Dataset Information

Encoded, click-reactive DNA-binding domains for programmable capture of specific chromatin segments.

ABSTRACT: Enrichment of chromatin segments from specific genomic loci of living cells is an important goal in chromatin biology, since it enables establishing local molecular compositions as the basis of locus function. A central enrichment strategy relies on the expression of DNA-binding domains that selectively interact with a local target sequence followed by fixation and isolation of the associated chromatin segment. The efficiency and selectivity of this approach critically depend on the employed enrichment tag and the strategy used for its introduction into the DNA-binding domain or close-by proteins. We here report chromatin enrichment by expressing programmable transcription-activator-like effectors (TALEs) bearing single strained alkynes or alkenes introduced via genetic code expansion. This enables in situ biotinylation at a defined TALE site via strain-promoted inverse electron demand Diels Alder cycloadditions for single-step, high affinity enrichment. By targeting human pericentromeric SATIII repeats, the origin of nuclear stress bodies, we demonstrate enrichment of SATIII DNA and SATIII-associated proteins, and identify factors enriched during heat stress.

SUBMITTER: Witte A

PROVIDER: S-EPMC8162481 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.

Project description:The genome adopts varied and specific 3D conformations in different cell types. The loop extruding factor, cohesin, is thought to play a key role in the formation of this 3D structure, however it is unknown how cell-type specificity is achieved. One proposed mechanism is that cis-regulatory elements, which are active in unique combinations in different cell types, recruit cohesin and therefore direct the formation of specific 3D conformation. We have identified genome-wide natural variation in enhancer sites in healthy human donors, by performing cohesin ChIP-seq in these cells we show that cohesin occupancy is directly correlated with enhancer activity. Identification of the genetic variants associated with the gain or loss of enhancer-like elements and associating this with the recruitment of cohesin provides evidence of a direct link between widespread genetic variation in the non-coding genome and cell-specific changes in 3D genome organization. Therefore, enhancer elements have evolved not only the ability to upregulate transcription at promoters, but also the ability to stimulate the mechanisms that insure robust interactions between them. Next, using enhancer KO models in the well characterised alpha globin locus, we show how cohesin recruitment and 3D genome organization correlates with changes in the local regulatory landscape. We then selected the strongest of the alpha globin enhancers and inserted it into a neutral region of the genome, to test if a single active enhancer was capable of recruiting cohesin and initiating the formation of a 3D chromatin domain when isolated from its native context. Our results show that an enhancer is able to direct the recruitment of cohesin when in an active state and that this is correlated with changes in the local chromatin environment, giving rise to open chromatin sites and de novo chromatin interactions in an otherwise “neutral” region of the genome. These results support the hypothesis that tissue-specific recruitment of cohesin is facilitated by enhancers and that this plays a role in establishing cell-specific changes in 3D genome conformation.

Dataset Information

Encoded, click-reactive DNA-binding domains for programmable capture of specific chromatin segments.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets