Biology subjects

van der Weide, R. H.

Publications and source records attributed to van der Weide, R. H..

5 recordsLinked to original sources

bioRxiv2023

Combinatorial single-cell profiling of all major chromatin types with MAbID

Gene expression programs result from the collective activity of many regulatory factors. To obtain insight into the mechanisms that govern gene regulation, it is imperative to study their combined mode of action and interconnectivity. However, it has been challenging to simultaneously measure a combination of these factors within one sample. Here, we introduce MAbID, a method that combines genomic profiling of many histone modifications and chromatin-binding proteins in a single reaction. MAbID employs antibody-DNA conjugates to enable genomic barcoding of chromatin at sites of epitope occupancy. This barcoding strategy allows for the combined incubation of multiple antibodies in a single sample to reveal the genomic distributions of many epigenetic states simultaneously. We used MAbID to profile both active and inactive chromatin types in human cell lines and multiplexed measurements in the same sample without loss of data quality. Moreover, we obtained joint measurements of six epitopes covering all major chromatin types in single cells during mouse in vitro neural differentiation and captured associated changes in multifactorial chromatin states. Thus, MAbID holds the potential to gain unique insights into the interplay between gene regulatory mechanisms, especially in settings with limited sample material and in single cells.

genomics↗

bioRxiv2021

Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the CDK-Mediator (CDK-MED) complex has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here we discover that CDK-MED contributes little to 3D genome organisation in ESCs, but has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CDK-MED, which facilitates binding of cPRC1 to its target sites. Importantly, through separation of function experiments, we reveal that this collaboration between CDK-MED and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CDK module supports core Mediator engagement with gene promoters to enable gene activation.

molecular biology↗

bioRxiv2021

Rapid depletion of CTCF and cohesin proteins reveals dynamic features of chromosome architecture.

The interphase genome is mainly shaped by cohesin-mediated loop extrusion and cohesin-independent compartmentalization. Extrusion is a dynamic process of cohesin loading, loop extension and release. Cohesin release is mediated by WAPL. Loss of WAPL leads to the formation of longer loops and counters compartmentalization. The dynamics of these changes in chromosome organization have been unclear. We have used acute depletion of WAPL to show that within six hours cohesin accumulates at CTCF-bound loop anchors and extended loops are formed. When we deplete WAPL and CTCF simultaneously, new loops are formed between active genes. Surprisingly, active gene clustering is independent of cohesin. Stabilization of cohesin on chromatin leads to a decrease in compartmentalization, which is rapidly restored by depletion of cohesin. Our analyses show that loop extrusion counters compartmentalization and plays a central role in many aspects of chromosome organization. HIGHLIGHTSO_LICohesin accumulates at CTCF-mediated chromatin loop anchors following WAPL depletion. C_LIO_LIActively transcribed genes form long-range gene clusters independent of the cohesin complex. C_LIO_LIPlumes are a novel architectural feature of juxtaposed DNA formed by cohesin at open chromatin islands. C_LIO_LIChromosome compartmentalization can be uncoupled from nuclear lamina interactions. C_LI

molecular biology↗

bioRxiv2021

Hi-C Analyses with GENOVA: a case study with cohesin variants

Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise conformation capture data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that {Delta}SA1 cells gain intra-TAD interactions and increase compartmentalisation. {Delta}SA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesinSA1 forms longer loops, while cohesinSA2 plays a role in forming and maintaining intra-TAD interactions. Our data supports the model that the genome is provided structure in 3D by the counter-balancing of loop formation on one hand, and compartmentalization on the other hand. By differentially controlling loops, cohesinSA1 and cohesinSA2 therefore also affect nuclear compartmentalization. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail.

genomics↗

bioRxiv2017

Condensin’s ATPase Machinery Drives and Dampens Mitotic Chromosome Condensation

Chromosome condensation by condensin is essential for faithful chromosome segregation. Metazoans have two complexes, named condensin I and II. Both are thought to act by creating looped structures in DNA, but how they do so is unknown. Condensins SMC subunits together form a composite ATPase with two pseudo-symmetric ATPase sites. We reveal that these sites have opposite functions in the condensation process. One site drives condensation, while the other site rather has a dampening function. Mutation of this dampener site hyperactivates both condensin I and II complexes. We find that hyperactive condensin I efficiently shortens chromosomes in the total absence of condensin II. The two complexes form loops with different lengths, and specifically condensin II is key to the decatenation of sister chromatids and the formation of a straight chromosomal axis.

molecular biology↗