Different Types Of Chromatin Research Articles

Mapping Replication Timing in Single Mammalian Cells.

Replication timing (RT) is the temporal order in which genomic DNA is replicated during S phase. Early and late replication correlate with transcriptionally active and inactive chromatin compartments, but mechanistic links between large-scale chromosome structure, transcription, and replication are still enigmatic. A proper RT program is necessary to maintain the global epigenome that defines cell identity, suggesting that RT is critical for epigenome integrity by facilitating the assembly of different types of chromatin at different times during S phase. RT is regulated during development and has been found to be altered in disease. Thus, RT can identify stable epigenetic differences distinguishing cell types, and can be used to help stratify patient outcomes and identify markers of disease. Most methods to profile RT require thousands of S-phase cells. In cases where cells are rare (e.g., early-stage embryos or rare primary cell types) or consist of a heterogeneous mixture of cell states (e.g., differentiation intermediates), or when the interest is in determining the degree of stable epigenetic heterogeneity within a population of cells, single-cell measurements of RT are necessary. We have previously developed single cell Repli-seq, a method to measure replication timing in single cells using DNA copy number quantification. To date, however, single-cell Repli-seq suffers from relatively low throughput and high costs. Here, we describe an improved single-cell Repli-seq protocol that uses degenerate oligonucleotide-primed PCR (DOP-PCR) for uniform whole-genome amplification and uniquely barcoded primers that permit early pooling of single-cell samples into a single library preparation. We also provide a bioinformatics platform for analysis of the data. The improved throughput and decreased costs of this method relative to previously published single-cell Repli-seq protocols should make it considerably more accessible to a broad range of investigators. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Whole Genome Amplification (WGA) of single cells and sequence library construction. Basic Protocol 2: Deriving and displaying single-cell replication timing data from whole genome sequencing.

Core Components of the Nuclear Pore Bind Distinct States of Chromatin and Contribute to Polycomb Repression.

Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. Here, we report high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. Our investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, we identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. Our findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93.

Beyond the Histone Code: A Physical Map of Chromatin States

Post-translational modifications of histones are widely used to discriminate between different types of chromatin. In a recent issue of Molecular Cell, Becker etal. (2017) delineate chromatin states based on physical properties, thereby expanding our understanding of chromatin function.

Targeting of P-Element Reporters to Heterochromatic Domains by Transposable Element 1360 in Drosophila melanogaster.

Heterochromatin is a common DNA packaging form employed by eukaryotes to constitutively silence transposable elements. Determining which sequences to package as heterochromatin is vital for an organism. Here, we use Drosophila melanogaster to study heterochromatin formation, exploiting position-effect variegation, a process whereby a transgene is silenced stochastically if inserted in proximity to heterochromatin, leading to a variegating phenotype. Previous studies identified the transposable element 1360 as a target for heterochromatin formation. We use transgene reporters with either one or four copies of 1360 to determine if increasing local repeat density can alter the fraction of the genome supporting heterochromatin formation. We find that including 1360 in the reporter increases the frequency with which variegating phenotypes are observed. This increase is due to a greater recovery of insertions at the telomere-associated sequences (∼50% of variegating inserts). In contrast to variegating insertions elsewhere, the phenotype of telomere-associated sequence insertions is largely independent of the presence of 1360 in the reporter. We find that variegating and fully expressed transgenes are located in different types of chromatin and that variegating reporters in the telomere-associated sequences differ from those in pericentric heterochromatin. Indeed, chromatin marks at the transgene insertion site can be used to predict the eye phenotype. Our analysis reveals that increasing the local repeat density (via the transgene reporter) does not enlarge the fraction of the genome supporting heterochromatin formation. Rather, additional copies of 1360 appear to target the reporter to the telomere-associated sequences with greater efficiency, thus leading to an increased recovery of variegating insertions.

RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

The maize genome is relatively large (∼ 2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼ 100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.

Application of histone modification-specific interaction domains as an alternative to antibodies.

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.

Plant biosystematics with the help of cytology and cytogenetics

The importance of cytological and cytogenetic information in the investigation of evolutionary history of species, i.e. biosystematics (cytotaxonomy) and cytogeography, is surveyed. The main issues considered are the numbers and morphology of the chromosomes, the distribution and amount of the different types of chromatin in chromosomes, nuclei at interphase stained with classical and molecular cytogenetic methods, e.g. fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and bacterial artificial chromosome (BAC), painting chromosomes, the isolation of large DNA segments using e.g. pulse-field gel electrophoresis, and chromosome disposition in the cell nucleus. A wide ranging biosystematic interest puts emphasis with the help of cytological and cytogenetic methods on getting to know chromosome variation within the family, genus and species connected with the phenomena of hybridization and polyploidization. Chromosome variation among taxa includes differences in the morphology and numbers of chromosomes, the amount of DNA, the sizes of chromosomes, inner chromosome structure, and DNA sequence, often shown in chromosome bands or various proportions of AT and GC pairs. On this basis we can track the course of evolution among taxa and determine reciprocal relationships. One of the main aims of our work, apart from getting to know evolutionary processes, is to create a taxonomic system of organisms.

Effete, a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.

Replication Timing and Pediatric Leukemia

Abstract 4609Abnormal temporal control of replication (“replication timing”) is a universal hallmark of cancer, but the significance of this relationship is not understood. During normal development, changes in replication timing are regulated in defined chromosomal units (“replication domains”) of 400–800 kb, with late replication generally associated with stable gene silencing. We have performed genome-wide replication timing mapping on pediatric acute leukemias and identified patterns of replication-timing mis-regulation (“leukemia fingerprints”) in common across multiple types of pediatric leukemia (“multi-lineage fingerprints”), as well as those linked to specific subtypes of acute leukemia and patient-specific fingerprints. Most of the mis-regulated regions are not associated with copy number variation or translocations and correspond to many of the same domains whose replication timing is regulated during normal development. The existence of multi-lineage fingerprints suggests an early origin of their mis-regulation while type-specific and patient-specific fingerprints suggest that mis-regulation continues to occur downstream of the initiating event. Our hypothesis is that there exists an hierarchy of replication-timing mis-regulation, initiating with frozen vestiges of normal hematopoiesis that are inappropriately maintained, followed by additional changes that evolve as the tumor differentiates. These changes may then affect the ability of downstream lineages to activate or silence genes. Since chromatin is assembled at the replication fork, and different types of chromatin are assembled at different times during S phase, changes in replication timing likely contribute to domain-wide changes in chromatin structure, similar to those observed in long range epigenetic silencing (LRES). In fact, we find that genes within leukemia fingerprint domains are significantly enriched for those transcribed during hematopoiesis and/or mis-regulated in cancers. Replication timing is an exceptionally stable epigenetic property that is not easily disturbed by loss or mis-expression of single master regulators of cell fate and is not directly related to transcription, but rather to the stability of gene silencing. Hence, events occurring early in hematopoiesis could have profound effects on the activation of transcriptional programs in downstream lineages. Elucidating the genesis and genealogy of leukemia-specific replication timing fingerprints and their relationship to transcriptional control will be fundamental to understanding their link to the development of acute leukemias. Disclosures:No relevant conflicts of interest to declare.

Gene Density, Transcription, and Insulators Contribute to the Partition of the Drosophila Genome into Physical Domains

The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.

Centromeric heterochromatin assembly in fission yeast—balancing transcription, RNA interference and chromatin modification

Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of centromeric heterochromatin in fission yeast that integrates our current understanding of the competing forces of transcription, replication, and RNA decay that influence its assembly and propagation.

The inner nuclear membrane proteins Man1 and Ima1 link to two different types of chromatin at the nuclear periphery in S. pombe

Metazoan chromatin at the nuclear periphery is generally characterized by lowly expressed genes and repressive chromatin marks and presents a sub-compartment with properties distinct from the nuclear interior. To test whether the S. pombe nuclear periphery behaves similarly, we used DNA adenine methyltransferase identification (DamID) to map the target loci of two inner nuclear membrane proteins, Ima1 and Man1. We found that peripheral chromatin shows low levels of RNA-Polymerase II and nucleosome occupancy, both characteristic of repressed chromatin regions. Consistently, lowly expressed genes preferentially associate with the periphery and highly expressed genes are depleted from it. When looking at peripheral intergenic regions (IGRs), we found that divergent IGRs are enriched compared with convergent IGRs, indicating that transcription preferentially points away from the periphery rather than toward it. Interestingly, we found that Ima1 and Man1 have common, but also separate target regions in the genome. Ima1-interacting loci were enriched for the RNAi components Dcr1 and Rdp1. This agrees with previous findings that Dcr1 is localized at the nuclear periphery. In contrast, Man1 target loci were bound by the heterochromatin protein Swi6, especially at subtelomeric regions. Subtelomeric chromatin was shown to form a unique chromatin type lacking both repressive and active chromatin features and containing low levels of the histone variant H2A.Z. Thus, we find that the fission yeast nuclear periphery shows similar properties to those of metazoan cells, despite the absence of a nuclear lamina. Our results point to a role of nuclear membrane proteins in organizing chromatin domains and loops.

Whole-genome views of chromatin structure

DNA in eukaryotes is packed into chromatin. The basic component of chromatin is the nucleosome consisting of DNA wrapped around a histone octamer. Inside the cell nucleus, chromatin is folded into higher-order structures through various mechanisms, including repositioning of nucleosomes along the DNA, packing of nucleosomes into more condensed 3-dimensional configurations, looping of chromatin fibres, and tethering of chromosomal regions to nuclear structures. Over the past few years, new microarray-based methods have been developed for the genome-wide mapping of various aspects of chromatin structure. These methods are beginning to provide insights into the different types of chromatin and the architectural principles that govern the 3-dimensional organisation of the genome inside the nucleus.

Distribution of sister chromatid exchanges in different types of chromatin in the X chromosome ofMicrotus cabrerae

We used the X chromosomes ofMicrotus cabrerae as a model to analyze the distribution of sister chromatid exchanges (SCEs) on different types of chromatin, because of the marked heterogeneity of the heterochromatin in the entire short arm and a portion of the long arm of this chromosome. Computer-simulated distributions, according to an algorithm that makes it possible to modify the distribution on the basis of any possible hypothesis, were compared with real distributions by log-linear models. We found that the frequency of SCEs in different types of heterochromatin was higher than that expected for a random distribution, and located an SCE hot-spot at the junction between euchromatin and heterochromatin. The possible relationship between the distribution of SCEs and base composition or chromatin accessibility are discussed.

Structural and functional properties of linker histones and high mobility group proteins in polytene chromosomes.

Variants of histone H1 and high mobility group (HMG) proteins and their genes in Dipteran insects are being studied in our laboratory and have revealed different properties of DNA binding and intrachromosomal distribution. One of the H1 variants of Chironomus is found only in a minority of polytene chromosome bands and differs from the other H1 proteins of the same organism by genomic organization and by an inserted structural motif, the KAPKAP repeat, that is present also in single H1 variants of other, evolutionarily remote organisms. NH2-terminal peptides containing the KAPKAP repeat were found in vitro to interact with DNA, whereas no DNA interaction was observed with the homologous peptide of another H1 variant that does not contain the inserted KAPKAP repeat. We assume that H1 variants containing the KAP motif may interact with a stretch of linker DNA and package chromatin more tightly than other H1 variants. A large series of antibodies directed against different sites in all regions of the H1 molecule is being applied in studying the sites of interaction of the H1 molecule with other molecules in interphase chromatin in terms of antibody epitope accessibility. A search for insect proteins that share properties of the mammalian HMG proteins resulted in isolation and sequencing of two different HMG1 proteins and an HMGI protein. The HMG1 protein of the midge, Chironomus tentans, show a differential distribution in chromosomes. The more abundant cHMG1a protein appears uniformly distributed, whereas the less abundant cHMG1b protein could be localized only in chromosomal puffs. This strongly indicates that these highly similar proteins have different functions in chromatin. The Chironomus HMGI protein and the intron/exon organization of its gene were found to be very similar to human HMGI/Y proteins that are highly abundant in rapidly proliferating cells. Common properties of HMG1 and HMGI proteins include high affinity interaction with AT-rich DNA, irregular DNA structures, and the capacity to bend DNA. These properties suggest that the HMG proteins may have an architectural role in assembling different types of chromatin.

Characterization of different chromatin types in Mytilus galloprovincialis L. after C-banding, fluorochrome and restriction endonuclease treatments

In this study, we have carried out the cytogenetical characterization of Mytilus galloprovincialis L. (2n = 28) using conventional staining and banding techniques such as fluorochromes and restriction endonucleases treatment. Chromosome digestion with trypsin enzyme resulted in a G-banding pattern which allowed us to clearly identify and classify the chromosome pairs of M. galloprovincialis. C-banding and chromomycin A3 staining confirmed the existence of small amounts of constitutive heterochromatin. The treatment of samples with AluI, HaeIII, DpnI, MspI, HpaII and HinfI restriction endonucleases produced specific banding patterns which demonstrate the potential of endonucleases for chromosome banding in mussels. The results obtained allow us to describe six different types of chromatin in M. galloprovincialis. The type is determined by the response of the chromosomes to the different treatments. Differential digestion by the enzyme pair HpaII-MspI of specific C-band positive heterochromatic areas in some of the chromosomes suggests the presence of methylation.

28] Reconstitution of chromatin from purified components

Publisher Summary This chapter focuses on the reconstitution of chromatin from purified components. Several useful methods have resulted from attempts to reconstitute purified histories and DNA into chromatin in vitro. Alternatively, core histories can be added very slowly to DNA at physiological salt concentrations to form nucleosomes. Both of these methods have been devised to circumvent the aggregation pathways that are encountered on directly mixing the components at physiological salt concentrations. The aggregation pathways can also be avoided by using a negatively charged third component as an assembly factor. The use of poly (glutamic acid) as a nucleosome and chromatin assembly factor is described in this chapter. It has been possible to generate spaced nucleosome arrays using histones from all species and cell types that have been examined thus far in our laboratory. However, reaction conditions must be modified in some cases, suggesting that different types of chromatin may have rather different stabilities.

Characterization of chromosome replication during S-phase with bromodeoxyuridine labelling in Chinese hamster ovary and HeLa cells

Chinese hamster ovary (CHO) and HeLa cells were successively pulse labelled at 1-h intervals after the cultures were synchronized at the end of G1 (monitored by flow cytometry). The metaphases analysed afterwards showed R-type replication patterns after 1-h pulses during the early S-phase (SE; from h 1-5 after release) and replication of G- and C-bands in late S-phase (SL: from h 6-8 after release). The transition from SE to SL is abrupt, constituting a sudden switch of replication between different types of chromatin as has been described for human lymphocytes. The differences between these two cell lines and earlier results reported on a V79 Chinese hamster cell line and on normal diploid human and Chinese hamster fibroblasts are discussed.

Morphometric analyses on the muscles of exercise trained and untrained dogs.

Nine adult dogs were subjected to an endurance training program consisting of gradually increasing periods of treadmill exercise. Biopsies of vastus lateralis muscles taken prior to an on completion of the training program were used to study the alterations in the capillary network and muscle-fiber nuclei. Combined morphometric and stereological analyses showed that the total length of capillaries (LV) and their total surface area (SV) per unit volume of muscle was significantly increased during training. Changes in the properties of myonuclei following training were also studied. Highly significant increases in the number of nuclei per unit volume of muscle (NV) were found. The increase in the numerical density of muscle-fiber nuclei would seem to indicate that the new nuclei were formed in the muscle during prolonged training. The relative amounts and distribution of heterochromatin and euchromatin were measured in trained and untrained dogs using a simple point-counting and random-transect technique. Their content of the different types of chromatin (volume functions and chromatin patch sizes in micrographs) were found to be altered significantly as a result of the training. The findings were consistent with the view that the amount of euchromatin within nuclei varies according to the degree of cellular activity.

Histochemical properties of spermatozoa and somatic cells. II. Differences in the Feulgen hydrolysis pattern induced through alterations of the nucleoprotein complex

In this investigation we have studied the influence of the protein components of different types of chromatin on the acid hydrolysis pattern of DNA. The addition of NaCl or mercaptoethanol or both to the hydrolysis bath was found to increase considerably both the depurination and the depolymerization speed. These treatments were also found to alter the form of the hydrolysis patterns obtained under standard conditions for a number of cell types. Thus salmon and bull spermatozoa, which are widely different in their BAO-hydrolysis patterns when hydrolysed in pure acid, exhibit close similarities in their pattern after the addition of NaCl and mercaptoethanol to the hydrolysis bath. The spermatozoan DNA seemed to be stabilized in a much simpler way than the somatic cells, e.g. ascites cells and avian erythrocytes, which were far more resistant to the treatments. The results point towards the existence of functional similarities in the examined types of spermatozoa.