Epigenetic mechanisms govern the quality, the stability, and the responsiveness of transcriptional programs to the environment. This regulation is ensured via the concerted action of different players (transcription factors, “reader” and “writer” enzymes, histone marks, structural proteins, noncoding regulatory RNAs) that flow in the 3D organization of the genome. Indeed, nuclear architecture participates in the punctual and cell-type-specific regulation of transcription. Hence, the fine dissection of these mechanisms will allow a deeper understanding of the gene expression machinery. In this chapter, we propose a challenging imaging-based method to study the reciprocal interactions between chromatin-associated RNAs, genomic loci, and chromatin compartment with a procedure of 3D COMBO chrRNA–DNA–ImmunoFISH, specifically developed to preserve the nuclear integrity and topology of human primary T cells. We believe that our protocol will contribute to the improvement of epigenetic studies on the 3D nuclear structure of T cell subsets, possibly shedding light on the still hidden epigenetic players responsible for the great plasticity and functional diversification exerted by T cells.

3d combo chrrna–dna–immunofish / F. Marasca, A. Cortesi, B. Bodega (METHODS IN MOLECULAR BIOLOGY). - In: Capturing Chromosome Conformation / [a cura di] B. Bodega, C. Lanzuolo. - [s.l] : Humana Press Inc., 2021. - ISBN 9781071606636. - pp. 281-297

3d combo chrrna–dna–immunofish

Marasca F.;Bodega B.
2021

Abstract

Epigenetic mechanisms govern the quality, the stability, and the responsiveness of transcriptional programs to the environment. This regulation is ensured via the concerted action of different players (transcription factors, “reader” and “writer” enzymes, histone marks, structural proteins, noncoding regulatory RNAs) that flow in the 3D organization of the genome. Indeed, nuclear architecture participates in the punctual and cell-type-specific regulation of transcription. Hence, the fine dissection of these mechanisms will allow a deeper understanding of the gene expression machinery. In this chapter, we propose a challenging imaging-based method to study the reciprocal interactions between chromatin-associated RNAs, genomic loci, and chromatin compartment with a procedure of 3D COMBO chrRNA–DNA–ImmunoFISH, specifically developed to preserve the nuclear integrity and topology of human primary T cells. We believe that our protocol will contribute to the improvement of epigenetic studies on the 3D nuclear structure of T cell subsets, possibly shedding light on the still hidden epigenetic players responsible for the great plasticity and functional diversification exerted by T cells.
3D COMBO RNA-DNA-Immuno FISH; Chromatin associated RNAs; Epigenetics; 3D genome organization; Human primary T cells; Transposable elements
Settore BIO/11 - Biologia Molecolare
Settore BIO/13 - Biologia Applicata
Settore BIO/18 - Genetica
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/825250
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