HiTIPS: High-Throughput Image Processing Software for the Study of Nuclear Architecture and Gene Expression.

High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software for image analysis workflows does not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new algorithms for existing analysis pipelines and to adding new analysis pipelines through separate plugins. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI analysis platform for a variety of cell biology applications.

BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome.

Histone-modifying systems play fundamental roles in gene regulation and the development of multicellular organisms. Histone modifications that are enriched at gene regulatory elements have been heavily studied, but the function of modifications found more broadly throughout the genome remains poorly understood. This is exemplified by histone H2A monoubiquitylation (H2AK119ub1), which is enriched at Polycomb-repressed gene promoters but also covers the genome at lower levels. Here, using inducible genetic perturbations and quantitative genomics, we found that the BAP1 deubiquitylase plays an essential role in constraining H2AK119ub1 throughout the genome. Removal of BAP1 leads to pervasive genome-wide accumulation of H2AK119ub1, which causes widespread reductions in gene expression. We show that elevated H2AK119ub1 preferentially counteracts Ser5 phosphorylation on the C-terminal domain of RNA polymerase II at gene regulatory elements and causes reductions in transcription and transcription-associated histone modifications. Furthermore, failure to constrain pervasive H2AK119ub1 compromises Polycomb complex occupancy at a subset of Polycomb target genes, which leads to their derepression, providing a potential molecular rationale for why the BAP1 ortholog in Drosophila has been characterized as a Polycomb group gene. Together, these observations reveal that the transcriptional potential of the genome can be modulated by regulating the levels of a pervasive histone modification.

Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells.

How chromosome organization is related to genome function remains poorly understood. Cohesin, loop extrusion, and CCCTC-binding factor (CTCF) have been proposed to create topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find, as in other cell types, that cohesin is required to create TADs and regulate A/B compartmentalization. However, in the absence of cohesin, we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system and are dependent on PRC1. Importantly, we discover that cohesin counteracts these polycomb-dependent interactions, but not interactions between super-enhancers. This disruptive activity is independent of CTCF and insulation and appears to modulate gene repression by the polycomb system. Therefore, we discover that cohesin disrupts polycomb-dependent chromosome interactions to modulate gene expression in embryonic stem cells.

PRC1 Catalytic Activity Is Central to Polycomb System Function.

The Polycomb repressive system is an essential chromatin-based regulator of gene expression. Despite being extensively studied, how the Polycomb system selects its target genes is poorly understood, and whether its histone-modifying activities are required for transcriptional repression remains controversial. Here, we directly test the requirement for PRC1 catalytic activity in Polycomb system function. To achieve this, we develop a conditional mutation system in embryonic stem cells that completely removes PRC1 catalytic activity. Using this system, we demonstrate that catalysis by PRC1 drives Polycomb chromatin domain formation and long-range chromatin interactions. Furthermore, we show that variant PRC1 complexes with DNA-binding activities occupy target sites independently of PRC1 catalytic activity, providing a putative mechanism for Polycomb target site selection. Finally, we discover that Polycomb-mediated gene repression requires PRC1 catalytic activity. Together these discoveries provide compelling evidence that PRC1 catalysis is central to Polycomb system function and gene regulation.

Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression.

The Polycomb system modifies chromatin and plays an essential role in repressing gene expression to control normal mammalian development. However, the components and mechanisms that define how Polycomb protein complexes achieve this remain enigmatic. Here, we use combinatorial genetic perturbation coupled with quantitative genomics to discover the central determinants of Polycomb-mediated gene repression in mouse embryonic stem cells. We demonstrate that canonical Polycomb repressive complex 1 (PRC1), which mediates higher-order chromatin structures, contributes little to gene repression. Instead, we uncover an unexpectedly high degree of synergy between variant PRC1 complexes, which is fundamental to gene repression. We further demonstrate that variant PRC1 complexes are responsible for distinct pools of H2A monoubiquitylation that are associated with repression of Polycomb target genes and silencing during X chromosome inactivation. Together, these discoveries reveal a new variant PRC1-dependent logic for Polycomb-mediated gene repression.

Polycomb repressive complex 1 shapes the nucleosome landscape but not accessibility at target genes.

Polycomb group (PcG) proteins are transcriptional repressors that play important roles in regulating gene expression during animal development. In vitro experiments have shown that PcG protein complexes can compact chromatin to limit the activity of chromatin remodeling enzymes and access of the transcriptional machinery to DNA. In fitting with these ideas, gene promoters associated with PcG proteins have been reported to be less accessible than other gene promoters. However, it remains largely untested in vivo whether PcG proteins define chromatin accessibility or other chromatin features. To address this important question, we examine the chromatin accessibility and nucleosome landscape at PcG protein-bound promoters in mouse embryonic stem cells using the assay for transposase accessible chromatin (ATAC)-seq. Combined with genetic ablation strategies, we unexpectedly discover that although PcG protein-occupied gene promoters exhibit reduced accessibility, this does not rely on PcG proteins. Instead, the Polycomb repressive complex 1 (PRC1) appears to play a unique role in driving elevated nucleosome occupancy and decreased nucleosomal spacing in Polycomb chromatin domains. Our new genome-scale observations argue, in contrast to the prevailing view, that PcG proteins do not significantly affect chromatin accessibility and highlight an underappreciated complexity in the relationship between chromatin accessibility, the nucleosome landscape, and PcG-mediated transcriptional repression.

RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes.

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation.

Early-late genes of the ecdysone cascade as models for transcriptional studies.

The DHR3 and Hr4 early-late genes of the ecdysone cascade are described as models for transcriptional studies in Drosophila cells. In a set of experiments, it became clear that these genes are a convenient and versatile system for research into the physiological conditions upon 20-hydroxyecdysone induction. DHR3 and Hr4 gene transcription is characterized by fast activation kinetics, which enables transcriptional studies without the influence of indirect effects. A limited number of activated genes (only 73 genes are induced one hour after treatment) promote the selectivity of transcriptional studies via 20-hydroxyecdysone induction. DHR3 and Hr4 gene expression is dose dependent, is completely controlled by the hormone titer and decreases within hours of 20-hydroxyecdysone withdrawal. The DHR3 and Hr4 gene promoters become functional within 20 minutes after induction, which makes them useful tools for investigation if the early activation process. Their transcription is controlled by the RNA polymerase II pausing mechanism, which is widespread in the genome of Drosophila melanogaster but is still underinvestigated. Uniform expression activation of the DHR3 and Hr4 genes in a cell population was confirmed at both the RNA and protein levels. Homogeneity of the transcription response makes DHR3/Hr4 system valuable for investigation of the protein dynamics during transcription induction.