The Determinants of Nucleosome Patterns and the Impact of Phosphate Starvation on Nucleosome Patterns and Gene Expression in Rice

In eukaryotic cells, DNA is a large molecule that must be greatly condensed to fit within the nucleus. DNA is wrapped around histone proteins to form nucleosomes, which facilitate DNA condensation, but on the other hand, may limit DNA processes. Organisms must respond to environmental stress in order to survive, and one strategy is by remodeling nucleosomes to promote changes in DNA accessibility to alter gene expression. Studies have demonstrated a clear correlation between nucleosome dynamics and transcriptional change in some eukaryotes, however factors that affect nucleosome positioning in plants are largely unknown, and the correlation between nucleosome dynamics and transcriptional changes in response to environmental perturbation remain unclear. We report a high-resolution map of nucleosome patterns in the rice (Oryza sativa) genome by deep sequencing of micrococcal nuclease digested chromatin. The results reveal that nucleosome patterns at rice genes were affected by both cis- and trans- determinants, including GC content and transcription. A negative correlation between nucleosome occupancy across the transcription start site (TSS) and transcription was observed, and the nucleosome patterns across the TSS were correlated with distinct functional categories of genes. A parallel experiment was done monitoring nucleosome dynamics and transcription changes in response to phosphate starvation for 24 hours. Phosphate starvation resulted in numerous instances of nucleosome dynamics across the genome which were enhanced at differentially expressed genes. This work demonstrates that rice nucleosome patterns are suggestive of gene functions, and reveal a link between chromatin remodeling and transcriptional changes in response to deficiency of a major macronutrient. The findings help to enhance the understanding towards eukaryotic gene regulation at the chromatin level

Weakly Positioned Nucleosomes Enhance the Transcriptional Competency of Chromatin

Background: Transcription is affected by nucleosomal resistance against polymerase passage. In turn, nucleosomal resistance is determined by DNA sequence, histone chaperones and remodeling enzymes. The contributions of these factors are widely debated: one recent title claims ‘‘… DNA-encoded nucleosome organization…’’ while another title states that ‘‘histone-DNA interactions are not the major determinant of nucleosome positions.’’ These opposing conclusions were drawn from similar experiments analyzed by idealized methods. We attempt to resolve this controversy to reveal nucleosomal competency for transcription. Methodology/Principal Findings: To this end, we analyzed 26 in vivo, nonlinked, and in vitro genome-wide nucleosome maps/replicates by new, rigorous methods. Individual H2A nucleosomes are reconstituted inaccurately by transcription, chaperones and remodeling enzymes. At gene centers, weakly positioned nucleosome arrays facilitate rapid histone eviction and remodeling, easing polymerase passage. Fuzzy positioning is not due to artefacts. At the regional level, transcriptional competency is strongly influenced by intrinsic histone-DNA affinities. This is confirmed by reproducing the high in vivo occupancy of translated regions and the low occupancy of intergenic regions in reconstitutions from purified DNA and histones. Regional level occupancy patterns are protected from invading histones by nucleosome excluding sequences and barrier nucleosomes at gene boundaries and within genes. Conclusions/Significance: Dense arrays of weakly positioned nucleosomes appear to be necessary for transcription. Weak positioning at exons facilitates temporary remodeling, polymerase passage and hence the competency for transcription. At regional levels, the DNA sequence plays a major role in determining these features but positions of individual nucleosomes are typically modified by transcription, chaperones and enzymes. This competency is reduced at intergenic regions by sequence features, barrier nucleosomes, and proteins, preventing accessibility regulation of untargeted genes. This combination of DNA- and protein-influenced positioning regulates DNA accessibility and competence for regulatory protein binding and transcription. Interactive nucleosome displays are offered at http://chromatin.unl.edu/cgi-bin/skyline.cgi

Nucleosome Positioning and Its Role in Gene Regulation in Yeast

Nucleosome, composed of a 147-bp segment of DNA helix wrapped around a histone protein octamer, serves as the basic unit of chromatin. Nucleosome positioning refers to the relative position of DNA double helix with respect to the histone octamer. The positioning has an important role in transcription, DNA replication and other DNA transactions since packing DNA into nucleosomes occludes the binding site of proteins. Moreover, the nucleosomes bear histone modifications thus having a profound effect in regulation. Nucleosome positioning and its roles are extensively studied in model organism yeast. In this chapter, nucleosome organization and its roles in gene regulation are reviewed. Typically, nucleosomes are depleted around transcription start sites (TSSs), resulting in a nucleosome-free region (NFR) that is flanked by two well-positioned H2A.Z-containing nucleosomes. The nucleosomes downstream of the TSS are equally spaced in a nucleosome array. DNA sequences, especially 10–11 bp periodicities of some specific dinucleotides, partly determine the nucleosome positioning. Nucleosome occupancy can be determined with high throughput sequencing techniques. Importantly, nucleosome positions are dynamic in different cell types and different environments. Histones depletions, histones mutations, heat shock and changes in carbon source will profoundly change nucleosome organization. In the yeast cells, upon mutating the histones, the nucleosomes change drastically at promoters and the highly expressed genes, such as ribosome genes, undergo more change. The changes of nucleosomes tightly associate the transcription initiation, elongation and termination. H2A.Z is contained in the +1 and −1 nucleosomes and thus in transcription. Chaperon Chz1 and elongation factor Spt16 function in H2A.Z deposition on chromatin. The chapter covers the basic concept of nucleosomes, nucleosome determinant, the techniques of mapping nucleosomes, nucleosome alteration upon stress and mutation, and Htz1 dynamics on chromatin

The interplay between transcription, histone variants, and chromatin structure in Eukaryotes

Transcription is a fundamental process necessary for life. In Eukaryotes this process is shaped and constrained, in part, by the 3D structure of chromatin –the assemblage of protein and DNA into which the genome is organized. Additionally, chromatin itself is reorganized as conditions change and different transcriptional programs are activated. Within this work, I present an exploration of the dynamic system created by this intricately intertwined regulation between chromatin structure and transcriptional outputs. In Chapter 1, I begin with a review of the determinants of direction in the initiation stage of Eukaryotic transcription. The process of initiation involves numerous forms of regulation, including chromatin based. The next three chapters investigate different aspects of the nucleosome, which has been the primary topic of my research. Chapter 2 presents an overview on researching the nucleosome in the yeast Saccharomyces cerevisiae. Chapter 3 examines the connections between H2A.Z and transcription. Here, I challenge the generally accepted model of H2A.Z incorporation at the +1 and -1 nucleosomes hedging the transcription start site. Chapter 4 focuses specifically on perturbations to nucleosomal structure produced either from gene deletions or in response to environmental changes. Finally, I conclude by summarizing my findings and with a general discussion of questions in the field that remain to be explored.Cellular and Molecular Biolog

Localization and function of histone methylation at active genes in "Drosophila"

In the eukaryotic nucleus, DNA is bound by an octamer of four core histones forming the fundamental repeating unit of chromatin, called the nucleosome. Presenting a barrier to virtually all DNA-templated events, nucleosomal packaging is subject to dynamic alterations. Nucleosomal histone modifications have emerged as a major determinant of chromatin structure and gene expression. Genome-wide and local profiling of chromatin structure in Drosophila cells reveals a complex landscape of histone methylation marks along the body of active genes. Methylation of lysine 4 and lysine 79 of histone H3 coincide at promoters and gradually decrease towards the 3’ end. Conversely, H3 lysine 36 methylation states show very different distribution patterns. Dimethylation of H3K36 peaks downstream of promoter-proximal K4 methylation, whereas trimethylation accumulates towards the 3’ end of genes. These topographic differences do not reflect deposition-coupled targeting by histone variant H3.3 but instead argue for discrete regulation and function of active methylation marks during transcription elongation. Indeed, H3K36 di- and trimethylation states rely on two distinct HMTs and display opposite effects on H4K16 acetylation at autosomal genes. This crosstalk is reminiscent of K36me3-dependent deacetylase recruitment in budding yeast, yet it is more intricate as dimethylation appears to signal for increased H4K16 acetylation. Apart from its autosomal function, H3K36me3 has a separate role to enhance H4K16 acetylation at the dosage-compensated X chromosome in male Drosophila cells. This additional function most likely involves MSL complex recruitment to dosage compensated genes. Together, our results reveal a complex pattern of histone methylation marks at active genes, which may enable dynamic chromatin changes during transcription elongation in higher eukaryotes. Furthermore, the context-dependent readout of H3K36me3 implies that methylation marks act as general signaling platforms, which impart their specificity by recruiting effector proteins to characteristic landmarks along the transcription unit

Chromatin remodeling and dna topology in transcription and genome stability

Eukaryotic DNA is wrapped around histone proteins to form nucleosomes, the fundamental repeati ng unit of chromatin. DNA packaging into chromatin both solves and creates problems. It allows the centimeters, or even meters, of DNA that constitute a eukaryotic genome to fit inside a micrometer - scale cell nucleus. Nucleosomes also block access to the D NA, necessitating complex rearrangements to allow for transcription, replication, recombination, or repair, while also providing a way to regulate these processes. ATP - dependent chromatin remodelers slide, assemble, disassemble, and alter nucleosomes to en able and regulate DNA - dependent processes. In parallel, topoisomerases relieve the tangles, torsional stress, and supercoils generated when DNA is exposed and unwound. Topoisomerases also enable efficient nucleosome remodeling. In this thesis, we use genom ewide and single - locus techniques to study the interplay between DNA topoisomerases, Snf2 family chromati n remodelers, and transcription in the fission yeast Schizosaccharomyces pombe. We find that topoisomerase activity is essential for transcription elon gation and for proper chromatin structure at genes, which in turn are required for efficient transcription initiation and termination. This is partially mediated by cooperation with chromatin remodelers. We also find that the fission yeast Chd1 subfamily r emodelers maintain correct gene body nucleosome positioning, which inhibits cryptic transcription initiation. Finally, we show that the Fun30 subfamily chromatin remodeler Fft2 is involved in centromere function and heterochromatic silencing, as well as th e full transcription of highly transcribed genes. Fft2 and its paralog Fft3 also regulate the transcriptional response to stress. As a part of this function, Fft2 and Fft3 repress retrotransposons by a novel mechanism, in which they enforce the use of an a lternative transcription start site