Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and spatial proximity

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation.These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one

Cohesin is required for long-range enhancer action at the Shh locus

The regulatory landscapes of developmental genes in mammals can be complex, with enhancers spread over many hundreds of kilobases. It has been suggested that three-dimensional genome organisation, particularly topologically associating domains formed by cohesin-mediated loop extrusion, are important for enhancers to act over such large genomic distances. By coupling acute protein degradation with synthetic activation by targeted transcription factor recruitment, here we show that cohesin, but not CTCF, is required for activation of a target gene – Shh - by distant enhancers in mouse embryonic stem cells. Cohesin is not required for activation directly at the promoter or from an enhancer located closer to the Shh gene. Our findings support the hypothesis that chromatin compaction mediated by cohesin-mediated loop extrusion allows for genes to be activated by enhancers that are located many hundreds of kilobases away in the linear genome but suggests that cohesin is dispensable for more genomically close enhancers

Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture

A Mechanism of Cohesin-Dependent Loop Extrusion Organizes Zygotic Genome Architecture

Abstract Fertilization triggers assembly of higher‐order chromatin structure from a condensed maternal and a naïve paternal genome to generate a totipotent embryo. Chromatin loops and domains have been detected in mouse zygotes by single‐nucleus Hi‐C (snHi‐C), but not bulk Hi‐C. It is therefore unclear when and how embryonic chromatin conformations are assembled. Here, we investigated whether a mechanism of cohesin‐dependent loop extrusion generates higher‐order chromatin structures within the one‐cell embryo. Using snHi‐C of mouse knockout embryos, we demonstrate that the zygotic genome folds into loops and domains that critically depend on Scc1‐cohesin and that are regulated in size and linear density by Wapl. Remarkably, we discovered distinct effects on maternal and paternal chromatin loop sizes, likely reflecting differences in loop extrusion dynamics and epigenetic reprogramming. Dynamic polymer models of chromosomes reproduce changes in snHi‐C, suggesting a mechanism where cohesin locally compacts chromatin by active loop extrusion, whose processivity is controlled by Wapl. Our simulations and experimental data provide evidence that cohesin‐dependent loop extrusion organizes mammalian genomes over multiple scales from the one‐cell embryo onward

Low level of expression of C-terminally truncated human FUS causes extensive changes in the spinal cord transcriptome of asymptomatic transgenic mice

A number of mutations in a gene encoding RNA-binding protein FUS have been linked to the development of a familial form of amyotrophic lateral sclerosis known as FUS-ALS. C-terminal truncations of FUS by either nonsense or frameshift mutations lead to the development of FUS-ALS with a particularly early onset and fast progression. However, even in patients bearing these highly pathogenic mutations the function of motor neurons is not noticeably compromised for at least a couple of decades, suggesting that until cytoplasmic levels of FUS lacking its C-terminal nuclear localisation signal reaches a critical threshold, motor neurons are able to tolerate its permanent production.In order to identify how the nervous system responds to low levels of pathogenic variants of FUS we produced and characterised a mouse line, L-FUS[1-359], with a low neuronal expression level of a highly aggregation-prone and pathogenic form of C-terminally truncated FUS. In contrast to mice that express substantially higher level of the same FUS variant and develop severe early onset motor neuron pathology, L-FUS[1-359] mice do not develop any clinical or histopathological signs of motor neuron deficiency even at old age. Nevertheless, we detected substantial changes in the spinal cord transcriptome of these mice compared to their wild type littermates. We suggest that at least some of these changes reflect activation of cellular mechanisms compensating for the potentially damaging effect of pathogenic FUS production. Further studies of these mechanism might reveal effective targets for therapy of FUS-ALS and possibly, other forms of ALS