Detection of gene loops by 3C in yeast

“Chromosome conformation capture” (3C) is a powerful method to detect physical interaction between any two genomic loci. 3C involves formaldehyde crosslinking to stabilize transient interactions, followed by restriction digestion, ligation and locus-specific PCR. Accordingly, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Here, we describe a modified 3C protocol designed for detection of transient chromatin interactions in the yeast Saccharomyces cerevisiae. Using this protocol, we are able to detect juxtaposition of promoter and terminator regions of genes with ORFs as short as 1 kb in length. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions and will facilitate the characterization of gene loops and their functional consequences.     

Chromatin structure in somatic nucleus of ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis>

The structural organization of macronuclear chromatin of the ciliate Didinium nasutum was studied. The macronuclear genome of D. nasutum is represented by DNA molecules of subchromosomal size. At interphase, macronuclear chromatin is organized into chromatin of 100–200-nm clumps. Some of these clumps form short, thick fibers that consist of several chromatin clumps. Using the differential staining of nucleic acids on ultrathin sections, we revealed perichromatin fibers and granules on the surface of many chromatin clumps. A 3D model of the spatial distribution of chromatin clumps in the macronucleus was built based on serial ultrathin sections and peculiar features of chromatin spatial organization were studied.     

Coexistence of two chromatin structures in sperm nuclei of the bivalve molluscProtothaca thaca

The chromatin of the spermatozoa from the bivalve molluscProtothaca thaca, has a peculiar composition in which coexist core histones with sperm-specific proteins H1 and Pt1, the latter being a protein exhibiting features intermediate between histones and protamines. In this paper, we report an analysis of chromatin organization using micrococcal nuclease digestion, salt fractionation of soluble chromatin derived from nuclease digestion and crosslinking experiments. The results obtained indicate that it is possible to obtain two types of chromatin, one which is soluble, more accessible to micrococcal nuclease action and which does not contain Pt1, and another insoluble type, more resistant to micrococcal nuclease and enriched in protein Pt1. The crosslinking experiments show that the protein Pt1 interacts with itself and with core histones but not with sperm-specific H1. These results have led us to propose a special structural arrangement for this chromatin. Based in the data reported here we propose the coexstence in the genome ofP. thaca of two interspersed chromatin domains, one nucleosomal and the other nonnucleosomal containing protein Pt1.     

ChIP‐based methods for the identification of long‐range chromatin interactions

Chromatin immunoprecipitation (ChIP) is an important technique for studying protein–DNA interactions. Whole genome ChIP methods have enjoyed much success, but are limited in that they cannot uncover important long‐range chromatin interactions. Chromosome conformation capture (3C) and related methods are capable of detecting remote chromatin interactions, but are tedious, have low signal‐to‐noise ratios, and are not genome‐wide. Although the addition of ChIP to 3C (ChIP–3C) would conceivably reduce noise and increase specificity for chromatin interaction detection, there are concerns that simple mixing of the ChIP and 3C protocols would lead to high levels of false positives. In this essay, we dissect current ChIP‐ and 3C‐based methodologies, discuss the models of specific as opposed to non‐specific chromatin interactions, and suggest approaches to separate specific chromatin complexes from non‐specific chromatin fragments. We conclude that the combination of sonication‐based chromatin fragmentation, ChIP‐based enrichment, chromatin proximity ligation and Paired‐End Tag ultra‐high‐throughput sequencing will be a winning implementation for genome‐wide, unbiased and de novo discovery of long‐range chromatin interactions, which will help to establish an emerging field for studying human chromatin interactomes and genome regulation networks in three‐dimensional spaces. J. Cell. Biochem. 107: 30–39, 2009. © 2009 Wiley‐Liss, Inc.     

Methods designed for the identification and characterization of<Emphasis Type="Italic">in vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> chromatin assembly mutants in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model systemSaccharomyces cerevisiae. Modifications to anin vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s):RSP5, and a subunit of the Anaphase Promoting Complex (APC),APC5. Additional modifications are described that allow for a rapid analysis and anin vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that thein vitro andin vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks. Published: July 3, 2003     

Genome-wide Mapping of Cellular Protein–RNA Interactions Enabled by Chemical Crosslinking

RNA–protein interactions influence many biological processes. Identifying the binding sites of RNA-binding proteins（RBPs） remains one of the most fundamental and important challenges to the studies of such interactions. Capturing RNA and RBPs via chemical crosslinking allows stringent purification procedures that significantly remove the non-specific RNA and protein interactions. Two major types of chemical crosslinking strategies have been developed to date, i.e., UV-enabled crosslinking and enzymatic mechanism-based covalent capture. In this review, we compare such strategies and their current applications, with an emphasis on the technologies themselves rather than the biology that has been revealed. We hope such methods could benefit broader audience and also urge for the development of new methods to study RNA RBP interactions.     

Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation

We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that “drive” chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase. Received: 4 September 1997; in revised form: 14 September 1997 /Accepted: 14 September 1997     

Actin‐induced dimerization of palladin promotes actin‐bundling

A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics.     

Tn5-FISH,a novel cytogenetic method to image chromatin interactions with sub-kilobase resolution

There is an increasing interest in understanding how three-dimensional (3D) organization of the genome is regulated. Different strategies have been employed to identify genome-wide chromatin interactions. However, due to current limitations in resolving genomic contacts, visualization and validation of these genomic loci with sub-kilobase resolution remain unsolved to date. Here, we describe Tn5 transposase-based Fluorescencein situhybridization (Tn5-FISH), a PCR-based, cost-effective imaging method, which can co-localize the genomic loci with sub-kilobase resolution, dissect genome architecture, and verify chromatin interactions detected by chromatin configuration capture (3C)-derived methods. To validate this method, short-range interactions in keratin-encoding gene (KRT) locus in topologically associated domain (TAD) were imaged by triple-color Tn5-FISH, indicating that Tn5-FISH is very useful to verify short-range chromatin interactions inside the contact domain and TAD. Therefore, Tn5-FISH can be a powerful molecular tool for the clinical detection of cytogenetic changes in numerous genetic diseases such as cancers.     

Characterization of genome-nucleoporin interactions in Drosophila links chromatin insulators to the nuclear pore complex

Nuclear pore complexes (NPCs) are multiprotein complexes consisting of nucleoporins and function in transport between the nucleus and the cytoplasm. In yeast, nucleoporins have also been linked to gene expression as well as to chromatin insulating activity. Recently, we identified genomic regions that interact with nucleoporins in Drosophila using DamID technology. We found that nucleoporins in the nucleoplasm interact with active genes and stimulate gene expression. However, genes interacting with nucleoporins at the NPC itself show average gene expression and it remains unclear why they interact with the NPC. Here, we further investigated the function of the genome-NPC interactions. First, to investigate whether a different technique would lead to similar results, we compared our nucleoporin DamID data to recently published nucleoporin chromatin immunoprecipitation (ChIP) data. Then, to further understand the function of interactions between the genome and NPCs, we analyzed the relationship between NPC-interacting genomic regions and chromatin insulators. We found that the insulator protein Su(Hw) was enriched within and near NPC-interacting genomic regions, suggesting a role of this protein in chromatin architecture close to the NPC. This suggests that the NPC may have a function in the structural organization of the genome.     

The basis of chromatin fiber assembly within chromosomes studied by histone-DNA crosslinking followed by trypsin digestion

To determine the structural basis of chromatin assembly that leads to chromosome formation in mitosis, crosslinks were introduced by formaldehyde between contiguous components within chromosomes. Crosslinked stable products were then observed by electronmicroscopy after non-cross-linked portions were briefly digested by trypsin to unfold chromosomes. — When the DNA-histone crosslink was the primary product, trypsin readily unfolded the whole chromosome structure while preserving the 250 Å unit chromatin fiber intact; only a single unit fiber was tracked within the centromere region connecting the arms of each chromatid. When a histone polymer was formed by a prolonged formaldehyde crosslinking, trypsin digestion gave rise to chromatin fibers interacting with others at certain distances, and the typical chromosome structure remained unchanged. Regardless of the degree of crosslinking, there were neither thick supercoiled unit fibers nor proteinaceous cores. — These results suggest that the fiber connection may represent, to some extent, the interacting sites of folded chromatin fibers in situ within chromosomes, and also that the 250 Å unit fibers are the sole, highest structural basis in chromosomes. Since virtually no appreciable histone digestion took place in the crosslinked chromosomes, the observation that even after DNA-histone crosslinking the fiber interacting sites were accessible to trypsin preferentially over other portions, may be consistent with our recent results that the exposed, lysine-rich tails of histones represent such interacting sites.