Using transgenic reporter assays to functionally characterize enhancers in animals

Kvon, Evgeny Z.

doi:10.1016/j.ygeno.2015.06.007

Cited by 67 publications

(53 citation statements)

References 139 publications

(206 reference statements)

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“…It is thus possible that Yellow chromatin marks housekeeping genes without contributing to their high expression. There is ample evidence that endogenous enhancers can activate integrated transgenes (Kvon 2015), so it is surprising that they do not show a preponderant role in this study. It is important to note that enhancer trap and STARR-seq are performed with minimal promoters (Kvon 2015;Zabidi et al 2015), whereas the promoters used here have not been truncated.…”

Section: Discussionmentioning

confidence: 64%

“…There is ample evidence that endogenous enhancers can activate integrated transgenes (Kvon 2015), so it is surprising that they do not show a preponderant role in this study. It is important to note that enhancer trap and STARR-seq are performed with minimal promoters (Kvon 2015;Zabidi et al 2015), whereas the promoters used here have not been truncated. It is possible that the effect of enhancers is visible on weak promoters but that their effects are less obvious on stronger promoters.…”

Section: Discussionmentioning

confidence: 64%

“…This is consistent with our findings but again raises the question of why contacts with enhancers poorly predict reporter expression. An issue may be the sensitivity of STARR-seq, since many promoters of active genes were not picked up as enhancers (Kvon 2015;Zabidi et al 2015). Unfortunately, it is unclear if the enhancer screens were performed to saturation.…”

Section: Discussionmentioning

confidence: 99%

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Clustering of Drosophila housekeeping promoters facilitates their expression

et al. 2017

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Housekeeping genes of animal genomes cluster in the same chromosomal regions. It has long been suggested that this organization contributes to their steady expression across all the tissues of the organism. Here, we show that the activity of Drosophila housekeeping gene promoters depends on the expression of their neighbors. By measuring the expression of ∼85,000 reporters integrated in Kc167 cells, we identified the best predictors of expression as chromosomal contacts with the promoters and terminators of active genes. Surprisingly, the chromatin composition at the insertion site and the contacts with enhancers were less informative. These results are substantiated by the existence of genomic "paradoxical" domains, rich in euchromatic features and enhancers, but where the reporters are expressed at low level, concomitant with a deficit of interactions with promoters and terminators. This indicates that the proper function of housekeeping genes relies not on contacts with long distance enhancers but on spatial clustering. Overall, our results suggest that spatial proximity between genes increases their expression and that the linear architecture of the Drosophila genome contributes to this effect.

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Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

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Clustering of Drosophila housekeeping promoters facilitates their expression

et al. 2017

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“…Candidate cis-regulatory modules identified using the methods mentioned above have to be coupled with approaches aimed at testing their functional significance, which range from in vitro massively parallel reporter assays to smaller scale in vivo reporter assays. [30,31] Although we anticipate the development of CRISPR/ Cas9-mediated genome editing tools in striped mice (see below), the laboratory mouse can be leveraged as a powerful tool to dissect enhancer function, either by performing reporter assays in one of the many murine cell lines already available or directly in vivo. Together, the profiling of chromatin landscapes in different stripes coupled to the functional characterization of the differences seen, will provide insights into the regulatory events controlling the differential expression of pigment pattern implementation factors.…”

Section: Implementing Periodic Patternsmentioning

confidence: 99%

Periodic patterns in Rodentia: Development and evolution

Johnson

Barsh

Mallarino

2019

Experimental Dermatology

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Mammalian periodic pigment patterns, such as spots and stripes, have long interested mathematicians and biologists because they arise from non-random developmental processes that are programmed to be spatially constrained, and can therefore be used as a model to understand how organized morphological structures develop. Despite such interest, the developmental and molecular processes underlying their formation remain poorly understood. Here, we argue that Arvicanthines, a clade of African rodents that naturally evolved a remarkable array of coat patterns, represent a tractable model system in which to dissect the mechanistic basis of pigment pattern formation. Indeed, we review recent insights into the process of stripe formation that were obtained using an Arvicanthine species, the African striped mouse (Rhabdomys pumilio), and discuss how these rodents can be used to probe deeply into our understanding of the factors that specify and implement positional information in the skin. By combining naturally evolved pigment pattern variation in rodents with classic and novel experimental approaches, we can substantially advance our understanding of the processes by which spatial patterns of cell differentiation are established during embryogenesis, a fundamental question in developmental biology.

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“…The study of mammalian enhancers is hampered by the observation that these elements are defined by criteria only partially or indirectly related to their role in vivo 9,10. We therefore used homologous recombination to make seven mouse models in which each constituent of the proposed α-globin super-enhancer is deleted, singly and in informative pairs, to dissect the its function.…”

mentioning

confidence: 99%

Genetic dissection of the α-globin super-enhancer in vivo

et al. 2016

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Many genes determining cell identity are regulated by clusters of mediator-bound enhancer elements collectively referred to as super-enhancers. These have been proposed to manifest higher-order properties important in development and disease. Here, we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer singly and in informative combinations, we demonstrate that each constituent enhancer appears to act independently and in an additive fashion with respect to hematologic phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

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Using transgenic reporter assays to functionally characterize enhancers in animals

Cited by 67 publications

References 139 publications

Clustering of Drosophila housekeeping promoters facilitates their expression

Clustering of Drosophila housekeeping promoters facilitates their expression

Periodic patterns in Rodentia: Development and evolution

Genetic dissection of the α-globin super-enhancer in vivo

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