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Transposable elements (TEs) regulate diverse biological processes, from early development to cancer. Expression of young TEs is difficult to measure with next-generation, single-cell sequencing technologies because their highly repetitive nature means that short complementary DNA reads cannot be unambiguously mapped to a specific locus. Single CELl LOng-read RNA-sequencing (CELLO-seq) combines long-read single cell RNA-sequencing with computational analyses to measure TE expression at unique loci. We used CELLO-seq to assess the widespread expression of TEs in two-cell mouse blastomeres as well as in human induced pluripotent stem cells. Across both species, old and young TEs showed evidence of locus-specific expression with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to the reference with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed large heterogeneity, with TEs within a class showing different patterns of correlation and suggesting distinct regulatory mechanisms.

The X inactive-specific transcript (Xist) gene is the master regulator of X chromosome inactivation in mammals. Xist produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in cis. Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.

CIZ1 forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner and is required for accurate maintenance of polycomb targets genome-wide. Here we address requirements for assembly formation and show that CIZ1 undergoes two direct interactions with Xist, via independent N- and C-terminal domains. Interaction with Xist, assembly at Xi, and complexity of self-assemblies formed in vitro are modulated by two alternatively spliced glutamine-rich prion-like domains (PLD1 and 2). PLD2 is dispensable for accumulation at existing CIZ1–Xi assemblies in wild-type cells but is required in CIZ1-null cells where targeting, assembly, and enrichment for H3K27me3 and H2AK119ub occur de novo. In contrast, PLD1 is required for both de novo assembly and accumulation at preexisting assemblies and, in vitro, drives formation of a stable fibrillar network. Together they impart affinity for RNA and a complex relationship with repeat E of Xist. These data show that alternative splicing of two PLDs modulates CIZ1’s ability to build large RNA–protein assemblies.

RNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5′ splice sites. m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5′ splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream from a 5′ splice site that is suppressed in the presence of m6A and upstream of a 5′ splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.

Xist RNA directs the process of X-chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, enabling us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation, and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a distinct function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique dynamic properties of Xist RNA.One Sentence SummaryVisualizing Xist RNA dynamics in single cells during X chromosome inactivation

SummaryCIP1-interacting zinc finger protein 1 (CIZ1) forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner. Here we address the requirements for assembly formation, and show that CIZ1 interacts directly with Xist via two independent domains in its N- and C-terminus. Interaction with Xist repeat E, assembly at Xi in cells, and the complexity of self-assemblies formed in vitro, are all modulated by alternatively-spliced exons that include two glutamine-rich prion-like domains (PLD1 and PLD2), both conditionally excluded from the N-terminal domain. Exclusion of PLD1 alone is sufficient to abrogate de novo establishment of new CIZ1 assemblies and Xi territories enriched for H3K27me3 in CIZ1-null fibroblasts. Together the data suggest that PLD1-driven CIZ1 assemblies form at Xi, are nucleated by interaction with Xist and amplified by multivalent interaction with RNA, so implicating a polyglutamine tract in the maintenance of epigenetic state.

Summary Transcriptional fidelity depends on accurate promoter selection and initiation from the correct sites. In yeast, H3K36me3-mediated recruitment of the Rpd3S HDAC complex to gene bodies suppresses spurious transcription initiation. Here we describe an equivalent pathway in metazoans. PWWP2A/B is an H3K36me3 reader that forms a stable complex with HDAC1/2. We used CAGE-seq to profile all transcription initiation sites in wild-type mESCs and cells lacking PWWP2A/B. Loss of PWWP2A/B enhances spurious initiation from intragenic sites present in wild-type mESCs, and this effect is associated with increased levels of initiating Pol-II and histone acetylation. Spurious initiation events in Pwwp2a/b DKO mESCs do not overlap in genomic location or chromatin features with spurious sites that arise in Dnmt3b KO mESCs, previously reported to function in the suppression of intragenic transcriptional initiation, suggesting these pathways function cooperatively in maintaining the fidelity of transcription initiation in metazoans., Graphical Abstract, Highlights • Loss of PWWP2A/B leads to increased levels of spurious transcription initiation • Spurious TSS sites are predominantly in the gene bodies of highly expressed genes • Spurious sites are marked with increased histone acetylation and initiating Pol II • PWWP2-spurious TSSs are distinct from those caused by DNMT3B loss, Molecular Biology; Molecular Mechanism of Gene Regulation; Stem Cells Research

Transposable elements (TEs) regulate diverse biological processes, from early development to cancer. Expression of young TEs is difficult to measure with next-generation, single-cell sequencing technologies because their highly repetitive nature means that short complementary DNA reads cannot be unambiguously mapped to a specific locus. Single CELl LOng-read RNA-sequencing (CELLO-seq) combines long-read single cell RNA-sequencing with computational analyses to measure TE expression at unique loci. We used CELLO-seq to assess the widespread expression of TEs in two-cell mouse blastomeres as well as in human induced pluripotent stem cells. Across both species, old and young TEs showed evidence of locus-specific expression with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to the reference with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed large heterogeneity, with TEs within a class showing different patterns of correlation and suggesting distinct regulatory mechanisms.

RNAN6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed co-transcriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells (mESCs). We find that around 10% m6A peaks are in introns, often close to 5’-splice sites. RNA m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Interestingly, acute dTAG depletion of METTL3 reveals that inclusion of m6A-bearing alternative introns/exons in the nascent transcriptome is disrupted. For terminal or variable-length exons, m6A peaks are generally located upstream of a repressed 5’-splice site, and downstream of an enhanced 5’-splice site. Intriguingly, genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Our findings demonstrate a direct crosstalk between m6A machinery and the regulation of RNA processing.

RNA N(6)-methyladenosine (m(6)A) modification plays important roles in multiple aspects of RNA regulation. m(6)A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m(6)A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m(6)A peaks are located in alternative introns/exons, often close to 5′ splice sites. m(6)A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5′ splice sites that are proximal to m(6)A peaks. For terminal or variable-length exons, m(6)A peaks are generally located on or immediately downstream from a 5′ splice site that is suppressed in the presence of m(6)A and upstream of a 5′ splice site that is promoted in the presence of m(6)A. Genes with the most immediate effects on splicing include several components of the m(6)A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m(6)A machinery and the regulation of RNA splicing.

Many intricate pathways contribute to the timely control of gene expression during development. Polycomb repressive complexes (PRC1 and PRC2) and long non-coding RNAs (lncRNAs) are players associated with gene repression in various developmental processes such as X chromosome inactivation (XCI) and genomic imprinting. Historically, lncRNAs were proposed to directly recruit PRC2. However, recent evidence suggests that promiscuous interactions between PRC2 and RNA fine-tune the function of the complex through a multiplicity of mechanisms. A PRC2-recruitment model was definitively overturned in the paradigm of XCI by Xist RNA, being replaced by a novel mechanism which puts PRC1 in the spotlight. This review focuses on these recent advances in understanding the interplay between RNA and Polycomb complexes for gene expression control.

Background: X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed. High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5’ end of the transcript, and also further 3’, in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist–mediated gene silencing. Methods: We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5’ m6A region in interspecific XX mouse embryonic stem cells (mESCs). Following induction of Xist RNA expression, we assay chromosome silencing using allelic RNA-seq and Xist m6A distribution using m6A-seq. Additionally, we use Xist RNA FISH to analyse the effect of deleting the 5’ m6A region on the function of the endogenous Xist promoter. We purify epitope tagged RBM15 from mESCs, and then apply MS/MS analysis to define the RBM15 interactome. Results: We show that a deletion encompassing the entire Xist 5’ m6A region results in a modest reduction in Xist-mediated silencing, and that the 5’ m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5’ m6A region without affecting the modification in exon VII. We show that in mESCs, RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism. Conclusions: Our findings support that RBM15 binding to the Xist A-repeat recruits the m6A complex to the 5’ Xist m6A region and that this region plays a role in Xist-mediated chromosome silencing.

Summary: We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation. : Jansz et al. report that the chromatin protein Smchd1 depends on polycomb repressive complex 1-mediated ubiquitylation of histone H2A for its recruitment to the inactive X chromosome and for its protein stability. These data have implications for Smchd1 targeting genome-wide. Keywords: Smchd1, X inactivation, Xist, PRC1, Hnrnpk, Ring1B

Polycomb steps to inactivate X XX females silence one of their X chromosomes. This involves a process whereby a noncoding RNA known as Xist coats one of the X chromosomes and recruits chromatin silencing factors. The Polycomb complexes PRC1 and PRC2 are also known to be involved in X chromosome inactivation. Almeida et al. elucidate a key role of a specific complex, PCGF3/5-PRC1, in initiating Polycomb recruitment by Xist RNA. They further demonstrate that Polycomb recruitment is critical for Xist-mediated chromosome silencing and female embryogenesis. Science , this issue p. 1081

The biology of non-coding RNA (ncRNA) and the regulation of mammalian gene expression is a rapidly expanding field. In this review, we consider how recent advances in technology, enabling the precise mapping of modifications to RNA transcripts, has provided new opportunities to dissect post-transcriptional gene regulation. With this has come the realisation that in the absence of translation, the modification of ncRNAs may play a fundamental role in their regulation, protein interactome and subsequent downstream effector functions. We focus upon modification of RNA by N6-methyladenosine (m6A); its readers, writers and erasers, before considering the differing role of m6A modified lncRNAs MALAT1 and Xist.This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

Chromosome silencing by Xist RNA occurs in two steps; localisation in cis within the nuclear matrix to form a domain that corresponds to the territory of the inactive X chromosome elect, and transduction of silencing signals from Xist RNA to the underlying chromatin. Key factors that mediate these processes have been identified in a series of recent studies that harnessed comprehensive proteomic or genetic screening strategies. In this review we discuss these findings in light of prior knowledge both of Xist-mediated silencing and known functions/properties of the novel factors.

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing., Xist RNA is the master regulator of X chromosome inactivation. Here the authors describe a systematic analysis of Xist-mediated allelic silencing in mouse ESC models and define the contribution of different pathways that regulate gene silencing.

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), and formation of two mega-domains. In this study we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 null cells revealed the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred in an acute SmcHD1 knockout model, but in this case, independent of Xi gene de-repression. We conclude that SmcHD1 is a key factor in antagonising TAD formation on Xi. NOTE: MRG and TBN contributed equally to this work.

In mammals, a dosage compensation mechanism exists to equalize gene expression levels between male and female. This process is initiated by Xist RNA, a long noncoding RNA that mediates the transcriptional silencing of a complete chromosome. The kinetics of events occurring on the future inactive X-chromosome has been described in detail over the last 20 years. More recently, parallel studies using advanced biochemical assays and genetic screens identified key factors critical for the silencing cascade. Here, we describe the procedure adopted in one of these studies, an shRNA-based loss-of-function screen in mouse embryonic stem cells (mESCs).The screen made use of a reporter cell line in which Xist-mediated silencing could be monitored by changes in GFP fluorescence. Loss of function was achieved using a custom made bar-coded pooled shRNA library. The screen aimed to identify shRNAs that lessen Xist mediated repression of the GFP reporter. The methods that were applied are of potential relevance for the development of related screens, for example to better understand how specific repressors silence one or several genes.

Identifying the critical RNA binding proteins (RBPs) that elicit Xist mediated silencing has been a key goal in X inactivation research. Early studies implicated the Polycomb proteins, a family of factors linked to one of two major multiprotein complexes, PRC1 and PRC2 (Wang 2001 Nat. Genet. 28, 371–375 (doi:10.1038/ng574); Silva 2003 Dev. Cell 4, 481–495 (doi:10.1016/S1534-5807(03)00068-6); de Napoles 2004 Dev. Cell 7, 663–676 (doi:10.1016/j.devcel.2004.10.005); Plath 2003 Science 300, 131–135 (doi:10.1126/science.1084274)). PRC1 and PRC2 complexes catalyse specific histone post-translational modifications (PTMs), ubiquitylation of histone H2A at position lysine 119 (H2AK119u1) and methylation of histone H3 at position lysine 27 (H3K27me3), respectively, and accordingly, these modifications are highly enriched over the length of the inactive X chromosome (Xi). A key study proposed that PRC2 subunits bind directly to Xist RNA A-repeat element, a region located at the 5′ end of the transcript known to be required for Xist mediated silencing (Zhao 2008 Science 322, 750–756 (doi:10.1126/science.1163045)). Subsequent recruitment of PRC1 was assumed to occur via recognition of PRC2 mediated H3K27me3 by the CBX subunit of PRC1, as has been shown to be the case at other Polycomb target loci (Cao 2002 Science 298, 1039–1043 (doi:10.1126/science.1076997)). More recently, several reports have questioned aspects of the prevailing view, both in relation to the mechanism for Polycomb recruitment by Xist RNA and the contribution of the Polycomb pathway to Xist mediated silencing. In this article I provide an overview of our recent progress towards resolving these discrepancies. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.

Summary The Polycomb-repressive complexes PRC1 and PRC2 play a key role in chromosome silencing induced by the non-coding RNA Xist. Polycomb recruitment is initiated by the PCGF3/5-PRC1 complex, which catalyzes chromosome-wide H2A lysine 119 ubiquitylation, signaling recruitment of other PRC1 complexes, and PRC2. However, the molecular mechanism for PCGF3/5-PRC1 recruitment by Xist RNA is not understood. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt sequence encompassing the Xist B-repeat element. Deletion of XR-PID abolishes Xist-dependent Polycomb recruitment, in turn abrogating Xist-mediated gene silencing and reversing Xist-induced chromatin inaccessibility. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking XR-PID is sufficient for Xist-dependent Polycomb recruitment. Our findings define a key pathway for Polycomb recruitment by Xist RNA, providing important insights into mechanisms of chromatin modification by non-coding RNA., Graphical Abstract, Highlights • A 600 nt element in Xist RNA, XR-PID, is required for Polycomb recruitment • Deletion of XR-PID abrogates Xist-mediated chromosome silencing • hnRNPK binds XR-PID to recruit the Polycomb-initiating complex PCGF3/5-PRC1 • Tethering hnRNPK to Xist RNA bypasses the requirement for XR-PID, This study advances our understanding of the molecular mechanism of X chromosome inactivation in mammals, defining XR-PID, the critical element in Xist RNA that recruits Polycomb complexes to the inactive X chromosome, and further demonstrating that the RNA binding protein hnRNPK bridges XR-PID with the initiating Polycomb complex, PCGF3/5-PRC1.

A plethora of noncoding (nc) RNAs has been revealed through the application of high-throughput analysis of the transcriptome, and this has led to an intensive search for possible biological functions attributable to these transcripts. A major category of functional ncRNAs that has emerged is for those that are implicated in coordinate gene silencing, either in cis or in trans. The archetype for this class is the well-studied long ncRNA Xist which functions in cis to bring about transcriptional silencing of an entire X chromosome in female mammals. An important step in X chromosome inactivation is the recruitment of the Polycomb repressive complex PRC2 that mediates histone H3 lysine 27 methylation, a hallmark of the inactive X chromosome, and recent studies have suggested that this occurs as a consequence of PRC2 interacting directly with Xist RNA. Accordingly, other ncRNAs have been linked to PRC2 targeting either in cis or in trans, and here also the mechanism has been proposed to involve direct interaction between PRC2 proteins and the different ncRNAs. In this review, I discuss the evidence for and against this hypothesis, in the process highlighting alternative models and discussing experiments that, in the future, will help to resolve existing discrepancies.

The use of whole-genome pooled shRNA libraries in loss-of-function screening in tissue culture models provides an effective means to identify novel factors acting in pathways of interest. Embryonic stem cells (ESCs) offer a unique opportunity to study processes involved in stem cell pluripotency and differentiation. Here, we report a genome-wide shRNA screen in ESCs to identify novel components involved in repression of the Gata6 locus, using a cell viability-based screen, which offers the benefits of stable shRNA integration and a robust and simple protocol for hit identification. Candidate factors identified were enriched for transcription factors and included known Polycomb proteins and other chromatin-modifying factors. We identified the protein Bcor, which is known to associate in complexes with the Polycomb protein Ring1B, and verified its importance in Gata6 repression in ESCs. Potential further applications of such a screening strategy could allow the identification of factors important for regulation of gene expression and pluripotency.

Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS), suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation, unveiling a new hallmark in ORI efficiency regulation in mammalian cells., Author Summary The duplication of the genetic information of a cell starts from specific sites on the chromosomes called DNA replication origins. Their number varies from a few hundred in yeast cells to several thousands in human cells, distributed along the genome at comparable distances in both systems. An important question in the field is to understand how origins of replication are specified and regulated in the mammalian genome, as neither their location nor their activity can be directly inferred from the DNA sequence. Previous studies at individual origins and, more recently, at large scale across 1% of the human genome, have revealed that most origins overlap with transcriptional regulatory elements, and specifically with gene promoters. To gain insight into the nature of the relationship between active transcription and origin specification we have combined a genomic mapping of origins at 0.4% of the mouse genome with detailed studies of activation efficiency. The data identify two types of origins with distinct regulatory properties: highly efficient origins map at CpG island-promoters and low efficient origins locate elsewhere in association with transcriptional units. We also find a remarkable parallel organisation of the replication initiation sites and transcription start sites at efficient promoter-origins that suggests a prominent role of transcription initiation in setting the efficiency of replication origin activation.