Chromatin protein complexes involved in gene repression in lamina-associated domains.

Lamina-associated domains (LADs) are large chromatin regions that are associated with the nuclear lamina (NL) and form a repressive environment for transcription. The molecular players that mediate gene repression in LADs are currently unknown. Here, we performed FACS-based whole-genome genetic screens in human cells using LAD-integrated fluorescent reporters to identify such regulators.

Widespread chromatin context-dependencies of DNA 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.

Predicting the DNA binding specificity of mutated transcription factors using family-level biophysically interpretable machine learning.

Sequence-specific interactions of transcription factors (TFs) with genomic DNA underlie many cellular processes. High-throughput binding assays coupled with computational analysis have made it possible to accurately define such sequence recognition in a biophysically interpretable yet mechanism-agonistic way for individual TFs. The fact that such sequence-to-affinity models are now available for hundreds of TFs provides new avenues for predicting how the DNA binding specificity of a TF changes when its protein sequence is mutated.

Defining the fine structure of promoter activity on a genome-wide scale with CISSECTOR.

Classic promoter mutagenesis strategies can be used to study how proximal promoter regions regulate the expression of particular genes of interest. This is a laborious process, in which the smallest sub-region of the promoter still capable of recapitulating expression in an ectopic setting is first identified, followed by targeted mutation of putative transcription factor binding sites. Massively parallel reporter assays such as survey of regulatory elements (SuRE) provide an alternative way to study millions of promoter fragments in parallel.

Nonlinear control of transcription through enhancer-promoter interactions.

Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs). However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity.

Protocol: A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break Repair.

DNA double-strand breaks (DSBs) can be repaired through various pathways. Understanding how these pathways are regulated is of great interest for cancer research and optimization of gene editing. The local chromatin environment can affect the balance between repair pathways, but this is still poorly understood.

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. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations.

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 toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood.

Promoter-Intrinsic and Local Chromatin Features Determine Gene Repression in LADs.

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.

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.

Automatically visualise and analyse data on pathways using PathVisioRPC from any programming environment.

Background: Biological pathways are descriptive diagrams of biological processes widely used for functional analysis of differentially expressed genes or proteins. Primary data analysis, such as quality control, normalisation, and statistical analysis, is often performed in scripting languages like R, Perl, and Python. Subsequent pathway analysis is usually performed using dedicated external applications.