Aspects Of Chromatin Biology Research Articles

The histone chaperoning pathway: from ribosome to nucleosome.

Nucleosomes represent the fundamental repeating unit of eukaryotic DNA, and comprise eight core histones around which DNA is wrapped in nearly two superhelical turns. Histones do not have the intrinsic ability to form nucleosomes; rather, they require an extensive repertoire of interacting proteins collectively known as ‘histone chaperones’. At a fundamental level, it is believed that histone chaperones guide the assembly of nucleosomes through preventing non-productive charge-based aggregates between the basic histones and acidic cellular components. At a broader level, histone chaperones influence almost all aspects of chromatin biology, regulating histone supply and demand, governing histone variant deposition, maintaining functional chromatin domains and being co-factors for histone post-translational modifications, to name a few. In this essay we review recent structural insights into histone-chaperone interactions, explore evidence for the existence of a histone chaperoning ‘pathway’ and reconcile how such histone-chaperone interactions may function thermodynamically to assemble nucleosomes and maintain chromatin homeostasis.

Chromatin Immunoprecipitation (ChIP) of Plasmid-Bound Proteins in Xenopus Egg Extracts.

Xenopus egg extracts provide a cell-free system to analyze various aspects of chromatin biology. Here we describe a modified method of chromatin immunoprecipitation (ChIP) to detect the interaction of proteins with plasmid DNA incubated in extract. The combination of ChIP and Xenopus egg extracts provides a highly versatile and tractable approach to analyze dynamic protein-DNA interactions with great spatial and temporal detail.

Recent Advances in Understanding Histone Modification Events

This review will discuss histone modification events that have been recently discovered or significant technical advancements made in the last year. The physiological state of the eukaryotic genome is chromatin. Chromatin is a complex of DNA and primarily histone proteins. The post-translational modification of histones and the occupancy of specific histone variants in a given nucleosome play a central role in the regulation of the genome. Histone variants and modifications exist in complex and unique combinations. The resulting diversity allows for histones to play key roles in almost all cellular processes and their dysregulation is often the root cause of many diseases. In particular, epigenetic alterations have been noted in cancer, are associated with its progression, aggressiveness, and metastasis, and are useful in its prognosis. Thus, a deep understanding of histone variants and modifications is needed to comprehend both normal cellular function and disease. Technological advancements have dramatically improved our quantitative understanding of histone modification events, decreased sample requirements, and revealed nuances about how these events work together. Specifically, the miniaturization of ChIP-seq and the continuing use and development of mass spectrometric approaches for histone analysis have revealed interesting and diverse new aspects of chromatin biology. This review will explore the causes, mechanisms, and downstream functions of these newly discovered events. We will conclude with an outlook on the future of histone modification analysis and its biological impact.

Improved Determination of Subnuclear Position Enabled by Three-Dimensional Membrane Reconstruction

Many aspects of chromatin biology are influenced by the nuclear compartment in which a locus resides, from transcriptional regulation to DNA repair. Further, the dynamic and variable localization of a particular locus across cell populations and over time makes analysis of a large number of cells critical. As a consequence, robust and automatable methods to measure the position of individual loci within the nuclear volume in populations of cells are necessary to support quantitative analysis of nuclear position. Here, we describe a three-dimensional membrane reconstruction approach that uses fluorescently tagged nuclear envelope or endoplasmic reticulum membrane marker proteins to precisely map the nuclear volume. This approach is robust to a variety of nuclear shapes, providing greater biological accuracy than alternative methods that enforce nuclear circularity, while also describing nuclear position in all three dimensions. By combining this method with established approaches to reconstruct the position of diffraction-limited chromatin markers—in this case, lac Operator arrays bound by lacI-GFP—the distribution of loci positions within the nuclear volume with respect to the nuclear periphery can be quantitatively obtained. This stand-alone image analysis pipeline should be of broad practical utility for individuals interested in various aspects of chromatin biology, while also providing, to our knowledge, a new conceptual framework for investigators who study organelle shape.

Scrutinizing the FTO locus: compelling evidence for a complex, long-range regulatory context.

Single nucleotide polymorphisms (SNPs) within a genetic region including the first two introns of the gene encoding FTO have consistently been shown to be the strongest genetic factors influencing body mass index (BMI). However, this same also contains several regulatory DNA elements that affect the expression of IRX3 and IRX5, which respectively, are located approximately 500 kb and 1.2 Mbp downstream from the BMI-associated FTO locus. Through these affected regulatory elements, genetic variation at the FTO locus influences adipocyte development leading to decreased thermogenesis and increased lipid storage. These findings provide a genomic model for the functional implications of genetic variations at this locus, and also demonstrate the importance of accounting for chromatin-chromatin interactions when constructing hypotheses for the mechanisms of trait and disease-associated common genetic variants. Several consortia have generated genome-wide datasets describing different aspects of chromatin biology which can be utilized to predict functionality and propose biologically relevant descriptions of specific DNA regions. Here, we review some of the publically available data resources on genome function and organization that can be used to gain an overview of genetic regions of interest and to generate testable hypotheses for future studies. We use the BMI- and obesity-associated FTO locus as a subject as it poses an illustrative example on the value of these resources. We find that public databases strongly support long-range interactions between regulatory elements in the FTO locus with the IRXB cluster genes IRX3 and IRX5. Chromatin configuration capture data also support interactions across a large region stretching across from the RPGRIP1L gene, FTO and the IRXB gene cluster.

Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq)

The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein–DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.

Identifying and mitigating bias in next-generation sequencing methods for chromatin biology.

Next-generation sequencing (NGS) technologies have been used in diverse ways to investigate various aspects of chromatin biology by identifying genomic loci that are bound by transcription factors, occupied by nucleosomes or accessible to nuclease cleavage, or loci that physically interact with remote genomic loci. However, reaching sound biological conclusions from such NGS enrichment profiles requires many potential biases to be taken into account. In this Review, we discuss common ways in which biases may be introduced into NGS chromatin profiling data, approaches to diagnose these biases and analytical techniques to mitigate their effect.

Proteomics in epigenetics: new perspectives for cancer research

The involvement of epigenetic processes in the origin and progression of cancer is now widely appreciated. Consequently, targeting the enzymatic machinery that controls the epigenetic regulation of the genome has emerged as an attractive new strategy for therapeutic intervention. The development of epigenetic drugs requires a detailed knowledge of the processes that govern chromatin regulation. Over the recent years, mass spectrometry (MS) has become an indispensable tool in epigenetics research. In this review, we will give an overview of the applications of MS-based proteomics in studying various aspects of chromatin biology. We will focus on the use of MS in the discovery and mapping of histone modifications and how novel proteomic approaches are being utilized to identify and study chromatin-associated proteins and multi-subunit complexes. Finally, we will discuss the application of proteomic methods in the diagnosis and prognosis of cancer based on epigenetic biomarkers and comment on their future impact on cancer epigenetics.

The Fission Yeast Jmj2 Reverses Histone H3 Lysine 4 Trimethylation

Histone methylation regulates transcription, chromatin structure, and the epigenetic state of the cell. Recent studies identified the JmjC domain as a catalytic module for histone demethylation. Schizosaccharomyces pombe contains seven JmjC proteins, but it was unclear whether any of them functioned as histone demethylases. In this report, we show that the JmjC protein Jmj2, which is evolutionarily conserved from yeast to human, reversed trimethylated H3-Lys-4 to di- and mono-but not unmethylated products. Overexpression of Jmj2 but not a catalytically inactive mutant reduced H3-Lys-4 trimethylation levels in vivo and suppressed the toxicity caused by overexpression of the H3-Lys-4-me3-binding protein Yng1 in budding yeast. Genome-wide analysis showed that the loss of jmj2 was associated with an increase in the H3-Lys-4-me3 signal, which was enriched near the transcriptional start sites and the coding regions. At the mating-type locus, the loss of jmj2 or substitution of jmj2 with a catalytically inactive form is correlated with increased reporter gene transcription and H3-Lys-4-me3/2 levels, suggesting that Jmj2 and its demethylase activity may play a role in heterochromatin biology. Our findings identified a novel S. pombe histone demethylase with specificity toward di- and trimethylated histone H3-Lys-4 and a possible role in heterochromatin regulation.