Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program.

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.

Enhancer loops appear stable during development and are associated with paused polymerase.

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the ß-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase.

A discrete transition zone organizes the topological and regulatory autonomy of the adjacent tfap2c and bmp7 genes.

Despite the well-documented role of remote enhancers in controlling developmental gene expression, the mechanisms that allocate enhancers to genes are poorly characterized. Here, we investigate the cis-regulatory organization of the locus containing the Tfap2c and Bmp7 genes in vivo, using a series of engineered chromosomal rearrangements. While these genes lie adjacent to one another, we demonstrate that they are independently regulated by distinct sets of enhancers, which in turn define non-overlapping regulatory domains. Chromosome conformation capture experiments reveal a corresponding partition of the locus in two distinct structural entities, demarcated by a discrete transition zone. The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation. This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one. Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.

A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia.

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

FourCSeq: analysis of 4C sequencing data.

MOTIVATION: Circularized Chromosome Conformation Capture (4C) is a powerful technique for studying the spatial interactions of a specific genomic region called the 'viewpoint' with the rest of the genome, both in a single condition or comparing different experimental conditions or cell types. Observed ligation frequencies typically show a strong, regular dependence on genomic distance from the viewpoint, on top of which specific interaction peaks are superimposed. Here, we address the computational task to find these specific peaks and to detect changes between different biological conditions. RESULTS: We model the overall trend of decreasing interaction frequency with genomic distance by fitting a smooth monotonically decreasing function to suitably transformed count data. Based on the fit, z-scores are calculated from the residuals, and high z-scores are interpreted as peaks providing evidence for specific interactions. To compare different conditions, we normalize fragment counts between samples, and call for differential contact frequencies using the statistical method DESEQ2: adapted from RNA-Seq analysis. AVAILABILITY AND IMPLEMENTATION: A full end-to-end analysis pipeline is implemented in the R package FourCSeq available at www.bioconductor.org. CONTACT: felix.klein@embl.de or whuber@embl.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A chemical-genetic interaction map of small molecules using high-throughput imaging in cancer cells.

Small molecules often affect multiple targets, elicit off-target effects, and induce genotype-specific responses. Chemical genetics, the mapping of the genotype dependence of a small molecule's effects across a broad spectrum of phenotypes can identify novel mechanisms of action. It can also reveal unanticipated effects and could thereby reduce high attrition rates of small molecule development pipelines. Here, we used high-content screening and image analysis to measure effects of 1,280 pharmacologically active compounds on complex phenotypes in isogenic cancer cell lines which harbor activating or inactivating mutations in key oncogenic signaling pathways. Using multiparametric chemical-genetic interaction analysis, we observed phenotypic gene-drug interactions for more than 193 compounds, with many affecting phenotypes other than cell growth. We created a resource termed the Pharmacogenetic Phenome Compendium (PGPC), which enables exploration of drug mode of action, detection of potential off-target effects, and the generation of hypotheses on drug combinations and synergism. For example, we demonstrate that MEK inhibitors amplify the viability effect of the clinically used anti-alcoholism drug disulfiram and show that the EGFR inhibitor tyrphostin AG555 has off-target activity on the proteasome. Taken together, this study demonstrates how combining multiparametric phenotyping in different genetic backgrounds can be used to predict additional mechanisms of action and to reposition clinically used drugs.

Gain of CTCF-Anchored Chromatin Loops Marks the Exit from Naive Pluripotency.

The genome of pluripotent stem cells adopts a unique three-dimensional architecture featuring weakly condensed heterochromatin and large nucleosome-free regions. Yet, it is unknown whether structural loops and contact domains display characteristics that distinguish embryonic stem cells (ESCs) from differentiated cell types. We used genome-wide chromosome conformation capture and super-resolution imaging to determine nuclear organization in mouse ESC and neural stem cell (NSC) derivatives. We found that loss of pluripotency is accompanied by widespread gain of structural loops. This general architectural change correlates with enhanced binding of CTCF and cohesins and more pronounced insulation of contacts across chromatin boundaries in lineage-committed cells. Reprogramming NSCs to pluripotency restores the unique features of ESC domain topology. Domains defined by the anchors of loops established upon differentiation are enriched for developmental genes. Chromatin loop formation is a pervasive structural alteration to the genome that accompanies exit from pluripotency and delineates the spatial segregation of developmentally regulated genes.

Polarization-splitting common-path interferometer based on a zero-twist liquid crystal display.

We present a compact optical polarization-splitting common-path interferometer based on a zero-twist liquid crystal display (LCD). The LCD is encoded with a diffraction grating pattern and illuminated with a polarization state with both horizontal and vertical components. The polarization component perpendicular to the director axis of the liquid crystal molecules is not affected by the LCD and forms the reference beam. However, the polarization component parallel to the director axis is diffracted at an angle determined by the period of the grating. By imposing an analyzer polarizer, these two beams create an interferogram that can either display retardance patterns encoded onto the LCD or analyze external birefringent optical elements. The programmability of the system allows new ways of increasing the utility of the interferograms. Experimental results are provided, including the visualization of optical vortices with different and opposite topological charges.