vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ21.038vavilov-3018Research ArticleАКТУАЛЬНЫЕ ТЕХНОЛОГИИMAINSTREAM TECHNOLOGIESМетоды высокопроизводительного репортерного анализа энхансеровMethods of massive parallel reporter assays for investigation of enhancershttps://orcid.org/0000-0002-5989-5756РомановС. Е.RomanovS. E.

Новосибирский национальный исследовательский государственный университет, лаборатория эпигенетики факультета естественных наук,

Институт молекулярной и клеточной биологии Сибирского отделения Российской академии наук, лаборатория геномики

Новосибирск

Novosibirsk State University Epigenetics Laboratory, Department of Natural Sciences,

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences Genomics Laboratory

Novosibirsk

https://orcid.org/0000-0002-0209-4616КалашниковаД. А.KalashnikovaD. A.

Новосибирский национальный исследовательский государственный университет, лаборатория эпигенетики факультета естественных наук,

Институт молекулярной и клеточной биологии Сибирского отделения Российской академии наук, лаборатория геномики

Новосибирск

Novosibirsk State University Epigenetics Laboratory, Department of Natural Sciences,

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences Genomics Laboratory

Novosibirsk

https://orcid.org/0000-0003-2174-6496ЛактионовП. П.LaktionovP. P.

Новосибирский национальный исследовательский государственный университет, лаборатория эпигенетики факультета естественных наук,

Институт молекулярной и клеточной биологии Сибирского отделения Российской академии наук, лаборатория геномики

Новосибирск

Novosibirsk State University Epigenetics Laboratory, Department of Natural Sciences,

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences Genomics Laboratory

Novosibirsk

laktionov@mcb.nsc.ruНовосибирский национальный исследовательский государственный университет; Институт молекулярной и клеточной биологии Сибирского отделения Российской академии наукРоссияNovosibirsk State University; Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of SciencesRussian Federation202102062021253344355Copyright © Романов С.Е., Калашникова Д.А., Лактионов П.П., 20212021Романов С.Е., Калашникова Д.А., Лактионов П.П.Romanov S.E., Kalashnikova D.A., Laktionov P.P.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3018

Корректное развертывание генетических программ развития и дифференцировки опирается на тонко координированную регуляцию экспрессии специфических наборов генов. Исключительную роль в управлении этим процессом играют регуляторные элементы генома, к которым относятся промоторы, энхансеры, инсуляторы и сайленсеры. Нарушения в их работе могут приводить к развитию различных патологий, включая онкологические заболевания, пороки развития и аутоиммунные заболевания. Развитие технологий высокопроизводительного геномного анализа позволило значительно ускорить накопление информации о специфичных эпигенетических характеристиках регуляторных элементов. В совокупности с полногеномными исследованиями распределения эпигенетических меток, регуляторных белков и пространственной структуры хроматина такие данные значительно расширяют представления о принципах эпигенетической регуляции генов и позволяют осуществлять поиск потенциальных регуляторных элементов in silico. Вместе с тем основные экспериментальные подходы, используемые для исследования локальных характеристик хроматина, имеют ряд технических ограничений, которые снижают достоверность биоинформатической идентификации регуляторных областей генома. В связи с этим, а также с учетом вариабельности функций эпигенетических детерминант и многокомпонентной регуляции работы элементов генома определение их регуляторной роли часто требует функциональной проверки. Разработано множество методов, позволяющих провести исследование функциональной роли регуляторных элементов в масштабе генома. В настоящем обзоре кратко описаны основные экспериментальные подходы для проведения идентификации регуляторных элементов in silico и присущие им технические ограничения. Рассмотрены оригинальные методы высокопроизводительного репортерного анализа активности энхансеров, которые используют для валидации предсказанных регуляторных элементов и de novo поиска. Описанные методы анализа дают возможность оценить функциональную роль нуклеотидной последовательности регуляторного элемента, определить его точные границы, а также оценить влияние локального состояния хроматина на активность энхансеров и экспрессию генов. Применение таких методологических подходов обеспечило значительный вклад в понимание фундаментальных принципов регуляции генной экспрессии.

The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation.

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The authors declare that there are no conflicts of interest present.