Inactive Chromatin Regions Research Articles

FoxA factors: the chromatin key and doorstop essential for liver development and function.

Pioneer factors are transcriptional regulators with the capacity to bind inactive regions of chromatin and induce changes in accessibility that underpin cell fate decisions. The FOXA family of transcription factors is well understood to have pioneer capacity. Indeed, researchers have uncovered numerous examples of FOXA-dependent epigenomic modulation in developmental and disease processes. Despite the presence of FOXA being essential for correct epigenetic patterning, the need for continued FOXA presence postchromatin modulation has been debated. In a recent study in this issue of Genes & Development, Reizel and colleagues (pp. 1039-1050) show that the tissue-specific ablation of FOXA1/2/3 in the adult mouse liver results in the collapse of the epigenetic profile that maintains the hepatic gene expression profile. Thus, FOXA functions as a key, opening regions of chromatin during development, and as a doorstep, maintaining the established euchromatic structure in adult tissue.

Epigenetic landscape influences the liver cancer genome architecture

The accumulations of different types of genetic alterations such as nucleotide substitutions, structural rearrangements and viral genome integrations and epigenetic alterations contribute to carcinogenesis. Here, we report correlation between the occurrence of epigenetic features and genetic aberrations by whole-genome bisulfite, whole-genome shotgun, long-read, and virus capture sequencing of 373 liver cancers. Somatic substitutions and rearrangement breakpoints are enriched in tumor-specific hypo-methylated regions with inactive chromatin marks and actively transcribed highly methylated regions in the cancer genome. Individual mutation signatures depend on chromatin status, especially, signatures with a higher transcriptional strand bias occur within active chromatic areas. Hepatitis B virus (HBV) integration sites are frequently detected within inactive chromatin regions in cancer cells, as a consequence of negative selection for integrations in active chromatin regions. Ultra-high structural instability and preserved unmethylation of integrated HBV genomes are observed. We conclude that both precancerous and somatic epigenetic features contribute to the cancer genome architecture.

Comparison of Fetal and Adult Erythroid Chromosomal Architectures Identifies a Novel Fetal Hemoglobin Regulatory Region

Chromatin structure is tightly intertwined with transcription regulation. The extent to which global chromatin architecture is subjected to alterations at different developmental stages within the same cell lineage has not been examined in great depth. Erythropoiesis offers an ideal model system to study the molecular mechanisms of gene regulation within the same cell lineage during development. Here, we comparatively defined via RNA-seq the transcriptomes, and via Hi-C and Capture-C the chromosome architectures of primary human fetal and adult erythroid cells.Overall, fetal and adult chromosomal conformations displayed a high degree of similarity. This includes the maintenance of A and B compartments representing active and inactive chromatin regions, respectively. Only ~5% of the genome switched compartments from A to B or vice versa, in agreement with the highly similar gene expression profiles. Moreover, topologically associating domains (TADs) were extensively preserved from fetal to adult stages. The developmentally regulated β-globin gene cluster is contained within one topologically associating domain (TAD) but folds into a three sub-TADs structure, the central one of which encompasses the β-globin locus. Notably, although the three sub-TAD structures are flanked by tissue invariant CTCF bound sites, they engage in looped contacts only in erythroid cells, indicating that erythroid specific transcription factors are required for CTCF mediated boundary contacts.At a finer scale, Capture-C detected distinct folding patterns at the developmentally controlled β-globin locus, including the expected stage-specific interactions between the enhancer (LCR) and the fetal γ-globin and adult β-globin genes. Importantly, we identified new developmental stage-specific chromatin contacts involving a region compassing a pseudogene (HBBP1) that resides between the fetal and adult globin genes. Specifically, HBBP1 engages in fetal stage-specific contacts with DNase hypersensitive sites HS5 and 3'HS1 while contacting the embryonic ε-globin gene at the adult stage. Deletion of a 2.3kb fragment encompassing HBBP1 (but not its transcriptional silencing) leads to strong reactivation of γ-globin gene expression in an adult erythroid cell line. This is accompanied by an architecturally restructured locus, including increased LCR-γ-globin chromatin interactions. Notably, the effects of HBBP1 deletion on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the HBBP1 region.In sum, our results identify a new segment, distinct from previously described regions linked to hereditary persistence of fetal hemoglobin, which engages in functionally important chromatin contacts. Since the HBBP1 region resides quite distantly from the structural globin genes, it might be a useful target for therapeutic genome editing without risking damage to the globin genes. Finally, our study highlights the power of high resolution chromosome architectural analysis to identify new regulatory regions. DisclosuresNo relevant conflicts of interest to declare.

Endothelial cell differentiation is encompassed by changes in long range interactions between inactive chromatin regions

Endothelial cells (ECs) differentiate from mesodermal progenitors during vasculogenesis. By comparing changes in chromatin interactions between human umbilical vein ECs, embryonic stem cells and mesendoderm cells, we identified regions exhibiting EC-specific compartmentalization and changes in the degree of connectivity within topologically associated domains (TADs). These regions were characterized by EC-specific transcription, binding of lineage-determining transcription factors and cohesin. In addition, we identified 1200 EC-specific long-range interactions (LRIs) between TADs. Most of the LRIs were connected between regions enriched for H3K9me3 involving pericentromeric regions, suggesting their involvement in establishing compartmentalization of heterochromatin during differentiation. Second, we provide evidence that EC-specific LRIs correlate with changes in the hierarchy of chromatin aggregation. Despite these rearrangements, the majority of chromatin domains fall within a pre-established hierarchy conserved throughout differentiation. Finally, we investigated the effect of hypoxia on chromatin organization. Although hypoxia altered the expression of hundreds of genes, minimal effect on chromatin organization was seen. Nevertheless, 70% of hypoxia-inducible genes situated within a TAD bound by HIF1α suggesting that transcriptional responses to hypoxia largely depend on pre-existing chromatin organization. Collectively our results show that large structural rearrangements establish chromatin architecture required for functional endothelium and this architecture remains largely unchanged in response to hypoxia.

A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment

Background: Huntington's Disease (HD) is an incurable disease of the adult brain. Massive changes in gene expression are a prominent feature. Epigenetic effects have been reported to be implicated in HD, but the role of chromatin is not well understood. We tested if the chromatin state of dysregulated genes in HD is affected at a genome-wide scale and examined how epigenetic processes are associated with CpG-island-mediated gene expression. Methods: Our general approach incorporates computational and functional analysis of public data before embarking on expensive wet-lab experiments. We compared the location in the genome of the genes that were deregulated in HD human brain, obtained from public gene expression data, to the location of particular chromatin marks in reference tissues using data from the ENCODE project. Results: We found that differentially expressed genes were enriched in the active chromatin state, but not enriched in the silent state. In the caudate nucleus, the most highly affected brain region in HD, genes in the active state were associated with transcription, cell cycle, protein transport and modification, RNA splicing, histone post-translational modifications and RNA processing. Genes in the repressed state were linked with developmental processes and responses related to zinc and cadmium stimulus. We confirmed that genes within CpG-islands are enriched among HD dysregulated genes in human and mouse in HD. Epigenetic processes were associated more with genes that overlap with CpG-islands than genes that do not. Conclusion: Our results suggest that massive transcriptional dysregulation in HD is not matched by large-scale relocation of gene activity, i.e. inactive chromatin regions are altered into actively expressed chromatin regions and vice versa. We expect that changes in epigenetic chromatin state might occur at the level of single genes (e.g. promoters, gene body) and scattered genomic sites (e.g. CTCF sites, enhancer regions) instead of large-scale genomic regions.

Attenuation of genome-wide 5-methylcytosine level is an epigenetic feature of cutaneous malignant melanomas.

Epigenetic modification of DNA, namely covalent changes of cytosine residues, plays a key role in the maintenance of inactive chromatin regions, both in health and in disease. In the vast majority of malignant melanomas, the most notable known epigenetic abnormality is the attenuation of 5-hydroxymethylcytosine (5-hmC) residues. However, it remains unknown whether a decrease in 5-hmC represents a primary defect of melanoma cancer epigenome or whether it is secondary to the loss of 5-methylcytosine (5-mC), a chemical substrate for 5-hmC. Here, we evaluated 5-mC levels in a spectrum of melanocytic proliferations. To study the epigenetic features of melanocytic nuclei, we began by measuring 5-mC levels in histologic specimens semiquantitatively by immunohistochemistry. We next treated established melanoma cell lines with S-adenosyl methionine (SAM), a universal methyl group donor, in an effort to cause changes in 5-mC levels. We detected a marked reduction in 5-mC levels in both primary and metastatic melanomas compared with 5-mC levels in benign melanocytic nevi. We also empirically induced changes in 5-mC in melanoma cell lines by incubation with SAM. To our surprise, we observed a significant cytoreductive effect of SAM on all melanoma cell lines examined. At subcytotoxic levels, SAM treatment is accompanied by a genome-wide increase in 5-mC. Moreover, we recorded a dose-dependent increase in genome-wide 5-mC levels in melanoma cell lines following SAM treatment. Taken together, we report that genome-wide attenuation of 5-mC is a hallmark of malignant melanomas. We propose that genome-wide attenuation of 5-mC is not merely an epiphenomenon as it is required for melanoma cell growth, albeit by an as of yet undetermined mechanism. Given its potential benefit in slowing down the growth of melanoma cells, SAM should be studied further to determine its role in epigenome modulation.

Using Markov chains of nucleotide sequences as a possible precursor to predict functional roles of human genome: a case study on inactive chromatin regions.

Recent advances in computational epigenetics have provided new opportunities to evaluate n-gram probabilistic language models. In this paper, we describe a systematic genome-wide approach for predicting functional roles in inactive chromatin regions by using a sequence-based Markovian chromatin map of the human genome. We demonstrate that Markov chains of sequences can be used as a precursor to predict functional roles in heterochromatin regions and provide an example comparing two publicly available chromatin annotations of large-scale epigenomics projects: ENCODE project consortium and Roadmap Epigenomics consortium.

Normal RUNX1 and Pathogenic ETV6/RUNX1 Compete Genome-Wide for Chromatin Binding in Pre-B Acute Lymphoblastic Leukemia

Introduction and objectives:The ETV6/RUNX1 fusion protein is a leukemia-initiating transcription factor that interferes with normal RUNX1 transcription factor function. Although RUNX1 can be either a repressor or an activator, ETV6/RUNX1 is mainly associated with transcriptional repression. Both RUNX1 and ETV6/RUNX1 proteins are expressed in pre-B acute lymphoblasts and bind the same DNA binding motif, which is normally recognized by the RUNX1 DNA binding domain, therefore competing for gene regulation with possible adverse transcriptional effects. Here, (we address the question of how) do the abnormal transcription factor ETV6/RUNX1 and the normal RUNX1 interplay at the chromatin level to modulate expression of genes, possibly giving rise to leukemia.Materials and methods:We used chromatin-immunoprecipitation coupled with sequencing (ChIP-seq) both in pre-B leukemic REH cells, which originate from a relapse of an ETV6/RUNX1 leukemic patient and lack the non-translocated allele of ETV6, and in pre-B leukemic Nalm6 cells that are ETV6/RUNX1-negative, to characterize the genomic distribution of ETV6/RUNX1 and RUNX1, as well as transcriptionnally active and inactive chromatin regions (ChIP-seq for methylated H3K4me3 and H3K4me1 histones, and acetylated H3K27achistones). In addition, we ran microarrays experiments to define the transcriptional programme associated with ETV6/RUNX1 and RUNX1 expression in pre-B cell lines. Data were compared to gene expression data from ETV6/RUNX1 pre-B acute lymphoblastic leukemia samples.Results:Through mapping the genome-wide distribution of RUNX1 et ETV6/RUNX1, we identified both common and exclusive DNA sites. We further characterized the DNA binding motifs of RUNX1 and ETV6/RUNX1, which were particularly enriched within particular transposon sequences. ChIP-seq of histone marks identified active and inactive chromatin in correlation with ETV6/RUNX1- and RUNX1-binding. Direct target genes for both transcription factors were defined and used for gene ontology analysis, allowing to uncover specific biological functions associated with ETV6/RUNX1 and RUNX1 binding events. Finally, we show that a fraction of the genes misregulated in pre-B acute lymphoblastic leukemia patients are directly controlled by ETV6/RUNX1 and RUNX1.Conclusion:Our data give new insights on the competition between the fusion protein ETV6/RUNX1 and normal RUNX1 in pre-B acute lymphoblastic leukemia at chromatin level genome-wide.This work was supported by CNRS, University of Rennes 1, University Hospital of Rennes, Ligue Régionale contre le Cancer (sections 22, 35 and 56) (MPA, VG, MBT), Région Bretagne (MBT, VG), Rennes Métropole (MBT), SFR Biosit UMS 3480 (VG, MBT), Société Française de Lutte contre les Cancers et les Leucémies de l’Enfant et de l’Adolescent and the Fédération Enfants et Santé (MBT), Association Laurette Fugain (VG) and the People Programme (Marie Curie Actions) of the European Union Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n°291851 (MBT). DisclosuresNo relevant conflicts of interest to declare.

Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome

Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein.

NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors

NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB and NF-YC) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5000–15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of the NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (nonmodified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively colocalizes with FOS in all genomic contexts, and this often occurs in the absence of JUN and the AP-1 motif. NF-Y also coassociates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly used proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve coassociation with FOS.

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10. We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments. Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

Inhibition of Histone Deacetylation Does Not Block Resilencing of p16 after 5-Aza-2′-Deoxycytidine Treatment

Epigenetic drugs are in use in clinical trials of various human cancers and are potent at reactivating genes silenced by DNA methylation and chromatin modifications. We report here the analysis of a set of normal fibroblast and cancer cell lines after combination treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA). Low doses of the drug combination caused cell cycle arrest, whereas high doses induced apoptosis in T24 bladder carcinoma cells. Both p16 (CDKN2A/INK4) and p21 (CIP1/SDI1/WAF1) expression were induced to similar levels in normal and cancer cells in a dose-dependent fashion after combination treatments. We detected a distinct increase of histone H3 acetylation at lysine 9/14 near the transcription start sites, in both LD419 normal fibroblasts and T24 bladder carcinoma cells, whereas the acetylation changes in the p21 locus were less apparent. Interestingly, the levels of trimethylation of histone H3 on lysine 9, which usually marks inactive chromatin regions and was associated with the p16 promoter in silenced T24 cells, did not change after drug treatments. Furthermore, we provide evidence that the remethylation of the p16 promoter CpG island in T24 cells after 5-aza-CdR treatment cannot be halted by subsequent continuous PBA treatment. The p16 gene is resilenced with kinetics similar to 5-aza-CdR only-treated cells, which is also marked by a localized loss of histone acetylation at the transcription start site. Altogether, our data provide new insights into the mechanism of epigenetic drugs and have important implications for epigenetic therapy.

Structural Characterization of Histone H2A Variants

protein to form chromatin, the fundamental unit of which is the nucleosome core particle (NCP). An NCP consists of two copies each of the four core histones H2A, H2B, H3, and H4. This histone octamer binds 147 base pairs of DNA around its outer surface in 1.65 tight superhelical turns (Fig. 1A) (Luger et al. 1997; Richmond and Davey 2003). Linker histones and other nonhistone proteins promote or stabilize the folding of nucleosomal arrays into superstructures of increasing complexity and largely unknown architecture (Hansen 2002). Covalent modification of the core histones and variations in the fundamental biochemical composition of nucleosomes distinguish transcriptionally active from inactive chromatin regions, by either changing the structure of the nucleosomes, altering their ability to interact with other protein factors, or modifying their propensity to fold into varying degrees of higher-order structures (or by any combination of the above). Studying the mechanism for establishing distinct chromatin domains is essential to understanding differential regulation of gene expression and all other DNA-dependent processes. Much progress has been made in this direction in the past few years. Substitution of one or more of the core histones with the corresponding histone variants has the potential to exert considerable influence on the structure and function of chromatin. Histone variants are distinct nonallelic forms of conventional, major-type histones that form the bulk of nucleosomes during replication and whose synthesis is tightly coupled to S phase. Histone variants are characterized by a completely different expression pattern that is not restricted to S phase. They are found in most eukaryotic organisms and are expressed in all tissue types (unlike some H2B isoforms that are found only in specialized tissues such as testes). Compared to their majortype counterparts, histone variants exhibit moderate to significant degrees of sequence homology (Fig. 1B). H2A.X (82%) and H3.3 (~96%) are the least divergent of all histone variants. H2A.Z (~60%), macroH2A (~65%), H2A.Bbd (40%), and CenpA, which has a 93 amino acid domain that is 62% identical to H3 (Palmer et al. 1991; Sullivan et al. 1994), are increasingly divergent in their Structural Characterization of Histone H2A Variants

Varied interactions between proviruses and adjacent host chromatin.

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.

Varied interactions between proviruses and adjacent host chromatin.

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.

Association of poly(adenosine diphosphate ribosylated) nucleosomes with transcriptionally active and inactive regions of chromatin.

We have investigated whether transcriptionally active or inactive gene sequences are associated in vivo with poly(adenosine diphosphate ribosylated) regions of chromatin. Soluble HeLa cell chromatin derived from nuclei treated either briefly or extensively with micrococcal nuclease was fractionated on an anti-poly(adenosine diphosphate ribose)-Sepharose column [Malik, N., Miwa, M., Sugimura, T., Thraves, P., & Smulson, M. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] to obtain fractions that were enriched or depleted in poly(ADP-ribosylated) chromatin. DNA obtained from these fractions was then probed for active and inactive gene sequences with a cDNA probe made from total cell mRNA and a probe for the beta-globin gene. Chromatin enriched in poly(ADP-ribosylated) nucleosomes contained both active and inactive gene sequences as detected by the probes and appeared to be more nuclease sensitive than that found in the fraction of chromatin depleted of poly(ADP-Rib). Poly(ADP-ribosylated) chromatin from nuclei digested briefly with nuclease showed an enrichment in both active and inactive genes while that treated extensively with nuclease showed either no enrichment or a depletion of active and inactive genes. Actively transcribed chromatin was digested at a rate several times that of the bulk or inactive chromatin. Nevertheless, the enrichment of active genes in poly(ADP-ribosylated) nucleosomes derived from brief nuclease digestion was greater than that of inactive genes. These results are interpreted as showing that some, but not all, of actively transcribed chromatin contains associated poly(ADP-ribosylated) proteins. However, since poly(ADP-ribosylated) proteins are also associated with inactive genes, the function of this modification cannot be assigned solely to transcription.

Susceptibility of Rous sarcoma virus-specific sequences integrated into SR-C3H/He mouse ascites sarcoma cell chromatin to DNase I and DNase II.

The susceptibility of Rous sarcoma virus (RSV) genomes integrated in mouse ascites sarcoma cells (SR-C3H/He cells) to DNase I and DNase II was investigated. Approximately half of the viral sequences were sensitive to DNase I and DNase II when 17% and 7.4% of the chromatin DNA was rendered acid soluble, respectively. The results suggest that newly acquired exogenous proviral sequences are integrated into both transcriptionally active and inactive regions of chromatin in cells lacking related endogenous viral sequences.

Transcriptionally Active and Inactive Regions of Nucleolar Chromatin in Amplified Nucleoli of Fully Grown Oocytes of Hibernating Frogs, Rana pipiens (Amphibia, Anura) : A Quantitative Electron Microscopic Study

Chromatin structures contained in nuclei of full-grown oocytes of hibernating northern leopard frogs, Rana pipiens, kept at 4 degrees C were examined by light and electron microscopy, with special emphasis on the morphology of spread nucleoli. Light microscopic inspection of the in situ arrangement revealed that at this stage of oogenesis the numerous amplified nucleoli, together with the chromosomes, were included in a centrally located aggregate. Characteristically, the nucleoli appeared spheroidal and showed large size differences; many of them revealed a caveolated interior and close association with large dense bodies of granular (GDB) or fibrillar (FDB) substructure. A spreading procedure involving limited dispersal of nucleolar chromatin allowed the analysis of a large number of individual nucleoli from a specific nucleus. Electron microscopy showed that different types of nucleoli which could be distinguished in terms of transcriptional activity occurred in the same nucleus: (i) Nucleoli in which nearly all pre-rRNA gene regions appeared as fully covered by lateral fibrils, indicative of active transcription ('active nucleoli'); (ii) nucleoli that contained exclusively or predominantly chromatin strands free of lateral fibrils and were characterized by a uniformly beaded appearance, interpreted as transcriptionally inactive, nucleosome-packed chromatin ('inactive nucleoli'); and (iii) nucleoli in which a number of typical arrays of lateral fibril gradients occurred besides other regions that were free of such fibrils and apparently non-transcribed ('partly inactive nucleoli'). Quantitative evaluations of the proportions of active and inactive nucleolar chromatin indicated that a minimum of 70% of nucleolar DNA is not transcribed at this stage of oogenesis. Details of the pattern and ultrastructural organization of active and inactive chromatin regions are presented. Lampbrush chromosome structures were observed in the same nuclei and also showed transcriptional structures, though often with reduced packing density of lateral fibrils. The observations show that a number of genes of both kinds, those coding for pre-rRNAs and those coding for proteins, are transcribed in full-grown oocytes of hibernating frogs at 4 degrees C. However, the data also indicate that the number of transcribed genes and the frequency of transcriptional events are greatly reduced at this stage of oogenesis, especially in the nucleolar chromatin.

Non-preferential localization of poly(ADP-ribose) polymerase activity in transcriptionally active chromatin

eukaryote nucleus, DNA exists as a highly ordered nucleoprotein complex, chromatin, whose organization regulates ultimately various functional states of the cell. To better understand this organiza- tion, it is of interest to know the subchromatin locali- zation of proteins which influence functional activity of chromatin. Poly(ADP-ribose) polymerase, which is a ubiquitous nuclear enzyme, provides a suitable probe for this purpose, because it is firmly integrated in eukaryotic chromatin structure [l] . A chromatin fractionation technique, which yielded a fraction greatly enriched in nascent RNA and in DNA sequences transcriptionally active in vivo was developed [2,3] . The technique involves selective shearing of chromatin with DNAase II followed by differential precipitation. The nucleolytic cleavage of chromatin DNA is a mild treatment and does not lead to detectable level of chromosomal protein rearrangement relative to specific DNA sequences, which is observed during mechanical shearing of chromatin [4,5]. In this report, using this procedure, we studied the distribution of poly(ADP-ribose) polymerase activity in transcriptionally active and inactive chromatin regions. It was shown that the enzyme is not preferen- tially localized in transcriptionally active chromatin region. 2. Materials and methods 2.1.