Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome.

yesEpigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo- Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics

Nucleosome-coupled expression differences in closely-related species

<p>Abstract</p> <p>Background</p> <p>Genome-wide nucleosome occupancy is negatively related to the average level of transcription factor motif binding based on studies in yeast and several other model organisms. The degree to which nucleosome-motif interactions relate to phenotypic changes across species is, however, unknown.</p> <p>Results</p> <p>We address this challenge by generating nucleosome positioning and cell cycle expression data for <it>Saccharomyces bayanus </it>and show that differences in nucleosome occupancy reflect cell cycle expression divergence between two yeast species, <it>S. bayanus </it>and <it>S. cerevisiae</it>. Specifically, genes with nucleosome-depleted MBP1 motifs upstream of their coding sequence show periodic expression during the cell cycle, whereas genes with nucleosome-shielded motifs do not. In addition, conserved cell cycle regulatory motifs across these two species are more nucleosome-depleted compared to those that are not conserved, suggesting that the degree of conservation of regulatory sites varies, and is reflected by nucleosome occupancy patterns. Finally, many changes in cell cycle gene expression patterns across species can be correlated to changes in nucleosome occupancy on motifs (rather than to the presence or absence of motifs).</p> <p>Conclusions</p> <p>Our observations suggest that alteration of nucleosome occupancy is a previously uncharacterized feature related to the divergence of cell cycle expression between species.</p

Complex transcription units in Saccharomyces cerevisiae

Overlapping antisense transcription, once thought to be an exceptional event, has become established as a common feature of transcription units. However, despite its prevalence, a bona fide biological consequence or function of overlapping transcription has yet to be ascribed, with the exception of a few locus-specific examples. In this analysis a multi-dimensional systematic approach was employed to interrogate the antisense-producing HMS2 locus. As a result, two additional layers of transcription unit complexity were described: temporal separation of sense and antisense transcription states and transcript-end heterogeneity. Additionally, by observing transcription state-switching in near real time, evidence was provided that links the two phenomena in which changes in the dominant antisense isoform correlate with sense/antisense state switching. Furthermore, in the case of HMS2 regulation, a gene displaying expression periodicity in the metabolic cycle, it was demonstrated that the adoption of sense or antisense states bears consequences at neighbouring loci, showing that modulating antisense transcription can coordinate expression of a cluster of genes. Taken together, this study supports a general model in which sense/antisense state switching at a single locus, modulated by transcriptional regulation of both 5’ and 3’-end promoters, as well as adopting alternative transcript isoforms, can have long-range effects at neighbouring genes, ultimately providing cells with the ability to tailor a wider transcriptional landscape, so that they may adjust their genetic responses to their specific environmental or physiological needs

Nucleosome positioning in Arabidopsis

The aim of this project was to test hypotheses relating to nucleosome positioning in Arabidopsis to provide a basis for better understanding of epigenetic transcriptional regulation in plants. Prior to this study, virtually no information existed regarding nucleosome positioning in plants. Eukaryote chromosomes consist of chromatin, composed of nucleosomes separated by linker DNA of variable lengths. Nucleosomes consist of 147 bp of DNA wrapped 1.7 times around a histone octamer. Whilst no consensus nucleosome positioning DNA sequence exists, sequence preferences influence positioning, and contribute to the complex epigenetic processes which act to control transcriptional activity. These details of the underlying mechanisms are known to differ between the plant and animal kingdoms. High-throughput sequencing technologies were utilised to generate large datasets of mono- and di-nucleosome sequences from wild-type Arabidopsis. These enabled genome-wide analysis and inference of plant-specific patterns of nucleosome positioning and sequence properties. Further data were generated from a methyltransferase antisense (MET1) which is depleted in methylated CG epigenetic marks. The internal distributions of dinucleotides within Arabidopsis nucleosomes were similar to those observed in non-plant eukaryotes. A unique periodicity in the distribution of linker lengths was detected in Arabidopsis wild type chromatin. In contrast, the MET1 antisense line displayed the expected periodicity, indicating systematic differences in chromatin organisation. There was a significant increase in nucleosome occupancy within exons compared with introns. However, this difference was less marked in the MET1 antisense. Specific patterns of nucleosome phasing were observed around transcription start sites. Linker lengths within rRNA gene clusters associated with nucleolar organiser regions (NORs) differed depending on chromosome of origin, suggesting differences in higher order chromatin structure between the NORs. Comparison of the nucleosome position and DNA methylation within the rRNA gene cluster revealed interesting differences between the two regions, which may reflect interactions affecting chromatin structure and transcriptional regulation

Fast, accurate and automatic ancient nucleosome and methylation maps with epiPALEOMIX

The first epigenomes from archaic hominins (AH) and ancient anatomically modern humans (AMH) have recently been characterized, based, however, on a limited number of samples. The extent to which ancient genome-wide epigenetic landscapes can be reconstructed thus remains contentious. Here, we present epiPALEOMIX, an open-source and user-friendly pipeline that exploits post-mortem DNA degradation patterns to reconstruct ancient methylomes and nucleosome maps from shotgun and/or capture-enrichment data. Applying epiPALEOMIX to the sequence data underlying 35 ancient genomes including AMH, AH, equids and aurochs, we investigate the temporal, geographical and preservation range of ancient epigenetic signatures. We first assess the quality of inferred ancient epigenetic signatures within well-characterized genomic regions. We find that tissue-specific methylation signatures can be obtained across a wider range of DNA preparation types than previously thought, including when no particular experimental procedures have been used to remove deaminated cytosines prior to sequencing. We identify a large subset of samples for which DNA associated with nucleosomes is protected from post-mortem degradation, and nucleosome positioning patterns can be reconstructed. Finally, we describe parameters and conditions such as DNA damage levels and sequencing depth that limit the preservation of epigenetic signatures in ancient samples. When such conditions are met, we propose that epigenetic profiles of CTCF binding regions can be used to help data authentication. Our work, including epiPALEOMIX, opens for further investigations of ancient epigenomes through time especially aimed at tracking possible epigenetic changes during major evolutionary, environmental, socioeconomic, and cultural shifts

The effect of negative supercoiling on the formation and positioning of nucleosome cores in vitro

Bibliography: pages 250-279.The effect of the negative supercoiling of DNA on the formation and positioning of nucleosome cores was investigated in a 1915bp plasmid (pHP2) containing a section of the early H1-H4 histone gene spacer of Psammechinus miliaris, previously shown to position the histone octamer (Retief et al., 1987, Biochemistry, 26, 4449-4453). It is shown for the first time, by determinations of the linking difference of reconstituted supercoiled plasmid following topoisomerase I relaxation and the yield and fragment size distribution of micrococcal nuclease digests of supercoiled and linearized plasmid, that nucleosome core reconstitution by urea/salt and salt dialysis proceeds cooperatively on both linearized and supercoiled plasmids. Evidence is further presented which indicates that the nucleosome core.reconstitutes more efficiently on negatively supercoiled plasmids compared to linearized plasmids. The free energy of supercoiling is shown to be sufficient to account for this difference, and may contribut·e to the observed preferential migration of the octamer to negatively supercoiled plasmid compared to linear fragments. This migration is facilitated by high ionic strength, but not by high concentrations of poly[L-glutamate] or 146bp core DNA. It is further shown for the first time, by DNase I digestion and primer extension, that identical translational and rotational positions are adopted by nucleosome cores on linearized plasmids and circular plasmids in the absence and presence of negative superhelical stress. This conservation of the positioning frames is -shown to persist, irrespective of the precision of the core placement, or alterations of the relative angular orientation of the positioning frames of adjacent cores. This finding suggests that the topological and geometric constraints of chromatin loops may be negligible in the determination of nucleosome positioning in vivo. The positioning of the core incorporating a d(A-G)₁₆.d(C-T)₁₆ stretch, shown not to adopt a H-DNA conformation in the reconstituted supercoiled plasmid, is analyzed in terms of known rotational determinants, and possible translational determinants proposed. The biological significance of the determined position of the core is discussed, and lastly, the conclusions related to other studies

Nucleosomal organisation over the ovine β-lactoglobulin gene

The genetic material of all higher organisms from yeast to mammals is organised in the cell nucleus as a nucleoprotein complex called chromatin. The fundamental repeating unit of chromatin, which covers nearly the entire DNA, is the nucleosome. Each one comprises eight highly conserved protein subunits that sequester approximately 146bp of DNA. Nucleosomes facilitate the highly condensed packaging of DNA, most obvious in metaphase chromosomes, and also permit non-histone protein factors access to the DNA in order to facilitate DNA replication, transcription and repair.For temporally and spatially specific gene activation to occur, chromatin remodeling factors, transcription factors and RNA polymerase and its associated factors must act in concert with the underlying nucleosome environment to effect transcription. In some instances, this has shown to be a complex relationship. Nucleosomes are stably positioned over transcription factor binding sites in some genes. This can prevent access and therefore repress gene activation. In other genes, a positioned nucleosome is required to wrap up DNA between separate transcription factor binding sites. Bringing the sites together allows the binding factors to act cooperatively in initiating transcription. Therefore, nucleosomes that are positioned over a specific DNA sequence can have an instrumental role in gene regulation.To date, there have only been limited studies on the nucleosomal organisation of genes in their natural environment. The majority of these studies have concentrated on short regions of positioned nucleosomes spanning either repetitive DNA or the promoter regions of specific genes. However, nucleosome positioning over an entire gene domain may have a significant impact on its regulation and compaction. I have mapped the nucleosomal organisation over lOkb of a tissue specific, temporally regulated gene using the enzymatic probe, micrococcal nuclease and the chemical probe, cuprous phenanthroline. The ovine p-lactoglobulin (BLG) gene studied has a well characterised developmental profile, a minimal transcriptional domain and has been used extensively as an expression cassette in transgenic animals to drive heterologous gene transcriptionWhen the gene is inactive, in the liver, it displays a tightly defined array of positioned nucleosomes that modulate between two specific phases over the gene domain. A similar, less tightly defined array is present when the gene is active, in the mammary gland, except over the promoter and actively transcribing regions. The same arrays arc present over the BLG promoter region in transgenic mice in both active and inactive states. A monomer extension reaction provides in vitro evidence of the positioning signals that are determined by DNA sequence alone. These show an interesting correlation with the in vivo results.A number of other milk protein genes have a similar pattern of key transcription factor binding sites over their promoter regions. If the nucleosome positions were conserved in these genes, with respect to these binding sites, it might suggest a role for positioned nucleosomes in their regulation. A total of three genes, each in two different organisms, have been analysed to test for a correlation

Genetic and Epigenetic Determinants of Transcription in the Divergent Eukaryote Leishmania major

Leishmania spp. and other trypanosomatid protozoa use a highly unusual mechanism to generate functional messenger RNAs (mRNAs) in which protein-coding genes are transcribed polycistronically. Here, transcription initiates primarily in divergent strand switch regions (dSSRs), where two polycistronic gene clusters are oriented head-to-head. These regions lack all known eukaryotic cis-regulatory elements, and it is not known how genetic and epigenetic factors cooperate to define dSSRs as regions of productive initiation. To quantitatively identify regulatory elements and to study the contribution of epigenetic factors to dSSR function, we combined genome-wide studies of chromatin structure with a focused interrogation of a single dSSR using a novel integrated bidirectional, dual-luciferase reporter. Chromatin-based studies demonstrated that Leishmania lack well-positioned nuclease-hypersensitive sites associated with promoters in other eukaryotes. Rather, nuclease-hypersensitive sites are positioned heterogeneously across broad regions associated with epigenetic marks indicative of active transcription, suggesting that transcription initiation events occur promiscuously within regions associated with a transcriptionally-permissive epigenetic state. Our studies using the bidirectional reporter validate these observations and strongly suggest that Leishmania do not require cis-regulatory elements for efficient bidirectional transcription initiating in dSSRs, as a large region of the dSSR can be replaced with unrelated sequences without altering bidirectional reporter gene expression. In addition to these genetic studies we also focused on epigenetic determinants of transcriptional activity in Leishmania, with respect to both transcription initiation and transcription termination. We showed that the histone variants H2A.Z and H2B.V, which are associated with transcriptionally permissive regions in T. brucei, are essential in L. major, while the transcription termination-associated histone variant H3.V is not. Interestingly, unlike Leishmania lacking the DNA modification base J, H3.V-null L. major shows no defects in transcription termination. Although the study of essential genes in Leishmania is challenging at this time, we present preliminary data describing elements of inducible gene expression systems which may improve our ability to study essential genes. Together, the data in this thesis show that transcription of protein-coding genes is primarily determined epigenetically, and suggest that chromatin-related processes may be an attractive target for therapeutic intervention