Identification of non-coding silencer elements and their regulation of gene expression

Pang, Baoxu; Weerd, Jan Hendrik van; Hamoen, Feija L; Snyder, M

doi:10.1038/s41580-022-00549-9

Cited by 45 publications

(40 citation statements)

References 174 publications

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“…Broadly, two mechanisms of silencer action have been proposed: 1) competing DNA binding between activating and repressing proteins and 2) recruitment of negative regulators, such as HDACs, with subsequent formation of heterochromatin domains. (66,67) Although we do observe local heterochromatin mark enrichment at functional NREs, use of an accessible chromatin library as input precludes analysis of functional NREs within expansive heterochromatin marked domains. Rather, we demonstrate that functional NREs can exist in open chromatin and even within 'activated' chromatin environments.…”

Section: Discussionmentioning

confidence: 86%

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Cis-Regulatory Atlas in Primary Human CD4+ T Cells

Stefan

Barski

2022

Preprint

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Cis–regulatory elements (CRE) are critical for coordinating gene expression programs that dictate cell–specific differentiation and homeostasis. Recently developed self–transcribing active regulatory region sequencing (STARR–Seq) has allowed for genome-wide annotation of functional CREs. Despite this, STARR–Seq assays are only employed in cell lines, in part, due to difficulties in delivering reporter constructs. Herein, we implemented and validated a STARR–Seq–based screen in human CD4+ T cells using a non–integrating lentiviral transduction system. Lenti–STARR–Seq is the first example of a genome–wide assay of CRE function in human primary cells, identifying thousands of functional enhancers and negative regulatory elements (NREs) in human CD4+ T cells. Results of the screen were validated using traditional luciferase assays. Genome-wide, we find clear differences between enhancers and NREs in nucleosome positioning, chromatin modification, eRNA production, and transcription factor binding. Our findings support the idea of silencer repurposing as enhancers in alternate cell types. Collectively, these data suggest that Lenti–STARR–Seq is a can be used for CRE screening in primary human cell types.

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Section: Discussionmentioning

confidence: 86%

“…Other silencer screens corroborate this widely varied transcription factor and motif utilization across the functional silencer elements; no unifying chromatin marks or transcription factor binding profile of functional NREs has been identified to date. (1–3, 45, 67)…”

Section: Discussionmentioning

confidence: 99%

Cis-Regulatory Atlas in Primary Human CD4+ T Cells

Stefan

Barski

2022

Preprint

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“…Регуляторные элементы генома представляют собой короткие участки генома, свободные от нуклеосом и доступные для связывания регуляторных белков [16]. В зависимости от регуляторного эффекта выделяют несколько типов ЦРЭ [17][18][19][20].…”

Section: анализ цис-регуляторных элементов генома основные классы рег...unclassified

“…Сайленсеры являются дистальными регуляторными элементами, функция которых заключается в подавлении экспрессии генов [19]. Такие элементы являются сайтами посадки белков-репрессоров транскрипции, которые реализуют функциональное влияние посредством установления репрессивных хроматиновых меток и конкуренции с активирующими белками [35][36][37][38][39][40].…”

Section: анализ цис-регуляторных элементов генома основные классы рег...unclassified

Practical application of massively parallel reporter assay in biotechnology and medicine

Romanov

Laktionov

2023

Journal of Clinical Practice

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The development and functioning of an organism relies on tissue-specific gene programs. Genome regulatory elements play a key role in the regulation of such programs, and disruptions in their function can lead to the development of various pathologies, including cancers, malformations and autoimmune diseases. The emergence of high-throughput genomic studies has led to massively parallel reporter analysis (MPRA) methods, which allow the functional verification and identification of regulatory elements on a genome-wide scale. Initially MPRA was used as a tool to investigate fundamental aspects of epigenetics, but the approach also has great potential for clinical and practical biotechnology. Currently, MPRA is used for validation of clinically significant mutations, identification of tissue-specific regulatory elements, search for the most promising loci for transgene integration, and is an indispensable tool for creating highly efficient expression systems, the range of application of which extends from approaches for protein development and design of next-generation therapeutic antibody superproducers to gene therapy. In this review, the main principles and areas of practical application of high-throughput reporter assays will be discussed.

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“…The billboard model implies that the context of a TFBS is determined primarily by additive effects of the surrounding TFBSs, with few specific interactions between sites. Other models of cis-regulatory grammar propose that individual TFBSs are weak on their own and depend on strong cooperative interactions [26], that particular TFBSs recruit specific, required transcriptional cofactors [11], or that the balance among sites for transcriptional activators and repressors determines whether a CREs is an enhancer or silencer [27][28][29]. The degree to which these proposed features of cisregulatory grammar modulate the local context within a CRE is not well understood.…”

Section: Introductionmentioning

confidence: 99%

Transcription factor interactions explain the context-dependent activity of CRX binding sites

Loell

Friedman

Myers

et al. 2023

Preprint

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The effects of transcription factor binding sites (TFBSs) on the activity of acis-regulatory element (CRE) depend on the local sequence context. In rod photoreceptors, binding sites for the transcription factor (TF) Cone-rod homeobox (CRX) occur in both enhancers and silencers, but the sequence context that determines whether CRX binding sites contribute to activation or repression of transcription is not understood. To investigate the context-dependent activity of CRX sites, we fit neural network-based models to the activities of synthetic CREs composed of photoreceptor TFBSs. The models revealed that CRX binding sites consistently make positive, independent contributions to CRE activity, while negative homotypic interactions between sites cause CREs composed of multiple CRX sites to function as silencers. The effects of negative homotypic interactions can be overcome by the presence of other TFBSs that either interact cooperatively with CRX sites or make independent positive contributions to activity. The context-dependent activity of CRX sites is thus determined by the balance between positive heterotypic interactions, independent contributions of TFBSs, and negative homotypic interactions. Our findings explain observed patterns of activity among genomic CRX-bound enhancers and silencers, and suggest that enhancers may require diverse TFBSs to overcome negative homotypic interactions between TFBSs.

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Identification of non-coding silencer elements and their regulation of gene expression

Cited by 45 publications

References 174 publications

Cis-Regulatory Atlas in Primary Human CD4+ T Cells

Cis-Regulatory Atlas in Primary Human CD4+ T Cells

Practical application of massively parallel reporter assay in biotechnology and medicine

Transcription factor interactions explain the context-dependent activity of CRX binding sites

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