Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, ...but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here-particularly the large-scale topological changes and hub transience-will apply to other biological networks, including complex sub-systems in higher eukaryotes.
IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that ...bends the DNA by over 160°. HU is the most conserved NAP, which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. The sequence-specific binding profile of IHF encompasses ∼30% of all operons, though the expression of <10% of these is affected by its deletion suggesting combinatorial control or a molecular backup. The binding profile for HU is reflective of relatively non-specific binding to the chromosome, however, with a preference for A/T-rich DNA. The HU regulon comprises highly conserved genes including those that are essential and possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments, where possible, of each subunit of IHF and HU in the absence of the other subunit, we define genome-wide maps of DNA binding of the proteins in their hetero- and homodimeric forms.
Canonical Wnt/beta-catenin signalling regulates self-renewal and lineage selection within the mammalian epidermis. Although the transcriptional response of keratinocytes that receive a Wnt signal is ...well characterized, little is known about the mechanism by which keratinocytes in proximity to the Wnt-receiving cell are co-opted to undergo a change in cell fate.
To address this, we perform single-cell RNA-sequencing on mouse keratinocytes co-cultured with and without beta-catenin-activated neighbouring cells. We identify five distinct cell states in cultures that had not been exposed to the beta-catenin stimulus and show that the stimulus redistributes wild-type subpopulation proportions. Using temporal single-cell analysis, we reconstruct the cell fate change induced by Wnt activation from neighbouring cells. Gene expression heterogeneity is reduced in neighbouring cells and this effect is most dramatic for protein synthesis-associated genes. Changes in gene expression are accompanied by a shift to a more proliferative stem cell state. By integrating imaging and reconstructed sequential gene expression changes during the state transition we identify transcription factors, including Smad4 and Bcl3, that are responsible for effecting the transition in a contact-dependent manner.
Our data indicate that non-cell autonomous Wnt/beta-catenin signalling decreases transcriptional heterogeneity. This furthers our understanding of how epidermal Wnt signalling orchestrates regeneration and self-renewal.
MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL ...and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP-seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP-seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication-related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription.
The 3′ untranslated regions (3′ UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect ...processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3′ UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3′ UTR and stable 3′ UTR-derived fragments of unknown function. Cleavage of the long 3′ UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3′ UTR-derived RNAs.
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•Axons and cell bodies of sympathetic neurons express distinct 3′ UTR isoforms•Axon-specific short 3′ UTR isoforms are generated by local cleavage of longer 3′ UTRs•A protein complex containing Ago2, Upf1, HuD, and Pabpc4 mediates the 3′ UTR cleavage
Andreassi et al. show widespread differential usage of 3′ UTR in axons and cell bodies of sympathetic neurons. In axons, the cleavage of a longer 3′ UTR of Impa1 generates a shorter isoform that is stable, polyadenylated, and necessary for maintaining axon integrity.
Indirect development with an intermediate larva exists in all major animal lineages
, which makes larvae central to most scenarios of animal evolution
. Yet how larvae evolved remains disputed. Here ...we show that temporal shifts (that is, heterochronies) in trunk formation underpin the diversification of larvae and bilaterian life cycles. We performed chromosome-scale genome sequencing in the annelid Owenia fusiformis with transcriptomic and epigenomic profiling during the life cycles of this and two other annelids. We found that trunk development is deferred to pre-metamorphic stages in the feeding larva of O. fusiformis but starts after gastrulation in the non-feeding larva with gradual metamorphosis of Capitella teleta and the direct developing embryo of Dimorphilus gyrociliatus. Accordingly, the embryos of O. fusiformis develop first into an enlarged anterior domain that forms larval tissues and the adult head
. Notably, this also occurs in the so-called 'head larvae' of other bilaterians
, with which the O. fusiformis larva shows extensive transcriptomic similarities. Together, our findings suggest that the temporal decoupling of head and trunk formation, as maximally observed in head larvae, facilitated larval evolution in Bilateria. This diverges from prevailing scenarios that propose either co-option
or innovation
of gene regulatory programmes to explain larva and adult origins.
DNA cytosine methylation regulates gene expression in mammals. In bacteria, its role in gene expression and genome architecture is less understood. Here we perform high-throughput sequencing of ...bisulfite-treated genomic DNA from Escherichia coli K12 to describe, for the first time, the extent of cytosine methylation of bacterial DNA at single-base resolution. Whereas most target sites (C(m)CWGG) are fully methylated in stationary phase cells, many sites with an extended CC(m)CWGG motif are only partially methylated in exponentially growing cells. We speculate that these partially methylated sites may be selected, as these are slightly correlated with the risk of spontaneous, non-synonymous conversion of methylated cytosines to thymines. Microarray analysis in a cytosine methylation-deficient mutant of E. coli shows increased expression of the stress response sigma factor RpoS and many of its targets in stationary phase. Thus, DNA cytosine methylation is a regulator of stationary phase gene expression in E. coli.
Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression ...and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.
The vertebrate 'neural plate border' is a transient territory located at the edge of the neural plate containing precursors for all ectodermal derivatives: the neural plate, neural crest, placodes ...and epidermis. Elegant functional experiments in a range of vertebrate models have provided an in-depth understanding of gene regulatory interactions within the ectoderm. However, these experiments conducted at tissue level raise seemingly contradictory models for fate allocation of individual cells. Here, we carry out single cell RNA sequencing of chick ectoderm from primitive streak to neurulation stage, to explore cell state diversity and heterogeneity. We characterise the dynamics of gene modules, allowing us to model the order of molecular events which take place as ectodermal fates segregate. Furthermore, we find that genes previously classified as neural plate border 'specifiers' typically exhibit dynamic expression patterns and are enriched in either neural, neural crest or placodal fates, revealing that the neural plate border should be seen as a heterogeneous ectodermal territory and not a discrete transitional transcriptional state. Analysis of neural, neural crest and placodal markers reveals that individual NPB cells co-express competing transcriptional programmes suggesting that their ultimate identify is not yet fixed. This population of 'border located undecided progenitors' (BLUPs) gradually diminishes as cell fate decisions take place. Considering our findings, we propose a probabilistic model for cell fate choice at the neural plate border. Our data suggest that the probability of a progenitor's daughters to contribute to a given ectodermal derivative is related to the balance of competing transcriptional programmes, which in turn are regulated by the spatiotemporal position of a progenitor.
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