Методы высокопроизводительного репортерного анализа энхансеров

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.

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