RESUMO
Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity.
RESUMO
Genome-wide ensemble sequencing methods improved our understanding of chromatin organization in eukaryotes but lack the ability to capture single-cell heterogeneity and spatial organization. To overcome these limitations, new imaging-based methods have emerged, giving rise to the field of spatial genomics. Here, we present pyHiM, a user-friendly python toolbox specifically designed for the analysis of multiplexed DNA-FISH data and the reconstruction of chromatin traces in individual cells. pyHiM employs a modular architecture, allowing independent execution of analysis steps and customization according to sample specificity and computing resources. pyHiM aims to facilitate the democratization and standardization of spatial genomics analysis.
Assuntos
Genômica , Software , Genômica/métodos , Cromatina , Cromossomos , DNARESUMO
Multiplexed sequential and combinatorial imaging enables the simultaneous detection of multiple biological molecules, e.g. proteins, DNA, or RNA, enabling single-cell spatial multi-omics measurements at sub-cellular resolution. Recently, we designed a multiplexed imaging approach (Hi-M) to study the spatial organization of chromatin in single cells. In order to enable Hi-M sequential imaging on custom microscope setups, we developed Qudi-HiM, a modular software package written in Python 3. Qudi-HiM contains modules to automate the robust acquisition of thousands of three-dimensional multicolor microscopy images, the handling of microfluidics devices, and the remote monitoring of ongoing acquisitions and real-time analysis. In addition, Qudi-HiM can be used as a stand-alone tool for other imaging modalities.