Biology subjects

van Steensel, B.

Publications and source records attributed to van Steensel, B..

36 records · Page 2Linked to original sources

bioRxiv2022

Widespread chromatin context-dependencies ofDNA double-strand break repair proteins

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. BRCA2 is an example of the former, which is corroborated by chromatin-dependent shifts in mutation patterns of BRCA2-/- cancer genomes. These results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner. ONE SENTENCE SUMMARYA multiplexed screen reveals how dozens of proteins sense the local chromatin context to tune the balance between two DNA repair pathways.

molecular biology↗

bioRxiv2022

Transgenerational transcriptional heterogeneity from cytoplasmic chromatin

Transcriptional heterogeneity from plasticity of the epigenetic state of chromatin is thought to contribute to tumor evolution, metastasis, and drug resistance 1-3. However, the mechanisms leading to nongenetic cell-to-cell variation in gene expression remain poorly understood. Here we demonstrate that heritable transcriptional changes can result from the formation of micronuclei, aberrations of the nucleus that are common in cancer4,5. Micronuclei have fragile nuclear envelopes (NE) that are prone to spontaneous rupture, which exposes chromosomes to the cytoplasm and disrupts many nuclear activities 6,7. Using a combination of long-term live-cell imaging and same-cell, single-cell RNA sequencing (Look-Seq2), we identified significant reduction of gene expression in micronuclei, both before and after NE rupture. Furthermore, chromosomes in micronuclei fail to normally recover histone 3 lysine 27 acetylation, a critical step for the reestablishment of normal transcription after mitosis 8-10. These transcription and chromatin defects can persist into the next generation in a subset of cells, even after these chromosomes are incorporated into normal daughter nuclei. Moreover, persistent transcriptional repression is strongly associated with, and may be explained by, surprisingly long-lived DNA damage to these reincorporated chromosomes. Therefore, heritable alterations in transcription can originate from aberrations of nuclear architecture.

cell biology↗

bioRxiv2021

Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome.

Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterised in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE - promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity for a single promoter to quantitative differences in activation across a broad set of promoters. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order TF motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.

genetics↗

bioRxiv2021

Dynamic chromosomal interactions and control of heterochromatin positioning by Ki67

Ki-67 is a chromatin-associated protein with a dynamic distribution pattern throughout the cell cycle, and is thought to be involved in chromatin organization. Lack of genomic interaction maps has hampered a detailed understanding of its roles, particularly during interphase. By pA-DamID mapping in human cell lines we found that Ki-67 associates with large genomic domains that overlap mostly with late-replicating regions. Early in interphase, when Ki-67 is present in pre-nucleolar bodies, it interacts with these domains on all chromosomes. However, later in interphase, when Ki-67 is confined to nucleoli, it shows a striking shift towards small chromosomes. Nucleolar perturbations indicate that these cell cycle dynamics correspond to nucleolar maturation during interphase, and suggest that nucleolar sequestration of Ki-67 limits its interactions with larger chromosomes. Furthermore, we demonstrate that Ki-67 does not detectably control chromatin-chromatin interactions during interphase, but it competes with the nuclear lamina for interaction with late-replicating DNA, and it controls replication timing of (peri)centromeric regions. Together, these results reveal a highly dynamic choreography of genome interactions and roles for Ki-67 in heterochromatin organization.

genomics↗

bioRxiv2021

CTCF and cohesin promote focal detachment of DNA from the nuclear lamina

Lamina associated domains (LADs) are large genomic regions that are positioned at the nuclear lamina (NL). It has remained largely unclear what drives the positioning and demarcation of LADs. Because the insulator protein CTCF is enriched at LAD borders, it was postulated that CTCF binding could position a subset of LAD boundaries, possibly through its function in stalling cohesin and hence preventing cohesin to invade into the LAD. To test this, we mapped genome - NL interactions in mouse embryonic stem cells after rapid depletion of CTCF and other perturbations of cohesin dynamics. CTCF and cohesin contribute to a sharp transition in NL interactions at LAD borders, whilst LADs are maintained after depletion of these proteins, also at borders marked by CTCF. CTCF and cohesin may thus reinforce LAD borders, but do not position these. CTCF binding sites within LADs are locally detached from the NL and enriched for accessible DNA and active histone modifications. Remarkably, even though NL positioning is strongly correlated with genome inactivity, this DNA remains accessible after the local detachment is lost following CTCF depletion. At a chromosomal scale, cohesin depletion and cohesin stabilization (depletion of the unloading factor WAPL) quantitatively affect NL interactions, indicative of perturbed chromosomal positioning in the nucleus. Finally, while H3K27me3 is locally enriched at CTCF-marked LAD borders, we find no evidence for an interplay between CTCF and H3K27me3 on NL interactions. Combined, these findings illustrate that CTCF and cohesin do not shape LAD patterns. Rather, these proteins mediate fine-tuning of NL interactions.

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

Perturbations in 3D genome organization can promote acquired drug resistance

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.

molecular biology↗

bioRxiv2020

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

molecular biology↗

bioRxiv2020

SPIN reveals genome-wide landscape of nuclear compartmentalization

Chromosomes segregate differentially relative to distinct subnuclear structures, but this genome-wide compartmentalization, pivotal for modulating genome function, remains poorly understood. New genomic mapping methods can reveal chromosome positioning relative to specific nuclear structures. However, computational methods that integrate their results to identify overall intranuclear chromo-some positioning have not yet been developed. We report SPIN, a new method to identify genome-wide nuclear spatial localization patterns. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to genome function (transcription and replication timing). Comparisons of SPIN states with Hi-C sub-compartments and lamina-associated domains (LADs) from multiple cell types suggest constitutive compartmentalization patterns. By integrating different readouts of higher-order genome organization, SPIN provides critical insights into nuclear spatial and functional compartmentalization.

bioinformatics↗

bioRxiv2019

Cell cycle dynamics of lamina associated DNA

In mammalian interphase nuclei more than one thousand large genomic regions are positioned at the nuclear lamina (NL). These lamina associated domains (LADs) are involved in gene regulation and may provide a backbone for the overall folding of interphase chromosomes. While LADs have been characterized in great detail, little is known about their dynamics during interphase, in particular at the onset of G1 phase and during DNA replication. To study these dynamics, we developed an antibody-based variant of the DamID technology (named pA-DamID) that allows us to map and visualize genome - NL interactions with high temporal resolution. Application of pA-DamID combined with synchronization and cell sorting experiments reveals that LAD - NL contacts are generally rapidly established early in G1 phase. However, LADs on the distal [~]25 Mb of most chromosomes tend to contact the NL first and then gradually detach, while centromere-proximal LADs accumulate gradually at the NL. Furthermore, our data indicate that S-phase chromatin shows transiently increased lamin interactions. These findings highlight a dynamic choreography of LAD - NL contacts during interphase progression, and illustrate the usefulness of pA-DamID to study the dynamics of genome compartmentalization.

molecular biology↗

bioRxiv2019

m6A methylation potentiates cytosolic dsDNA recognition in a sequence-specific manner

Nucleic acid sensing through pattern recognition receptors is critical for immune recognition of microbial infections. Microbial DNA is frequently methylated at the N6 position of adenines (m6A), a modification that is rare in mammalian host DNA. We show that m6A-methylation of 5-GATC-3 motifs augments the immunogenicity of double stranded (ds)DNA in macrophages and dendritic cells. Transfection with m6A-methylated DNA increased the expression of the activation markers CD69 and CD86, and of Ifn{beta}, iNos and Cxcl10 mRNA. Recognition of m6A DNA occurs independently of TLR and RIG-I signaling but requires STING, a key mediator of cytosolic DNA sensing. Intriguingly, the response to m6A DNA is sequence-specific. m6A is immunostimulatory in some motifs, but immunosuppressive in others, a feature that is conserved between mouse and human. In conclusion, epigenetic alterations of bacterial DNA are differentially perceived by innate cells, a feature that could potentially be used for the design of immune-modulating therapeutics.

immunology↗

bioRxiv2019

Local rewiring of genome - nuclear lamina interactions by transcription.

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning towards the nuclear interior. How this process works and how it impacts flanking chromosomal regions is poorly understood. We addressed these questions by systematic manipulation of gene activity and detailed analysis of NL interactions. Activation of genes inside LADs typically causes detachment of the entire transcription unit but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. These findings show how NL interactions can be shaped locally by transcription and point to a remarkable flexibility of interphase chromosomes.

genomics↗

bioRxiv2018

Promoter-intrinsic and local chromatin features determine gene repression in lamina-associated domains

It is largely unclear whether genes that are naturally embedded in lamina associated domains (LADs) are inactive due to their chromatin environment, or whether LADs are merely secondary to the lack of transcription. We show that hundreds of human promoters become active when moved from their native LAD position to a neutral context in the same cells, indicating that LADs form a repressive environment. Another set of promoters inside LADs is able to \"escape\" repression, although their transcription elongation is attenuated. By inserting reporters into thousands of genomic locations, we demonstrate that these escaper promoters are intrinsically less sensitive to LAD repression. This is not simply explained by promoter strength, but by the interplay between promoter sequence and local chromatin features that vary strongly across LADs. Enhancers also differ in their sensitivity to LAD chromatin. This work provides a general framework for the systematic understanding of gene regulation by repressive chromatin.\n\nHighlightsO_LITwo promoter transplantation strategies elucidate the regulatory role of LAD chromatin\nC_LIO_LILADs are generally repressive, but also highly heterogeneous\nC_LIO_LILADs can impede both promoter activity and transcription elongation\nC_LIO_LIPromoters vary intrinsically in their sensitivity to LAD repression\nC_LI

genomics↗

bioRxiv2018

Systematic identification of human SNPs affecting regulatory element activity

Most of the millions of single-nucleotide polymorphisms (SNPs) in the human genome are non-coding, and many overlap with putative regulatory elements. Genome-wide association studies have linked many of these SNPs to human traits or to gene expression levels, but rarely with sufficient resolution to identify the causal SNPs. Functional screens based on reporter assays have previously been of insufficient throughput to test the vast space of SNPs for possible effects on enhancer and promoter activity. Here, we have leveraged the throughput of the SuRE reporter technology to survey a total of 5.9 million SNPs, including 57% of the known common SNPs. We identified more than 30 thousand SNPs that alter the activity of putative regulatory elements, often in a cell-type specific manner. These data indicate that a large proportion of human non-coding SNPs may affect gene regulation. Integration of these SuRE data with genome-wide association studies may help pinpoint SNPs that underlie human traits.

genomics↗

bioRxiv2018

TSA-Seq Mapping of Nuclear Genome Organization

While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. Here we describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription \"hot-zones\". Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, house-keeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for nuclear spatial organization of transcription.

genomics↗

bioRxiv2017

Easy quantification of template-directed CRISPR/Cas9 editing

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies.

genetics↗

bioRxiv2017

CHRAC/ACF Contribute to the Repressive Ground State of Chromatin

The chromatin remodeling complexes CHRAC and ACF combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression is unclear. Here we explored the influence of Drosophila CHRAC/ACF on transcription by complementary gain- and loss-of-function approaches.\n\nTargeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of a Acf knock-out allele showed that only lowly expressed genes are de-repressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains.\n\nTaken together our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.

molecular biology↗

bioRxiv2017

Quantitative Analysis Shows That Repair Of Cas9-Induced Double-Strand DNA Breaks Is Slow And Error-Prone

The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals a relatively slow repair rate (~6h). Furthermore, the double strand break is predominantly repaired in an error-prone fashion (at least 70%). Both classical and microhomology-mediated end-joining pathways are active and contribute to the repair in a stochastic manner. However, the balance between these two pathways changes over time and can be altered by chemical inhibition of DNAPKcs or additional ionizing radiation. Our strategy is generally applicable to study DSB repair kinetics and fidelity in single loci, and demonstrates that Cas9-induced DSBs are repaired in an unusual manner.

molecular biology↗