{{context|date=July 2021}} {{cs1 config|name-list-style=vanc|display-authors=6}} '''Shadow enhancers''' are groups of DNA regulatory sequences that function alongside primary enhancers to regulate gene expression. Originally discovered in ''Drosophila'', shadow enhancers have since been identified in a wide range of organisms, including insects, plants, and mammals.<ref name="Hong_2008" /><ref name=":1">{{cite journal | vauthors = Cannavò E, Khoueiry P, Garfield DA, Geeleher P, Zichner T, Gustafson EH, Ciglar L, Korbel JO, Furlong EE | title = Shadow Enhancers Are Pervasive Features of Developmental Regulatory Networks | journal = Current Biology | volume = 26 | issue = 1 | pages = 38–51 | date = January 2016 | pmid = 26687625 | pmc = 4712172 | doi = 10.1016/j.cub.2015.11.034 | bibcode = 2016CBio...26...38C | author-link9 = Eileen Furlong }}</ref><ref>{{cite journal | vauthors = Osterwalder M, Barozzi I, Tissières V, Fukuda-Yuzawa Y, Mannion BJ, Afzal SY, Lee EA, Zhu Y, Plajzer-Frick I, Pickle CS, Kato M, Garvin TH, Pham QT, Harrington AN, Akiyama JA, Afzal V, Lopez-Rios J, Dickel DE, Visel A, Pennacchio LA | title = Enhancer redundancy provides phenotypic robustness in mammalian development | journal = Nature | volume = 554 | issue = 7691 | pages = 239–243 | date = February 2018 | pmid = 29420474 | pmc = 5808607 | doi = 10.1038/nature25461 | bibcode = 2018Natur.554..239O }}</ref><ref>{{cite journal | vauthors = Garnett AT, Square TA, Medeiros DM | title = BMP, Wnt and FGF signals are integrated through evolutionarily conserved enhancers to achieve robust expression of Pax3 and Zic genes at the zebrafish neural plate border | journal = Development | volume = 139 | issue = 22 | pages = 4220–4231 | date = November 2012 | pmid = 23034628 | pmc = 4074300 | doi = 10.1242/dev.081497 }}</ref><ref>{{cite journal | vauthors = Bomblies K, Dagenais N, Weigel D | title = Redundant enhancers mediate transcriptional repression of AGAMOUS by APETALA2 | journal = Developmental Biology | volume = 216 | issue = 1 | pages = 260–264 | date = December 1999 | pmid = 10588876 | doi = 10.1006/dbio.1999.9504 | doi-access = free }}</ref> Shadow enhancers work alongside primary enhancers to drive overlapping gene expression patterns, which stabilizes gene expression against genetic and environmental fluctuations.<ref name="Waymack">{{cite journal | vauthors = Waymack R, Fletcher A, Enciso G, Wunderlich Z | title = Shadow enhancers can suppress input transcription factor noise through distinct regulatory logic | journal = eLife | volume = 9 | article-number = e59351 | date = August 2020 | pmid = 32804082 | pmc = 7556877 | doi = 10.7554/eLife.59351 | veditors = Wittkopp PJ, Crocker J | doi-access = free }} {{CC-notice|cc=by4}}</ref><ref name="Hong_2008">{{cite journal | vauthors = Hong JW, Hendrix DA, Levine MS | title = Shadow enhancers as a source of evolutionary novelty | journal = Science | volume = 321 | issue = 5894 | page = 1314 | date = September 2008 | pmid = 18772429 | pmc = 4257485 | doi = 10.1126/science.1160631 | bibcode = 2008Sci...321.1314H }}</ref><ref name = "Barolo_2012">{{cite journal | vauthors = Barolo S | title = Shadow enhancers: frequently asked questions about distributed cis-regulatory information and enhancer redundancy | journal = BioEssays | volume = 34 | issue = 2 | pages = 135–141 | date = February 2012 | pmid = 22083793 | pmc = 3517143 | doi = 10.1002/bies.201100121 }}</ref><ref name="Kvon_2021">{{cite journal | vauthors = Kvon EZ, Waymack R, Gad M, Wunderlich Z | title = Enhancer redundancy in development and disease | journal = Nature Reviews. Genetics | volume = 22 | issue = 5 | pages = 324–336 | date = May 2021 | pmid = 33442000 | pmc = 8068586 | doi = 10.1038/s41576-020-00311-x }}</ref> Shadow enhancers can act at a large genomic range, are highly evolutionarily conserved and interact with many molecules to drive gene expression patterns.<ref name="Barolo_2012" /><ref name="Kvon_2021" /><ref name=":3" /> Shadow enhancers play a crucial role in development and early embryogenesis by maintaining stable expression of a variety of genes.<ref name="Kvon_2021" /><ref name="Waymack" />
== Discovery == Shadow enhancers were first described in 2008 by Michael Levine and his research group at the University of California, Berkeley.<ref name="Hong_2008" /><ref name="Barolo_2012" /> Their research in Drosophila investigated the transcription factor Dorsal and its target genes. Through characterization of enhancers using ChIP-chip assays, they found that some enhancers appeared to produce gene expression patterns that overlap with those produced by the primary enhancer. Initially, shadow enhancers were believed to act redundantly to the function of the primary enhancer to ensure proper gene expression, despite environmental or genetic variability.<ref name="Hong_2008" /> thumb|450x450px|How shadow enhancers modulate overlapping expression patterns with the primary enhancer, ensuring proper developmental patterning. Shadow enhancers act as a layer of redundancy to ensure that any mutation to the primary enhancer does not affect development of an organism.
== Function == Shadow enhancers are regulatory DNA elements that play a critical role in stabilizing gene expression and minimizing variability. They work alongside primary enhancers to ensure consistent transcriptional activity, even under fluctuating environmental conditions or genetic disturbances.<ref name=":2">{{cite journal | vauthors = Fletcher A, Wunderlich Z, Enciso G | title = Shadow enhancers mediate trade-offs between transcriptional noise and fidelity | journal = PLOS Computational Biology | volume = 19 | issue = 5 | article-number = e1011071 | date = May 2023 | pmid = 37205714 | pmc = 10234526 | doi = 10.1371/journal.pcbi.1011071 | doi-access = free | bibcode = 2023PLSCB..19E1071F }}</ref><ref name="Dresch_2025">{{cite journal | vauthors = Dresch JM, Nourie LL, Conrad RD, Carlson LT, Tchantouridze EI, Tesfaye B, Verhagen E, Gupta M, Borges-Rivera D, Drewell RA | title = Two coacting shadow enhancers regulate twin of eyeless expression during early Drosophila development | journal = Genetics | volume = 229 | issue = 1 | pages = 1–43 | date = January 2025 | article-number = iyae176 | pmid = 39607769 | pmc = 11708921 | doi = 10.1093/genetics/iyae176 }}</ref> One of their primary functions is to provide a backup mechanism for gene regulation; if a primary enhancer is mutated or damaged, shadow enhancers can compensate and maintain proper gene expression patterns.<ref name="Perry_2010">{{cite journal | vauthors = Perry MW, Boettiger AN, Bothma JP, Levine M | title = Shadow enhancers foster robustness of Drosophila gastrulation | journal = Current Biology | volume = 20 | issue = 17 | pages = 1562–1567 | date = September 2010 | pmid = 20797865 | pmc = 4257487 | doi = 10.1016/j.cub.2010.07.043 | bibcode = 2010CBio...20.1562P }}</ref> Therefore, genes that are regulated by these redundant enhancer regions are more resistant to mutations within their non-coding regions.<ref name="Kvon_2021" />
Shadow enhancers, like any enhancer, do not directly interact with the promoter of a gene to regulate gene expression. Shadow enhancers instead directly bind transcription factors, which can then interact with the promoter.<ref name="Barolo_2012" /> Different shadow enhancers can interact with many different transcription factors in order to indirectly interact and affect the promoter of a gene. Shadow enhancers are a part of a multi-enhancer complex, therefore they can compete with one another to influence a single promoter. In contrast, multiple shadow enhancers can also have an additive effect on a single promoter, therefore boosting its intensity or activity. As well, shadow enhancers can work on multiple non-connected promoters in order to influence development at its different stages.<ref>{{Cite journal |last1=Uyehara |first1=Christopher M. |last2=Apostolou |first2=Effie |date=April 2023 |title=3D enhancer-promoter interactions and multi-connected hubs: Organizational principles and functional roles |journal=Cell Reports |language=en |volume=42 |issue=4 |article-number=112068 |doi=10.1016/j.celrep.2023.112068 |pmc=10556201 |pmid=37059094}}</ref>
=== Redundancy === A key characteristic of shadow enhancers is their functional redundancy, which arises from their ability to functionally overlap with primary enhancers in controlling gene expression. This redundancy enhances the strength of gene regulation by ensuring that multiple enhancers contribute to the expression of a single gene. If a gene is regulated not only by a primary enhancer but also by two or more shadow enhancers, then the gene has additional protection against the failure of a single regulatory element. In contrast, genes regulated solely by a primary enhancer lack this redundancy, making them more vulnerable to regulatory disruptions.<ref name="Kvon_2021" /> Shadow enhancers exhibit varying levels of redundancy across different contexts and timeframes. Some shadow enhancers' redundant function can be restricted to a small timeframe or a small number of cells, while others can have a more extensive overlap and thus are more functionally redundant.<ref name=":1" />
=== Non-redundancy === While shadow enhancers' primary function is to drive overlapping gene expression patterns in order to fine-tune gene expression patterns, some shadow enhancers also play important non-redundant roles. Shadow enhancers may be redundant in one developmental stage or tissue type and non-redundant in another, indicating they can have their own essential functions.<ref name=":1" /> Some shadow enhancers are redundant under normal conditions but non-redundant under extreme conditions, highlighting their importance in stabilizing gene expression in unfavourable conditions.<ref name=":1" /><ref name=":2" /><ref name="Dresch_2025" />
== Characteristics ==
=== Location === Shadow enhancers can be positioned at various distances from their target genes, often farther away compared to primary enhancers.<ref name="Barolo_2012" /> Shadow enhancers are cis-acting regulatory elements, thus they are located on the same DNA molecule as the gene they regulate.<ref name=":3">{{Cite journal |last1=Plank |first1=Jennifer L. |last2=Dean |first2=Ann |date=2014-07-03 |title=Enhancer function: mechanistic and genome-wide insights come together |journal=Molecular Cell |volume=55 |issue=1 |pages=5–14 |doi=10.1016/j.molcel.2014.06.015 |issn=1097-4164 |pmc=8441652 |pmid=24996062}}</ref> They may reside within intronic regions or beyond adjacent genes, exerting their regulatory influence over a broad genomic range.<ref name="Barolo_2012" /> Although both shadow and primary enhancers contribute to gene expression, shadow enhancers tend to operate from more distal genomic locations.<ref name="Kvon_2021" />
=== Evolutionary Conservation === Shadow enhancers are evolutionary conserved sequences that are present in a wide range of organisms, including both vertebrate and invertebrate species.<ref name="Kvon_2021" /> Shadow enhancers have been shown to be more conserved than non-redundant enhancers, which suggests their function is crucial not only in the development of an organism but also throughout evolutionary time. Genes important in development have complex and highly conserved regulation, which explains why shadow enhancers that regulate these genes are highly conserved. Shadow enhancers' partial redundancy also explains why they are maintained over evolutionary time, as they serve important redundant and non-redundant functions that contributes to the proper development of organisms.<ref name=":1" /> The conservation of shadow enhancers across taxonomic groups showcases just how important shadow enhancers are in the viability of organisms.<ref name="Kvon_2021" />
=== Functional sites === Shadow enhancers must interact with many factors in order to regulate and reduce variability in gene expression.<ref name="Hong_2008" /><ref name=":1" /><ref name="Waymack" /><ref name="Barolo_2012" /> Shadow enhancers contain clustered binding sites, and the binding of a transcription factor to these sites can either activate or repress gene expression.<ref name="Barolo_2012" /> Shadow enhancers have a higher proportion of functional sites than non-redundant enhancers, suggesting they are involved in complex regulation of gene expression.<ref name=":1" />
== Role in development == Shadow enhancers play a critical role in embryogenesis, particularly in defining body patterning.<ref name="Whitney_2022">{{cite journal | vauthors = Whitney PH, Shrestha B, Xiong J, Zhang T, Rushlow CA | title = Shadow enhancers modulate distinct transcriptional parameters that differentially effect downstream patterning events | journal = Development | volume = 149 | issue = 21 | article-number = dev200940 | date = November 2022 | pmid = 36264246 | pmc = 9687063 | doi = 10.1242/dev.200940 }}</ref><ref name=":0">{{cite journal | vauthors = El-Sherif E, Levine M | title = Shadow Enhancers Mediate Dynamic Shifts of Gap Gene Expression in the Drosophila Embryo | language = English | journal = Current Biology | volume = 26 | issue = 9 | pages = 1164–1169 | date = May 2016 | pmid = 27112292 | pmc = 4957242 | doi = 10.1016/j.cub.2016.02.054 | bibcode = 2016CBio...26.1164E }}</ref> By modulating transcriptional activation strength, timing, and location, they ensure precise control of gene expression during development.<ref name="Whitney_2022" /> Their ability to fine-tune gene expression levels helps maintain stability, allowing organisms to grow and develop properly despite environmental stresses.<ref name="Wunderlich_2015">{{cite journal |vauthors=Wunderlich Z, Bragdon MD, Vincent BJ, White JA, Estrada J, DePace AH |date=September 2015 |title=Krüppel Expression Levels Are Maintained through Compensatory Evolution of Shadow Enhancers |journal=Cell Reports |volume=12 |issue=11 |pages=1740–1747 |doi=10.1016/j.celrep.2015.08.021 |pmc=4581983 |pmid=26344774}}</ref><ref name="Waymack" /><ref name="Perry_2010" /> Shadow enhancers can compensate for mutations in primary enhancers, acting as a buffer against genetic and environmental fluctuations.<ref name="Kvon_2021" /><ref name="Dresch_2025" /><ref name="Perry_2010" /> This buffering capacity ensures consistent and precise gene expression patterns, which are crucial for proper body development and overall developmental stability.<ref name="Kvon_2021" /><ref name="Waymack" /> Mutations to shadow enhancers confer a higher fitness consequence than mutations to non-redundant enhancers, highlighting their importance in proper development and thus viability of organisms.<ref name=":1" /> Shadow enhancers are important in the regulation of many genes involved in development and have been well characterized in ''Drosophila''.<ref name="Wunderlich_2015" /><ref name="Dresch_2025" /><ref name="Perry_2010" /><ref name=":0" />
=== ''Twin of eyeless'' regulation in ''Drosophila'' === Shadow enhancers are important in the regulation of the twin of eyeless (''toy'') gene in ''Drosophila.'' The ''toy'' gene is crucial for eye development, therefore these shadow enhancers work together to drive ''toy'' expression during early embryogenesis. Their overlapping yet distinct expression patterns ensures consistent ''toy'' expression during critical developmental processes.<ref name="Dresch_2025" />
=== ''snail'' regulation in ''Drosophila'' === Shadow enhancers play a crucial role in regulation of the ''snail'' gene during ''Drosophila'' embryogenesis. The ''snail'' gene encodes a transcription factor that is essential for epithelial-mesenchymal transitions in many developmental processes. Shadow enhancers maintain ''snail'' expression under environmental and genetic disturbances in order to ensure proper gastrulation.<ref name="Perry_2010" />
=== ''Krüppel'' regulation in ''Drosophila'' === Shadow enhancers are important in the regulation of the ''Krüppel'' (''Kr'') gene in ''Drosophila.'' The ''Krüppel'' gene is involved in early segmentation and creates precise patterning along the anterior-posterior axis of the ''Drosophila'' embryo. Shadow enhancers are crucial in facilitating Kr expression, ultimately ensuring accurate spacial and temporal expression of ''Krüppel''. Shadow enhancers are important in fine-tuning gene expression patterns during critical developmental stages.<ref name=":0" /><ref name="Wunderlich_2015" />
=== ''Sonic hedgehog'' regulation in mice === Two shadow enhancers have been identified to be involved in the regulation of the ''sonic hedgehog'' (SHH) gene in mice.<ref name="Hong_2008" /> In vertebrates, SHH is important in forming the ventral midline of the central nervous system. Without shadow enhancers to ensure proper expression of Shh, the brain can become malformed. Therefore, shadow enhancers are crucial in ensuring proper development and stabilizing gene expression of important developmental genes, like SHH.<ref>{{cite journal | vauthors = Jeong Y, El-Jaick K, Roessler E, Muenke M, Epstein DJ | title = A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers | journal = Development | location = Cambridge, England | volume = 133 | issue = 4 | pages = 761–772 | date = February 2006 | pmid = 16407397 | doi = 10.1242/dev.02239 }}</ref>
== References == {{Reflist}}
Category:Gene expression