Role of the nuclear lamina in genome organization and gene expression.

The nuclear lamina is a major structural component of metazoan nuclei that has long been thought to provide an anchoring site for interphase chromosomes and have a role in gene regulation. Recent genome-wide mapping studies and functional experimental data strongly support these roles of the nuclear lamina. Here, we discuss new insights into various aspects of genome-nuclear lamina interactions, with emphasis on the links with gene regulation and with dynamics during cellular differentiation.

Chromatin profiling using targeted DNA adenine methyltransferase.

Chromatin is the highly complex structure consisting of DNA and hundreds of associated proteins. Most chromatin proteins exert their regulatory and structural functions by binding to specific chromosomal loci. Knowledge of the identity of these in vivo target loci is essential for the understanding of the functions and mechanisms of action of chromatin proteins. We report here large-scale mapping of in vivo binding sites of chromatin proteins, using a novel approach based on a combination of targeted DNA methylation and microarray technology. We show that three distinct chromatin proteins in Drosophila melanogaster cells each associate with specific sets of genes. HP1 binds predominantly to pericentric genes and transposable elements. GAGA factor associates with euchromatic genes that are enriched in (GA)n motifs. A Drosophila homolog of Saccharomyces cerevisiae Sir2p is associated with several active genes and is excluded from heterochromatin. High-resolution, genome-wide maps of target loci of chromatin proteins ('chromatin profiles') provide new insights into chromatin structure and gene regulation.

Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase.

We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.

Control of human telomere length by TRF1 and TRF2.

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.

Heterochromatic deposition of centromeric histone H3-like proteins.

Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. The functional significance of this centromere-heterochromatin relationship, if any, is unknown. One possibility is that heterochromatin provides a suitable environment for assembly of centromere components, such as special centromeric nucleosomes that contain distinctive histone H3-like proteins. We describe a Drosophila H3-like protein, Cid (for centromere identifier) that localizes exclusively to fly centromeres. When the cid upstream region drives expression of H3 and H2B histone-green fluorescent protein fusion genes in Drosophila cells, euchromatin-specific deposition results. Remarkably, when the cid upstream region drives expression of yeast, worm, and human centromeric histone-green fluorescent protein fusion proteins, localization is preferentially within Drosophila pericentric heterochromatin. Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells. Preferential localization to heterochromatin in heterologous systems is unexpected if centromere-specific or site-specific factors determine H3-like protein localization to centromeres. Rather, the heterochromatic state itself may help localize centromeric components.

TRF2 protects human telomeres from end-to-end fusions.

The mechanism by which telomeres prevent end-to-end fusion has remained elusive. Here, we show that the human telomeric protein TRF2 plays a key role in the protective activity of telomeres. A dominant negative allele of TRF2 induced end-to-end chromosome fusions detectable in metaphase and anaphase cells. Telomeric DNA persisted at the fusions, demonstrating that TTAGGG repeats per se are not sufficient for telomere integrity. Molecular analysis suggested that the fusions represented ligation of telomeres that have lost their single-stranded G-tails. Therefore, TRF2 may protect chromosome ends by maintaining the correct structure at telomere termini. In addition, expression of mutant forms of TRF2 induced a growth arrest with characteristics of senescence. The results raise the possibility that chromosome end fusions and senescence in primary human cells may be caused by loss by TRF2 from shortened telomeres.

Control of telomere length by the human telomeric protein TRF1.

Human telomeres, the nucleoprotein complexes at chromosome ends, consist of tandem arrays of TTAGGG repeats bound to specific proteins. In normal human cells, telomeres shorten with successive cell divisions, probably due to the terminal sequence loss that accompanies DNA replication. In tumours and immortalized cells, this decline is halted through the activation of telomerase, a reverse transcriptase that extends the telomeric TTAGGG-repeat arrays. Telomere length is stable in several immortal human-cell lines, suggesting that a regulatory mechanism exists for limiting telomere elongation by telomerase. Here we show that the human telomeric-repeat binding factor TRF1 (ref. 8) is involved in this regulation. Long-term overexpression of TRF1 in the telomerase-positive tumour-cell line HT1080 resulted in a gradual and progressive telomere shortening. Conversely, telomere elongation was induced by expression of a dominant-negative TRF1 mutant that inhibited binding of endogenous TRF1 to telomeres. Our results identify TRF1 as a suppressor of telomere elongation and indicate that TRF1 is involved in the negative feedback mechanism that stabilizes telomere length. As TRF1 does not detectably affect the expression of telomerase, we propose that the binding of TRF1 controls telomere length in cis by inhibiting the action of telomerase at the ends of individual telomeres.

Comparison of the human and mouse genes encoding the telomeric protein, TRF1: chromosomal localization, expression and conserved protein domains.

Mammalian chromosome ends contain long arrays of TTAGGG repeats that are complexed to a telomere specific protein, the TTAGGG repeat binding factor, TRF1. Here we describe the characterization of genes encoding the human and mouse TRF1 proteins, hTRF1 and mTRF1. The mTRF1 cDNA was isolated based on sequence similarity to the hTRF1 cDNA and the mTRF1 mRNA was shown to be ubiquitously expressed as a single 1.9 kb polyadenylated transcript in mouse somatic tissues. High levels of a 2.1 kb transcript were found in testes. In vitro translation of the mTRF1 cDNA resulted in a 56 kDa protein that binds to TTAGGG repeat arrays. mTRF1 displayed the same sequence specificity as hTRF1, preferring arrays of TTAGGG repeats as a binding substrate over TTAGGC and TTGGGG repeats. Expression of an epitope-tagged version of mTRF1 showed that the protein is located at the ends of murine metaphase chromosomes. In agreement, conceptual translation indicated that mTRF1 and hTRF1 are similarly-sized proteins with nearly identical C-terminal Myb-related DNA binding motifs. In addition, comparison of the predicted mTRF1 and hTRF1 amino acid sequences showed that the acidic nature of the N-terminus of TRF1 is conserved and revealed a highly conserved novel domain of approximately 200 amino acids in the middle of the proteins. However, other regions of the proteins are poorly conserved (<35% identity) and the overall level of identity of the mTRF1 and hTRF1 amino acid sequences is only 67%. The TRF1 genes are not syntenic; the hTRF1 gene localized to human chromosome 8 band q13 while the mTRF1 gene localized to mouse chromosome 17 band E3. The data indicate that the genes for mammalian telomeric proteins evolve rapidly.

PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components.

The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D-distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual-labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2-snRNP, or transcription factors (glucocorticoid receptor, TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle-late S-phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RAR alpha gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML-RAR fusion protein, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non-APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bodies are consistent with the idea that PML bodies are associated with specific genomic loci.

Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex.

Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TTAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TTAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix-associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.

Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons.

The glucocorticoid receptor and the mineralocorticoid receptor are hormone-dependent transcription factors. They regulate the excitability of rat hippocampus CA1 neurons in a coordinated fashion. We studied the spatial distribution of these transcription factors in nuclei of CA1 neurons by dual labeling immunocytochemistry and confocal microscopy, combined with novel image restoration and image analysis techniques. We found that both receptors are concentrated in about one thousand clusters within the nucleus. Some clusters contain either mineralocorticoid receptors or glucocorticoid receptors, but a significant number of clusters contains both receptors. These results indicate that the two receptor types are targeted to specific compartments in the nucleus. The coordinated action of the glucocorticoid and mineralocorticoid receptor on gene expression may be established in a specific set of nuclear domains that contain both receptors.

A human telomeric protein.

Telomeres are multifunctional elements that shield chromosome ends from degradation and end-to-end fusions, prevent activation of DNA damage checkpoints, and modulate the maintenance of telomeric DNA by telomerase. A major protein component of human telomeres has been identified and cloned. This factor, TRF, contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. Immunofluorescent labeling shows that TRF specifically colocalizes with telomeric DNA in human interphase cells and is located at chromosome ends during metaphase. The presence of TRF along the telomeric TTAGGG repeat array demonstrates that human telomeres form a specialized nucleoprotein complex.

Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus.

The cell nucleus is highly organized. Many nuclear functions are localized in discrete domains, suggesting that compartmentalization is an important aspect of the regulation and coordination of nuclear functions. We investigated the subnuclear distribution of the glucocorticoid receptor, a hormone-dependent transcription factor. By immunofluorescent labeling and confocal microscopy we found that after stimulation with the agonist dexamethasone the glucocorticoid receptor is concentrated in 1,000-2,000 clusters in the nucleoplasm. This distribution was observed in several cell types and with three different antibodies against the glucocorticoid receptor. A similar subnuclear distribution of glucocorticoid receptors was found after treatment of cells with the antagonist RU486, suggesting that the association of the glucocorticoid receptor in clusters does not require transformation of the receptor to a state that is able to activate transcription. By dual labeling we found that most dexamethasone-induced receptor clusters do not colocalize with sites of pre-mRNA synthesis. We also show that RNA polymerase II is localized in a large number of clusters in the nucleus. Glucocorticoid receptor clusters did not significantly colocalize with these RNA polymerase II clusters or with domains containing the splicing factor SC-35. Taken together, these results suggest that most clustered glucocorticoid receptor molecules are not directly involved in activation of transcription.

Domains of the human androgen receptor and glucocorticoid receptor involved in binding to the nuclear matrix.

Steroid receptors have been reported to bind to the nuclear matrix. The nuclear matrix is operationally defined as the residual nuclear structure that remains after extraction of most of the chromatin and all soluble and loosely bound components. To obtain insight in the molecular mechanism of the interaction of steroid receptors with the nuclear matrix, we studied the binding of several deletion mutants of the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) to the nuclear matrix. Receptor binding was tested for two different nuclear matrix preparations: complete matrices, in which most matrix proteins are retained during the isolation procedure, and depleted matrices, which consist of only a subset of these proteins. The results show that the C-terminal domain of the hAR binds tightly to both depleted and complete matrices. In addition, at least one other domain of the hAR binds to complete matrices but not to depleted matrices. In contrast to the hAR, the hGR binds only to complete matrices. For this interaction both the DNA-binding domain and the C-terminal domain of the hGR are required, whereas the N-terminal domain is not. We conclude that specific protein domains of the hAR and the hGR are involved in binding to the nuclear matrix. In addition, our results indicate that the hAR and the hGR are attached to the nuclear matrix through different molecular interactions.

Nuclear domains and the nuclear matrix.

This overview describes the spatial distribution of several enzymatic machineries and functions in the interphase nucleus. Three general observations can be made. First, many components of the different nuclear machineries are distributed in the nucleus in a characteristic way for each component. They are often found concentrated in specific domains. Second, nuclear machineries for the synthesis and processing of RNA and DNA are associated with an insoluble nuclear structure, called nuclear matrix. Evidently, handling of DNA and RNA is done by immobilized enzyme systems. Finally, the nucleus seems to be divided in two major compartments. One is occupied by compact chromosomes, the other compartment is the space between the chromosomes. In the latter, transcription takes place at the surface of chromosomal domains and it houses the splicing machinery. The relevance of nuclear organization for efficient gene expression is discussed.

Pharmacological and functional characterization of human mineralocorticoid and glucocorticoid receptor ligands.

We characterized the pharmacological profiles of the human mineralocorticoid and glucocorticoid receptor for 11 natural and synthetic steroids regarding binding pharmacology, intracellular localization of hormone-receptor complexes, and agonistic or antagonistic properties at the gene expression level. The sex steroid progesterone bound with an affinity (ki < 0.01 nM) even higher than that of aldosterone to the human mineralocorticoid receptor and effectively antagonized the effect of aldosterone via the human mineralocorticoid receptor in functional co-transfection assays. This indicates that progesterone has potent antimineralocorticoid properties, while its antiglucocorticoid effects were less pronounced. The partial agonistic activities of antihormones in this assay suggest a direct interaction of antihormone-receptor complexes with the response elements on the DNA. These results are supported by immunofluorescence studies, in which both unliganded human mineralocorticoid and glucocorticoid receptors were distributed throughout the cytoplasm and nucleus, whereas agonist- as well as antagonist-receptor complexes showed an exclusively nuclear localization. These results contribute to the understanding of antihormone pharmacology and increase our understanding of the role of human mineralocorticoid and glucocorticoid receptors in physiological processes during different endocrine states.

Progesterone receptor-mediated effects of neuroactive steroids.

Several 3 alpha-hydroxysteroids accumulate in the brain after local synthesis or after metabolization of steroids that are provided by the adrenals. The 3 alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone are believed not to interact with intracellular receptors, but enhance GABA-mediated chloride currents. The present study shows that these neuroactive steroids can regulate gene expression via the progesterone receptor. The induction of DNA binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neuroactive steroids into progesterone receptor active 5 alpha-pregnane steroids. Thus, at physiological concentrations, these neuroactive steroids regulate neuronal function through their effects on both transmitter-gated ion channels and steroid receptor-regulated gene expression.

Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus.

Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of well-defined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that contranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.

Binding of corticosteroid receptors to rat hippocampus nuclear matrix.

In rat hippocampus, the mineralocorticoid receptor and the glucocorticoid receptor bind corticosterone with high affinity. We have studied the association of these receptors with the nuclear matrix both after in vivo and in vitro administration of radiolabelled corticosterone to hippocampus cells. It was found that in vivo 100% and in vitro 60% of the corticosterone that specifically bound to rat hippocampus nuclei was attached to the nuclear matrix. A selective glucocorticoid receptor agonist did not compete for corticosterone binding. This indicates that this binding was mediated by the mineralocorticoid receptor rather than the glucocorticoid receptor.