Chromatin arranges in filaments of blobs with nanoscale functional zonation, bioRxiv, 2019-03-05
Three-dimensional (3D) chromatin organisation plays a key role in regulating genome function in higher eukaryotes. Despite recognition that the genome partitions into ~1Mb-sized topological associated domains (TADs) based on ensemble Hi-C measurements, many features of the physical organisation at the single cell level remain underexplored. Using 3D super-resolution microscopy, we reveal a sequential curvilinear arrangement of globular chromatin domains with viscoelastic properties (‘blobs’) juxtaposed to an RNA-populated interchromatin (IC) network. Quantitative mapping of genome function markers uncovers a zonal distribution, with RNA-binding factors concentrated in the IC, confinement of structural proteins and transcriptionally activepermissive marks to chromatin domain surfaces, and enrichment of repressive marks towards the interior. This correlation between nanoscale topology and genome function is relaxed in postreplicative chromatin, accentuated in replicative senescence, persists upon ATP depletion and hyperosmolarity induced chromatin condensation and, remarkably, after inactivation of cohesin. Our findings support a model of a higher-order chromatin architecture on the size level of TADs that creates and modulates distinct functional environments through a combination of biophysical parameters such as density and ATP-driven processes such as replication and transcription, but independent of cohesin.
biorxiv genomics 0-100-users 2019Functional dissection of TADs reveals non-essential and instructive roles in regulating gene expression, bioRxiv, 2019-03-05
AbstractThe genome is organized in megabase-sized three-dimensional units, called Topologically Associated Domains (TADs), that are separated by boundaries. TADs bring distant cis-regulatory elements into proximity, a process dependent on the cooperative action of cohesin and the DNA binding factor CTCF. Surprisingly, genome-wide depletion of CTCF has little effect on transcription, yet structural variations affecting TADs have been shown to cause gene misexpression and congenital disease. Here, we investigate TAD function in vivo in mice by systematically editing components of TAD organization at the Sox9Kcnj locus. We find that TADs are formed by a redundant system of CTCF sites requiring the removal of all major sites within the TAD and at the boundary for two neighboring TADs to fuse. TAD fusion resulted in leakage of regulatory activity from the Sox9 to the Kcnj TAD, but no major changes in gene expression. This indicates that TAD structures provide robustness and precision, but are not essential for developmental gene regulation. Gene misexpression and resulting disease phenotypes, however, were attained by re-directing regulatory activity through inversions andor the re-positioning of boundaries. Thus, efficient re-wiring of enhancer promoter interaction and aberrant disease causing gene activation is not induced by a mere loss of insulation but requires the re-direction of contacts.
biorxiv genetics 0-100-users 2019Reconstructing the ecology of a Jurassic pseudoplanktonic megaraft colony, bioRxiv, 2019-03-05
AbstractPseudoplanktonic crinoid megaraft colonies are an enigma of the Jurassic. They are among the largest in-situ invertebrate accumulations ever to exist in the Phanerozoic fossil record. These megaraft colonies and are thought to have developed as floating filter-feeding communities due to an exceptionally rich relatively predator free oceanic niche, high in the water column enabling them to reach high densities on these log rafts. However, this pseudoplanktonic hypothesis has never actually been quantitatively tested and some researchers have cast doubt that this mode of life was even possible. The ecological structure of the crinoid colony is resolved using spatial point process techniques and its longevity using moisture diffusion models. Using spatial analysis we found that the crinoids would have trailed preferentially positioned at the back of migrating structures in the regions of least resistance, consistent with a floating, not benthic ecology. Additionally, we found using a series of moisture diffusion models at different log densities and sizes that ecosystem collapse did not take place solely due to colonies becoming overladen as previously assumed. We have found that these crinoid colonies studied could have existed for greater than 10 years, even up to 20 years exceeding the life expectancy of modern documented megaraft systems with implications for the role of modern raft communities in the biotic colonisation of oceanic islands and intercontinental dispersal of marine and terrestrial species.Significance statementTransoceanic rafting is the principle mechanism for the biotic colonisation of oceanic island ecosystems. However, no historic records exist of how long such biotic systems lasted. Here, we use a deep-time example from the Early Jurassic to test the viability of these pseudoplanktonic systems, resolving for the first time whether these systems were truly free floating planktonic and viable for long enough to allow its inhabitants to grow to maturity. Using spatial methods we show that these colonies have a comparable structure to modern marine pesudoplankton on maritime structures, whilst the application of methods normally used in commercial logging is used to demonstrate the viability of the system which was capable of lasting up to 20 years.
biorxiv paleontology 0-100-users 2019Exploiting evolutionary herding to control drug resistance in cancer, bioRxiv, 2019-03-04
AbstractDrug resistance mediated by clonal evolution is arguably the biggest problem in cancer therapy today. However, evolving resistance to one drug may come at a cost of decreased growth rate or increased sensitivity to another drug due to evolutionary trade-offs. This weakness can be exploited in the clinic using an approach called ‘evolutionary herding’ that aims at controlling the tumour cell population to delay or prevent resistance. However, recapitulating cancer evolutionary dynamics experimentally remains challenging. Here we present a novel approach for evolutionary herding based on a combination of single-cell barcoding, very large populations of 108–109 cells grown without re-plating, longitudinal non-destructive monitoring of cancer clones, and mathematical modelling of tumour evolution. We demonstrate evolutionary herding in non-small cell lung cancer, showing that herding allows shifting the clonal composition of a tumour in our favour, leading to collateral drug sensitivity and proliferative fitness costs. Through genomic analysis and single-cell sequencing, we were also able to determine the mechanisms that drive such evolved sensitivity. Our approach allows modelling evolutionary trade-offs experimentally to test patient-specific evolutionary herding strategies that can potentially be translated into the clinic to control treatment resistance.
biorxiv cancer-biology 0-100-users 2019Genomic decoding of neuronal depolarization by stimulus-specific NPAS4 heterodimers, bioRxiv, 2019-03-04
Cells regulate gene expression in response to salient external stimuli. In neurons, depolarization leads to the expression of inducible transcription factors (ITFs) that direct subsequent gene regulation. Depolarization encodes both neuronal action potential (AP) output and synaptic inputs, via excitatory postsynaptic potentials (EPSPs). However, it is unclear if different types of depolarizing signals can be transformed by an ITF into distinct modes of genomic regulation. Here, we show that APs and EPSPs in the murine hippocampus trigger two spatially segregated and molecularly distinct mechanisms that lead to the expression of the ITF NPAS4. These two pathways culminate in the assembly of unique, stimulus-specific NPAS4 heterodimers that exhibit distinctive DNA binding patterns. Thus, NPAS4 independently communicates increases in neuronal spiking output and synaptic inputs to the nucleus, enabling gene regulation to be tailored to the type of depolarizing activity experienced by a neuron.
biorxiv neuroscience 0-100-users 2019Compositional Data Analysis is necessary for simulating and analyzing RNA-Seq data, bioRxiv, 2019-03-02
Seq techniques (e.g. RNA-Seq) generate compositional datasets, i.e. the number of fragments sequenced is not proportional to the total RNA present. Thus, datasets carry only relative information, even though absolute RNA copy numbers are often of interest. Current normalization methods assume most features are not changing, which can lead to misleading conclusions when there are large shifts. However, there are few real datasets and no simulation protocols currently available that can directly benchmark methods when such large shifts occur.We present absSimSeq, an R package that simulates compositional data in the form of RNA-Seq reads. We tested several tools used for RNA-Seq differential analysis sleuth, DESeq2, edgeR, limma, sleuth and ALDEx2 (which explicitly takes a compositional approach). For these tools, we compared their standard normalization to either “compositional normalization”, which uses log-ratios to anchor the data on a set of negative control features, or RUVSeq, another tool that directly uses negative control features.We show that common normalizations result in reduced performance with current methods when there is a large change in the total RNA per cell. Performance improves when spike-ins are included and used by a compositional approach, even if the spike-ins have substantial variation. In contrast, RUVSeq, which normalizes count data rather than compositional data, has poor performance. Further, we show that previous criticisms of spike-ins did not take into account the compositional nature of the data. We conclude that absSimSeq can generate more representative datasets for testing performance, and that spike-ins should be more broadly used in a compositional manner to minimize misleading conclusions from differential analyses.
biorxiv bioinformatics 0-100-users 2019