Transcriptional Activity Of Chromatin Research Articles

Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin

The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.

Nuclear pore complexes

What are they? Nuclear pore complexes (NPCs) are often referred to as the ‘gatekeepers of the nucleus’, being responsible for regulating the molecular traffic – from ions to ribosomes – between the nucleoplasm and the cytoplasm. A typical vertebrate nucleus has about 5,000 NPCs scattered over its surface.What do they look like? A picture is worth twice the length of this article (Figure 1Figure 1). All eukaryotes have these impressive structures: vertebrates have the Cadillac model (∼125 MDa) and yeast a sportier version (∼50 MDa). In spite of their immense size, these structures are made of a relatively small number of proteins – about 30 – the so-called nucleoporins or nups. Defining an NPC ‘interactome’ is paramount to understanding its function. A number of groups are contributing to this effort, so check back for updates in the near future.Figure 1A model of the vertebrate NPC within a cross sectional view of the nuclear envelope membrane (gray).The NPC is constructed with a central core consisting of ring structures (light blue and purple) from which eight spokes radiate to surround a central transporter (pink). Extending from the core are cytoplasmic filaments (red) and a nuclear basket (blue). (Model kindly provided by M. Rout and J. Aitchison.)View Large Image | View Hi-Res Image | Download PowerPoint SlideHow do they work? In the last 10 years, a clearer picture has developed for the NPC's role in nuclear transport. A family of soluble proteins called karyopherins or kaps – a.k.a. importins or exportins – recognize signal-containing cargoes and then bind to the NPC. Translocation then ensues with directionality and the energy for repeated rounds of transport provided by the GTPase Ran.How do cargoes move through the NPC? This remains a major unresolved issue. Models addressing this must consider the massive number of kap–cargo complexes that go through the NPC every second, and the NPC's large number of kap-binding sites. The field has settled on the idea that NPCs transport cargoes by a binding-diffusion mechanism rather than, for example, a molecular motor. One model suggests kaps are entrapped by nup-based tentacles, counteracting the entropically unfavorable process of diffusion through the NPC. The kaps are proposed to ping-pong between multiple low-affinity sites until reaching higher-affinity sites in the target compartment. Another model proposes that the nups form a hydrophobic meshwork that is selectively permeable to kaps. Still to be explained is why some kaps preferentially interact with certain nups. This suggests that the trip through the NPC is not the same for all the kaps and raises the possibility that these different traffic lanes can be differentially controlled.Are NPCs just for transport? No. Recently, a number of intriguing observations have opened our eyes to other functions of the NPC. Some reports suggest that traveling through the NPC gate may not necessarily be a passive event for the cargo molecules, and that some are ‘stamped’ with the ubiquitin-related modifier SUMO as they travel through. This modification likely helps determine the fate of the cargo after it enters the nucleus, including its destination and half-life.Anything else? NPCs also appear to control the spatial orientation and transcriptional activity of chromatin. In yeast, NPCs, like puppeteers, influence telomeric silencing via fiber-forming proteins emanating from their nucleoplasmic face. Recent studies have also shown that tethering a gene to the NPC can prevent its repression by creating a boundary between the gene and surrounding silenced heterochromatin. Both observations lend support to the enduring idea that the NPC, through association with the underlying chromatin, regulates gene expression.More Madness? Nups have begun to show up at some surprising locations. During mitosis in vertebrates, a number of nups are recruited to kinetochores. The link to kinetochores goes beyond this. At least two proteins that function in the spindle assembly checkpoint, Mad1 and Mad2, reside at NPCs during interphase. What they're doing there is still anyone's guess. Like the examples mentioned above, these observations show that our understanding of how the NPC influences nuclear physiology is just beginning to blossom.

Packaging principle: How DNA methylation and histone acetylation control the transcriptional activity of chromatin

Packaging principle: How DNA methylation and histone acetylation control the transcriptional activity of chromatin

Role of Nucleosome Remodeling Factor NURF in Transcriptional Activation of Chromatin

The Drosophila nucleosome remodeling factor (NURF) is a protein complex of four subunits that assists transcription factor-mediated perturbation of nucleosomes in an ATP-dependent manner. We have investigated the role of NURF in activating transcription from a preassembled chromatin template and have found that NURF is able to facilitate transcription mediated by a GAL4 derivative carrying both a DNA binding and an activator domain. Interestingly, once nucleosome remodeling by the DNA binding factor is accomplished, a high level of NURF activity is not continuously required for recruitment of the general transcriptional machinery and transcription for at least 100 nucleotides. Our results provide direct evidence that NURF is able to assist gene activation in a chromatin context, and identify a stage of NURF dependence early in the process leading to transcriptional initiation.

Transcriptional activity and DNA supercoiling during early germination in maize

The possible link between the ability of DNA to be transcribed and its three-dimensional organization in the eukaryotic nucleus has been studied during germination of the plant embryo. In this model system there is a gradual increase in the transcriptional activity of chromatin and no nuclear DNA replication takes place for at least the first 20–24 h following water imbibition. Maize embryo nucleoids derived by nonionic detergent and high salt treatment from nuclei isolated at different time points of germination were analysed by sedimentation in a series of sucrose gradients containing increasing amounts of the intercalating agent ethidium bromide. The sedimentation of the dry embryo nucleoids shows no dependence on the concentration of ethidium bromide while the 16-h germinated embryo possesses the titration curve characteristic of the active plant genomes. On the other hand, DNA isolated from both the dry and the germinated material behaved upon EthBr intercalation as expected on theoretical grounds, i.e. its sedimentation rate decreased with increase of the EthBr concentration. The lack of a biphasic response to EthBr titration we interpret as an indication of a lack of supercoiled DNA loops in the dry embryo nuclei.

Extensive purification and characterization of chromatin-bound histone acetyltransferase from Saccharomyces cerevisiae.

A strong correlation has been established between reversible acetylation of histones and transcriptional activation of chromatin. However, the function of histone acetylation remains unknown. We have approached this question by purifying histone acetyltransferase 15,000-fold from yeast and characterizing it enzymatically. Biochemical properties, including the pH and temperature optima and the Michaelis-Menten constants for both acetyl coenzyme A and histones, are similar to those reported for histone acetyltransferases from higher eukaryotes. Yeast histone acetyltransferase has a native molecular weight of 110,000 as determined by gel filtration and is tightly bound to chromatin. It displays high-substrate specificity for histones. It acetylates all four core histones in the order: H4 greater than H2B greater than H2A. 10-fold higher histone acetyltransferase activity is observed for free histones when compared to yeast polynucleosomes as a substrate.

DNA methylation in differentiating mouse teratocarcinoma and erythroleukemia cells.

We have measured the content of 5-methylcytosine (5MC) in the genomic DNA of differentiated and undifferentiated cultures of murine embryonal carcinoma (EC) and murine erythroleukemia (MEL) cells. A large proportion of deoxycytosine residues were methylated in EC cells (4.6%) and this proportion dropped significantly (4.1%) following differentiation. The alpha-1 globin genes were heavily methylated at HpaII sites in EC cells and in differentiated derivatives of these EC cells. The MEL cells had only 3.3% of their genomic deoxycytidine methylated and no significant change occurred following differentiation. The alpha-1 globin genes of MEL cells were much less methylated than the same genes in EC cells and no change in this methylation pattern accompanied the induction of hemoglobin synthesis. In EC X MEL cell hybrids which express the alpha-1 globin genes from both parents, the EC-derived genes had become demethylated. These results are consistent with the general model that DNA hypomethylation is correlated with expression of that DNA and that gene activation is accompanied by DNA demethylation. We have also measured the 5MC content of DNA isolated from nuclease-treated EC nuclei. Unexpectedly, the DNase I sensitive chromatin contained a large proportion of 5MC. This result, along with the work of others, suggests that nuclease sensitivity may often reflect the transcriptional activity of chromatin in somatic cells, but is not indicative of the active state in pluripotent EC cells.

Transcriptional activity of chromatin in developing and germinating triticale embryos

Rythmical changes in the transcriptional activity of embryo chromatin during development and maturation of grain of triticale (cv. MT‐3) have been observed. The template availability and RNA polymerase (EC 2.7.7.6) 1 and II activities were high at the beginning of embryo development (20 to 30 days) and lowest at days 35 to 40 after anthesis, after which they increased to the end of maturation (60 days). Grains from three important periods of development (20, 40 and 60 days after anthesis; that is milk, wax and full ripeness, respectively) were used for germination, and chromatin was isolated from embryos and seedlings. A dramatic increase in the activity of both RNA polymerases and template availability was observed after 48 h of germination. The increase in template availability depended also on the ripeness stage of the grain used for germination.

Lack of correlation between histone H4 acetylation and transcription during the Physarum cell cycle.

The interaction between nucleosomal proteins and DNA is expected to change during DNA replication as well as during transcription. A possible way of achieving the necessary structural changes is the modification of histones and high mobility group (HMG) proteins. The acetylation of core histones has been studied in various systems (for a review see ref. 1) and a correlation between histone acetylation and transcriptional activity of chromatin has frequently been proposed. In particular, Bradbury and co-workers have reported a cell cycle dependence of histone H4 acetylation in Physarum polycephalum which revealed two correlations: (1) tetraacetylated H4 (H4Ac4) correlated with the rate of transcription and (2) H4 acetylation was inversely correlated with H1 phosphorylation in mitosis. We present evidence here that H4 acetylation does not fit these correlations. Our data clearly show that the acetate content of H4 is high during the S phase, but low during later stages of the cell cycle. H4Ac4 remains at a nearly constant level during the whole cycle, with an elevation during the S phase. Furthermore, experiments with the deacetylase inhibitor sodium-n-butyrate do not support the proposed connection between diacetylated H4 (H4Ac2) and DNA replication. Our data imply that a correlation of H4 acetylation and transcription is unlikely during the cell cycle of Physarum. The conclusions of Bradbury and co-workers are therefore invalid.

Transcriptional activation and structural alteration of wheat chromatin during germination and seedling‐growth

The chromatin was isolated from embryos or young seedlings of wheat, Triticum aestivum L. cv. Mukakomugi, and the template activity, the endogenous RNA polymerase activity, the susceptibility to DNase 11 and polylysine binding capacity were assayed. Both transcriptional activities of chromatin were very low for the first 12 h of germination, increased from 12 to 24 h, and then increased markedly during the next 48 h of post‐germination. During the 72 h of germination, the template activity and the endogenous RNA polymerase activity increased 10‐fold and 40‐fold, respectively, while they decreased from 72 h to 96 h of seedling growth. Seventy‐two h seedling chromatin was more susceptible to endonuclease and was bound with more polylysine than the germ chromatin. These results suggest that the structure of the chromatin available for transcription changes during seed germination and seedling‐growth.

Influence of Kinetin on Histone Composition and Endogenous RNA Level in Differentiating Metaxylem ofZea MaysRoot Tips

SUMMARYCytochemical studies were undertaken to analyze the effect of kinetin on quantitative and qualitative changes in histones and endogenous RNA content in differentiating metaxylem cells of Zea mays roots. Microspectrophotometric analysis revealed close association between increase in the DNA: histone ratio and increase in RNA content of differentiating metaxylem cells of both, control and kinetin treated root apices. The kinetin treatment caused a larger increase in RNA content per cell but the increase in the DNA: histone ratio did not differ significantly from the developmentally correlated increase observed in the controls. However, the kinetin induced amplification of RNA is associated with an elevation of the arginine: lysine ratio of the histones. It is suggested that kinetin may bring about removal or acetylation of the lysine rich fraction and that this event is a factor in the elevation of the transcriptional activity of chromatin.

The Role of Cytokinins in the Development and Metabolism of Barley Leaves

Summary During the first six hours after illumination of etiolated barley leaves the transcriptional activity of chromatin rapidly increased. The intensity of this light-induced reaction was greatly influenced by leaf age. Kinetin treatment affected chromatin activity, but the response differed also with leaf age. In younger leaves (7-days old) kinetin treatment diminished the effect of light, while in older leaves (9 to l0-days old) kinetin additionally stimulated the activity of chromatin. The observed responses to light and kinetin were primarily due to the α-amanitin-resistant activity, so it is concluded that they were connected with the transcription controlled by RNA polymerase I responsible for the rRNA synthesis. These findings are discussed in relation to the greening process of etiolated leaves and involvement of cytokinins in this process.

Nucleosome—nucleosome interaction in chromatin

The available data suggest that transcriptional activity of chromatin is connected with its structural changes [ 1,2]. According to the electron microscope evidence, the elementary 100 A chromatin thread can exist in two main states, i.e., in a compact state, which is characterized by tight packing of nucleosomes and in an extended state when the connecting intemucleosomal DNA is clearly seen between the nucleosomes (beads-on-a-string) [3]. It is still unknown which factors are responsible for the structural transition from the closely packed state into the unfolded thread state which is probably indispensable for activation of transcription. In the presence of divalent metal ions nucleosomes have been shown tightly packed in chromatin [3,4]. Under these conditions intemucleosom~ DNA interacts with the nucleosome body which provides to it an ordered arrangement in a chromatin thread. In this work we used an enzymatic approach to the study of nucleosome packing in chromatin. Trypsin treatment of chromatin with subsequent nuclease digestion enabled us to characterise in more detail the interactions of intemucleosomal DNA with histone complexes of nucleosomes. The data obtained are interpreted to mean that internucleosomal DNA of compact chromatin interacts with the N-terminal parts of nucleosomal histones. Thus the intemucleosomal DNA plays a great part in histone-DNA interactions which are important in the close association of nucleosomes. It is supposed that modifications of amino acid residues in N-terminal regions of nucleosomal histones weakens the histone interactions with the internucleosomal DNA, leading to release of inter-

Morphology of transcriptional units at different states of activity.

The morphology of two forms of transcriptionally active chromatin, the nucleoli and the loops of lampbrush chromosomes, has been examined after fixation in situ or after isolation and dispersion of the material in media of low ionic strengths, using a variety of electron microscopic preparation techniques (e.g. spread preparations with positive or negative staining or without any staining at all, with bright and dark field illumination, with autoradiography, after pretreatment of the chromatin with specific detergents such as Sarkosyl NL-30; transmission and scanning transmission electron microscopy of ultrathin sections). Nucleolar chromatin and chromosomes from oocytes of various amphibia and insects as well as from green algae of the family of the Dasycladaceae were studied in particular detail. The morphology of transcriptional units that are densely packed with lateral ribonucleoprotein fibrils, indicative of great transcriptional activity, was compared with that of chromatin of reduced lateral fibril density, including stages of drug-induced inhibition. The micrographs showed that under conditions which preserve the nucleosomal organization in condensed chromatin studied in parallel, nucleosomes are not recognized in transcriptionally active chromatin. This holds for the transcribed regions as well as for apparently untranscribed (i.e. fibril-free) regions interspersed between (‘spacer’) and/or adjacent to transcribed genes and for the fibril-free regions within transcriptional units of reduced fibril density. In addition, comparison of lengths of repeating units of isolated rDNA with those observed in spread nucleolar chromatin indicated that this DNA is not foreshortened and packed into nucleosomal structures. Granular particles which were observed, at irregular frequencies and in variable patterns, in some spacer regions, did not result in a proportional shortening of the spacer axis, and were found to be resistant to detergent treatment effective in removing most of the chromatin associated proteins including histones. Thus, these particles behave like RNA polymerases rather than nucleosomes. It is suggested that structural changes from nucleosomal packing to an extended form of DNA are involved in the transcriptional activation of chromatin.