Biology subjects

Lenhard, B.

Publications and source records attributed to Lenhard, B..

29 records · Page 2Linked to original sources

bioRxiv2020

Widespread conservation and lineage-specific diversification of genome-wide DNA methylation patterns across arthropods

Cytosine methylation is an ancient epigenetic modification yet its function and extent within genomes is highly variable across eukaryotes. In mammals, methylation controls transposable elements and regulates the promoters of genes. In insects, DNA methylation is generally restricted to a small subset of transcribed genes, with both intergenic regions and transposable elements (TEs) depleted of methylation. The evolutionary origin and the function of these methylation patterns are poorly understood. Here we characterise the evolution of DNA methylation across the arthropod phylum. While the common ancestor of the arthropods had low levels of TE methylation and did not methylate promoters, both of these functions have evolved independently in centipedes and mealybugs. In contrast, methylation of the exons of a subset of transcribed genes is ancestral and widely conserved across the phylum, but has been lost in specific lineages. Remarkably the same set of genes are likely to be methylated in all species that retained exon-enriched methylation. We show that these genes have characteristic patterns of expression correlating to broad transcription initiation sites and well-positioned nucleosomes, providing new insights into potential mechanisms driving methylation patterns over hundreds of millions of years. Author SummaryAnimals develop from a single cell to form a complex organism with many specialised cells. Almost all of the fantastic variety of cells must have the same sequence of DNA, and yet they have distinct identities that are preserved even when they divide. This remarkable process is achieved by turning different sets of genes on or off in different types of cell using molecular mechanisms known as "epigenetic gene regulation". Surprisingly, though all animals need epigenetic gene regulation, there is a huge diversity in the mechanisms that they use. Characterising and explaining this diversity is crucial in understanding the functions of epigenetic pathways, many of which have key roles in human disease. We studied how one particular type of epigenetic regulation, known as DNA methylation, has evolved within arthropods. Arthropods are an extraordinarily diverse group of animals ranging from horseshoe crabs to fruit flies. We discovered that the levels of DNA methylation and where it is found within the genome changes rapidly throughout arthropod evolution. Nevertheless, there are some features of DNA methylation that seem to be the same across most arthropods-in particular we found that there is a tendency for a similar set of genes to acquire methylation of DNA in most arthropods, and that this is conserved over 350 million years. We discovered that these genes have distinct features that might explain how methylation gets targeted. Our work provides important new insights into the evolution of DNA methylation and gives some new hints to its essential functions.

genetics↗

bioRxiv2020

Germ plasm localisation dynamics mark distinct phases of transcriptional and post-transcriptional regulation control in primordial germ cells

In many animal models, primordial germ cell (PGC) development depends on maternally-deposited germ plasm to avoid somatic cell fate. Here, we show that PGCs respond to regulatory information from the germ plasm in two distinct phases and mechanisms in zebrafish. We show that PGCs commence zygotic genome activation together with the rest of the embryo with no demonstrable differences in transcriptional and chromatin accessibility levels. Thus, cytoplasmic germ plasm determinants only affect post-transcriptional stabilisation of RNAs to diverge transcriptome from somatic cells, which, unexpectedly, also activate germ cell-specific genes. Perinuclear relocalisation of germ plasm is coupled to dramatic divergence in chromatin opening and transcriptome from somatic cells characterised by PGC-specific chromatin topology. Furthermore, we reveal Tdrd7, regulator of germ plasm localisation, as crucial determinant of germ fate acquisition.

developmental biology↗

bioRxiv2019

Zebrafish embryonic tissue differentiation is marked by concurrent cell cycle dynamic and gene promoter regulatory changes

The core promoter, a stretch of DNA surrounding the transcription start site (TSS) is a major integration point for regulatory signals controlling gene transcription. The process of cell differentiation is accompanied by a marked divergence in transcriptional repertoire between cells of different fates, accompanied by changes in cellular behaviour, in particular their proliferative activity. Investigation of divergent core promoter architectures suggest distinct regulatory networks act on the core promoter, modulating cell behavior through transcriptional profile changes, which ultimately drives key transitions in cellular behaviour during embryonic development. The role that promoter-associated gene regulatory networks play in development associated transitions in cell cycle dynamics (e.g. during differentiation) however, is poorly understood. In this study we demonstrate in a developing in vivo model, how core promoter variations play a key role in defining transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. The FUCCI transgenic system, differentially marks cells in G1 and S/G2/M phases of the cell cycle and can therefore be used to separate rapidly and slowly cycling cells in vivo, by virtue of the cell cycle stage they primarily inhabit. Longitudinal assessment of cell proliferation rate during zebrafish embryo development, using this system, revealed a spatial and lineage-specific separation in cell cycling behaviour across post-gastrulation embryos. In order to investigate the role differential promoter usage plays in this process, cap analysis of gene expression (CAGE) was performed on fluorescent associated cell sorted (FACS) FUCCI zebrafish embryos going through somitogenesis, separating cells in accordance with the rate of their cell cycling. This analysis revealed a dramatic increase in lineage and tissue-specific gene expression, concurrent with a slowing of their cell cycling. Core promoters associated with rapidly cycling cells, showed broad distribution of transcription start site usage, featuring positionally constrained CCAAT-box, while slowly cycling cells favoured sharp TSS usage coupled with canonical TATA-box utilisation and enrichment of Sp1 binding sites. These results demonstrate the regulatory role of core promoters in cell cycle-dependent transcription regulation, during somitogenesis stages of embryo development.

developmental biology↗

bioRxiv2019

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

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 remodelling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However, they still present 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 to 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 emphasizes the role of transcription in the establishment of chromatin structure.

genomics↗

bioRxiv2019

CNEr: a toolkit for exploring extreme noncoding conservation

Conserved Noncoding Elements (CNEs) are elements exhibiting extreme noncoding conservation in Metazoan genomes. They cluster around developmental genes and act as long-range enhancers, yet nothing that we know about their function explains the observed conservation levels. Clusters of CNEs coincide with topologically associating domains (TADs), indicating ancient origins and stability of TAD locations. This has suggested further hypotheses about the still elusive origin of CNEs, and has provided a comparative genomics-based method of estimating the position of TADs around developmentally regulated genes in genomes where chromatin conformation capture data is missing. To enable researchers in gene regulation and chromatin biology to start deciphering this phenomenon, we developed CNEr, a R/Bioconductor toolkit for large-scale identification of CNEs and for studying their genomic properties. We apply CNEr to two novel genome comparisons - fruit fly vs tsetse fly, and two sea urchin genomes - and report novel insights gained from their analysis. We also show how to reveal interesting characteristics of CNEs by coupling CNEr with existing Bioconductor packages. CNEr is available at Bioconductor (https://bioconductor.org/packages/CNEr/) and maintained at github (https://github.com/ge11232002/CNEr).

genomics↗

bioRxiv2018

Intertwined canonical and non-canonical initiation in dual promoters are pervasive and differentially regulate Polymerase II transcription

The diversity and complexity of transcription start site (TSS) selection reflects variation of preinitiation complexes, divergent function of promoter-binding proteins and underlies not only transcriptional dynamics but may also impact on post-transcriptional fates of RNAs. The majority of metazoan genes are transcribed by RNA polymerase II from a canonical initiation motif having an YR dinucleotide at their TSSs. In contrast, translation machinery-associated genes carry promoters with polypyrimidine initiator (known as 5-TOP or TCT) with cytosine replacing the R nucleotide. The functional significance of start site choice in promoter architectures is little understood. To get insight into the developmental regulation of start site selection we profiled 5 ends of transcripts during zebrafish embryogenesis. We uncovered a novel class of dual-initiation (DI) promoters utilized by thousands of genes. In DI promoters non-canonical YC-initiation representing 5-TOP/TCT initiators is intertwined with canonical YR-initiation. During maternal to zygotic transition, the two initiation types are divergently used in hundreds of DI promoters, demonstrating that the two initiation systems are distinctly regulated. We show via the example of snoRNA host genes and translation interference experiments that dual-initiation from shared promoters can lead to divergent spatio-temporal expression dynamics generating distinct sets of RNAs with different post-transcriptional fates. Thus utilization of DI promoters in large number of genes suggests two transcription initiation mechanisms targeting these promoters. DI promoters are conserved within human and fruit fly and reflect an evolutionary conserved mechanism for switching transcription initiation to adapt to the changing developmental context. Thus, our findings highlight a novel level of complexity of core promoter regulation in metazoans and broaden the scope for identification and characterization of alternative RNA products generated at shared core promoters.

genomics↗

bioRxiv2018

Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions

One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of transcription start sites at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the shape index, a characteristic feature of each TSS describing the spatial distribution of transcription initiation events, has a surprisingly strong negative correlation with the measured expression levels. Our analysis supplies a set of high quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.

systems biology↗

bioRxiv2018

SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA

Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of rules of transcription initiation, led to discovery of new core promoter sequence features and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 micrograms), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods by generating a complex, high quality library from mouse embryonic day (E) 11.5 primordial germ cells.

genomics↗

bioRxiv2018

HOTAIR ancient sequence suggests regulatory roles both in cis and trans

HOTAIR is a long noncoding RNA transcribed between HOXC11 and HOXC12 in mammals. The proposed function(s) of HOTAIR lacks consensus as to whether it regulates HoxD cluster genes in trans or HoxC cluster genes in cis. We have identified a 32-nucleotide long conserved noncoding element (CNE) as HOTAIR ancient sequence which has a paralogous copy embedded in HOXD11 noncoding transcript. All vertebrates except teleosts have two copies of CNE and the paralogous CNEs exhibit sequence complementarity in the transcribed orientation. Moreover, paralogous CNEs underwent compensatory mutations suggesting they co-evolved and might hybridize. In both human and mouse, HOTAIR CNE exhibits characteristic features of a poised enhancer in HOTAIR-unexpressed stem cells and of an active enhancer in HOTAIR-expressed cells. Tight correlation between the transcriptional activity of the CNE and HOTAIR promoter suggests HOTAIR transcription is crucial for enhancer activity. In HOTAIR-expressed cells, HOTAIR expression is positively correlated with HOXC11 in cis and negatively correlated with HOXD11 in trans, suggesting a dual modality of HOTAIR ancient sequence.

genomics↗

bioRxiv2017

Integrated analysis sheds light on evolutionary trajectories of young transcription start sites in the human genome

Previous studies revealed widespread transcription initiation and fast turnover of transcription start sites (TSSs) in mammalian genomes. Yet how new TSSs originate and how they evolve over time remain poorly understood. To address these questions, we analyzed [~]200,000 human TSSs by integrating evolutionary and functional genomic data, particularly focusing on TSSs that emerged in the primate lineages. We found that intrinsic factors of repetitive sequences and their proximity to established regulatory modules (extrinsic factors) contribute significantly to origin of new TSSs. In early periods, young TSSs experience rapid sequence evolution driven by endogenous mutational mechanisms that reduce the instability of associated repetitive sequences. In later periods, the regulatory functions of young TSSs are gradually modified, and with evolutionary changes subject to temporal (fewer regulatory changes in younger TSSs) and spatial constraints (fewer regulatory changes in more isolated TSSs). These findings advance our understanding of how regulatory innovations arise in the genome throughout evolution and highlight the roles of repetitive sequences in these processes.

genomics↗

bioRxiv2017

Distinct Core Promoter Codes Drive Transcription Initiation At Key Developmental Transitions In A Marine Chordate

Development is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of Oikopleura dioica, a member of the closest living relatives to vertebrates. Our TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70% of transcription in the testis, including the male-specific use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. During oogenesis the cell cycle regulator, E2F1, has been co-opted in regulating maternal transcription in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in vertebrates and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box, which indicates that this ancient promoter motif may play a role in selecting DNA for transcription-associated methylation in invertebrate genomes.

genomics↗