Acknowledgementsmentioning

confidence: 99%

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Multi-enhancer transcriptional hubs confer phenotypic robustness

Tsai¹,

Alves

Crocker³

2019

Preprint

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9We had previously shown in Drosophila melanogaster embryos that low-affinity Ultrabithorax 10 (Ubx)-responsive shavenbaby (svb) enhancers drive robust expression using localized 11 transcriptional environments and that active svb enhancers tended to colocalize, even when placed 12 on different chromosomes (Tsai et al., 2017). Here, we test the hypothesis that these multi-13 enhancer "hubs" improve robustness by increasing transcription factor retention near transcription 14 sites. Deleting a redundant enhancer from the svb locus led to reduced trichome numbers in 15 embryos raised at elevated temperatures. Using high-resolution fluorescence microscopy, we 16 observed lower Ubx concentration and transcriptional output in this deletion allele. Transcription 17 sites of the full svb cis-regulatory region inserted into a different chromosome colocalized with the 18 svb locus, increasing Ubx concentration, the transcriptional output of svb, and partially rescuing 19 the phenotype. Thus, multiple enhancers could reinforce a local transcriptional hub to buffer 20 against environmental stresses and genetic perturbations, providing a mechanism for phenotypical 21 robustness. 22 Impact statement 23Multiple enhancers in physical proximity can reinforce shared transcriptional "hubs" to retain 24 transcription factors, providing a buffer during environmental stresses and genetic perturbations 25 to preserve phenotypic robustness. 30During embryogenesis, transcriptional regulation controls the expression of genes that 31 determine the fate of specific cells, ultimately leading to the correct patterning of the animal body 32 plan (Long et al., 2016; Mallo and Alonso, 2013; Reiter et al., 2017; Spitz and Furlong, 2012). 33 This involves coordinating a complex series of interactions between transcription factors and their 34 target binding sites on DNA, leading to the recruitment or exclusion of active RNA polymerases, 35 which determines the transcriptional state of the gene. Live imaging experiments have shown that 36 73 microenvironment, here we examined the transcriptional robustness of the svb locus to 74 temperature-induced stress in flies harboring the wild-type allele or one containing a deletion 75 (Df(X)svb 108 ) that includes the ventral svb enhancer DG3. When embryos were raised at high 76 temperatures, we observed phenotypical defects in ventral trichome formation for the DG3-77 3 deletion svb allele but not for the wild-type. At the molecular level, Ubx concentrations around 78 transcription sites of the DG3-deletion allele decreased. The transcriptional output of svb without 79 DG3 also decreased. To test the hypothesis that shared microenvironments modulate 80 transcriptional output and provide buffering under stress, we sought to rescue the DG3-deletion 81 allele through inserting the complete svb cis-regulatory region on a BAC (svbBAC) on a different 82 chromosome. We observed that Ubx concentration around active transcription sites of the DG3-83 deletion allele and their transcriptional output increased whe...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Multi-enhancer transcriptional hubs confer phenotypic robustness

Tsai¹,

Alves

Crocker³

2019

Preprint

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“…Surprisingly, Enh-89, a k-Cluster-6 element accessible from the epiblast stage, is active at 8ss, predominantly in the hindbrain (Fig. 4I), providing further evidence of the role of enhancer pleiotropy (Preger-Ben Noon et al, 2018) in NC gene regulation. Conversely, enh-87, a k-Cluster-3 element, is active in a complimentary domain (anterior cranial region, Fig.…”

Section: Dynamics Of Cis-regulatory Landscapesmentioning

confidence: 71%

Reconstruction of the global neural crest gene regulatory networkin vivo

Williams

Candido-Ferreira

Repapi

et al. 2018

Preprint

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Precise control of developmental processes is encoded in the genome in the form of gene regulatory networks (GRNs). Such multi-factorial systems are difficult to decode in vertebrates owing to their complex gene hierarchies and transient dynamic molecular interactions. Here we present a genome-wide in vivo reconstruction of the GRN underlying development of neural crest (NC), an emblematic embryonic multipotent cell population. By coupling NC-specific epigenomic and single-cell transcriptome profiling with genome/epigenome engineering in vivo, we identify multiple regulatory layers governing NC ontogeny, including NC-specific enhancers and super-enhancers, novel trans-factors and cis-signatures. Assembling the NC regulome has allowed the comprehensive reverse engineering of the NC-GRN at unprecedented resolution. Furthermore, identification and dissection of divergent upstream combinatorial regulatory codes has afforded new insights into opposing gene circuits that define canonical and neural NC fates. Our integrated approach, allowing dissection of cell-type-specific regulatory circuits in vivo, has broad implications for GRN discovery and investigation.

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“…Our lack of understanding is highlighted by the fact that even the most well-studied developmental enhancers cannot yet be built synthetically 7,8 . Furthermore, while we now have many examples of how individual nucleotide changes have altered enhancer function during evolution [9][10][11][12][13][14][15] , we have almost no understanding of what kinds of mutational changes are evolutionarily accessible or how the distribution of transcription factor binding sites may constrain enhancer evolvability.…”

Section: Introductionmentioning

confidence: 99%

Dense encoding of developmental regulatory information may constrain evolvability

Fuqua

Jordan

Breugel

et al. 2020

Preprint

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Gene regulatory changes underlie much of phenotypic evolution. However, the evolutionary potential of regulatory evolution is unknown, because most evidence comes from either natural variation or limited experimental perturbations. Surveying an unbiased mutation library for a developmental enhancer in Drosophila melanogaster using an automated robotics pipeline, we found that most mutations alter gene expression. Our results suggest that regulatory information is distributed throughout most of a developmental enhancer and that parameters of gene expression-levels, location, and state-are convolved. The widespread pleiotropic effects of most mutations and the codependency of outputs may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse drosophilids reveal mainly stasis and apparent biases in the phenotypes influenced by this enhancer. Developmental enhancers may encode a much higher density of regulatory information than has been appreciated previously, which may impose constraints on regulatory evolution.

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Comprehensive Analysis of a cis-Regulatory Region Reveals Pleiotropy in Enhancer Function

Cited by 85 publications

References 35 publications

Multi-enhancer transcriptional hubs confer phenotypic robustness

Multi-enhancer transcriptional hubs confer phenotypic robustness

Reconstruction of the global neural crest gene regulatory networkin vivo

Dense encoding of developmental regulatory information may constrain evolvability

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