PubMed Health⌕ Search

Biomedical subjects

Andrew S Belmont

Publications and source records attributed to Andrew S Belmont.

At least 19 recordsLinked to original sources
PubMed2026

Acidic transcription factors position the genome at nuclear speckles through transcription-dependent and -independent mechanisms.

A small fraction of the genome reproducibly positions near nuclear speckles (NSs), increasing the expression and/or splicing efficiency of NS-associated genes. How specific genomic regions in mammalian cells are targeted to NSs remains unclear. Here, we demonstrate the establishment of genome-wide NS association without active transcription. We show that DNA sequences derived from NS-associated regions, when integrated as transgenes, are autonomously targeted to NSs. By systematically dissecting one such genomic locus, the COL1A1-SGCA locus, we identified redundant NS-targeting cis-regulatory elements, including an ∼600-bp fragment with 17 binding motifs for 8 transcription factors (TFs). Four NS-targeting TFs within this fragment contain acidic activation domains (AADs) that provide both chromatin-context and transcription-dependent NS targeting, properties that appear to be common among several other tested AADs. A subset of acidic activator TFs contains an additional, transcription-independent NS-targeting activity. Our findings establish diverse and partially redundant NS-targeting activities, which may facilitate dynamic gene positioning at the NS periphery for context-specific transcriptional responses.

Transcription, Genetic↗
PubMed2006

Mitotic chromosome structure and condensation.

Mitotic chromosome structure has been the cell biology equivalent of a 'riddle, wrapped in a mystery, inside an enigma'. Observations that genetic knockout or knockdown of condensin subunits or topoisomerase II cause only minimal perturbation in overall chromosome condensation, together with analysis of early stages of chromosome condensation and effects produced by histone H1 depletion, suggest a need to reconsider textbook models of mitotic chromosome condensation and organization.

Adenosine Triphosphatases↗
PubMed2006

Long-range directional movement of an interphase chromosome site.

Increasing evidence suggests functional compartmentalization of interphase nuclei. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions , association of some active genes with nuclear speckles or transcription "factories", and association of transcriptionally repressed genes with heterochromatic regions. Dynamic changes in chromosome compartmentalization imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility, described as "constrained diffusion". None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1-2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1-0.9 microm/min over distances of 1-5 microm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

Actins↗
PubMed2005

Truncated HP1 lacking a functional chromodomain induces heterochromatinization upon in vivo targeting.

Packaging of the eukaryotic genome into higher order chromatin structures is tightly related to gene expression. Pericentromeric heterochromatin is typified by accumulations of heterochromatin protein 1 (HP1), methylation of histone H3 at lysine 9 (MeH3K9) and global histone deacetylation. HP1 interacts with chromatin by binding to MeH3K9 through the chromodomain (CD). HP1 dimerizes with itself and binds a variety of proteins through its chromoshadow domain. We have analyzed at the single cell level whether HP1 lacking its functional CD is able to induce heterochromatinization in vivo. We used a lac-operator array-based system in mammalian cells to target EGFP-lac repressor tagged truncated HP1alpha and HP1beta to a lac operator containing gene-amplified chromosome region in living cells. After targeting truncated HP1alpha or HP1beta we observe enhanced tri-MeH3K9 and recruitment of endogenous HP1alpha and HP1beta to the chromosome region. We show that CD-less HP1alpha can induce chromatin condensation, whereas the effect of truncated HP1beta is less pronounced. Our results demonstrate that after lac repressor-mediated targeting, HP1alpha and HP1beta without a functional CD are able to induce heterochromatinization.

Animals↗
PubMed2005

Close encounters between active genes in the nucleus.

A recent paper demonstrates that coregulated genes on different chromosomes show surprisingly high frequencies of colocalization within the nucleus; this complements similar results found previously for genes localized tens of megabases apart on a single chromosome. Colocalization could be related to the earlier observation of active genes associating with foci where RNA polymerase II is concentrated.

Animals↗
PubMed2005

Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope.

Increasing evidence points to a dynamical compartmentalization of the cell nucleus, yet the mechanisms by which interphase chromatin moves and is positioned within nuclei remain unclear. Here, we study the dynamics of chromatin in vivo applying a novel particle-tracking method in a two-photon microscope that provides approximately 10-fold higher spatial and temporal resolutions than previous measurements. We followed the motion of a chromatin sequence containing a lac-operator repeat in cells stably expressing lac repressor fused with enhanced green fluorescent protein, observing long periods of apparent constrained diffusion interrupted by relatively abrupt jumps of approximately 150 nm lasting 0.3-2 s. During these jumps, the particle moved an average of four times faster than in the periods between jumps and in paths more rectilinear than predicted for random diffusion motion. Additionally, the jumps were sensitive to the temperature and absent after ATP depletion. These experimental results point to an energy-dependent mechanism driving fast motion of chromatin in interphase cells.

Animals↗
PubMed2005

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation.

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.

Cells, Cultured↗
PubMed2005

Common effects of acidic activators on large-scale chromatin structure and transcription.

Large-scale chromatin decondensation has been observed after the targeting of certain acidic activators to heterochromatic chromatin domains. Acidic activators are often modular, with two or more separable transcriptional activation domains. Whether these smaller regions are sufficient for all functions of the activators has not been demonstrated. We adapted an inducible heterodimerization system to allow systematic dissection of the function of acidic activators, individual subdomains within these activators, and short acidic-hydrophobic peptide motifs within these subdomains. Here, we demonstrate that large-scale chromatin decondensation activity is a general property of acidic activators. Moreover, this activity maps to the same acidic activator subdomains and acidic-hydrophobic peptide motifs that are responsible for transcriptional activation. Two copies of a mutant peptide motif of VP16 (viral protein 16) possess large-scale chromatin decondensation activity but minimal transcriptional activity, and a synthetic acidic-hydrophobic peptide motif had large-scale chromatin decondensation activity comparable to the strongest full-length acidic activator but no transcriptional activity. Therefore, the general property of large-scale chromatin decondensation shared by most acidic activators is not simply a direct result of transcription per se but is most likely the result of the concerted action of coactivator proteins recruited by the activators' short acidic-hydrophobic peptide motifs.

Amino Acid Motifs↗
PubMed2004

Upstream binding factor association induces large-scale chromatin decondensation.

The function of upstream binding factor (UBF), an essential component of the RNA polymerase (pol) I preinitiation complex, is unclear. Recently, UBF was found distributed throughout ribosomal gene repeats rather than being restricted to promoter regions. This observation has led to the speculation that one role of UBF binding may be to induce chromatin remodeling. To directly evaluate the impact of UBF on chromatin structure, we used an in vivo assay in which UBF is targeted via a lac repressor fusion protein to a heterochromatic, amplified chromosome region containing lac operator repeats. We show that the association of UBF with this locus induces large-scale chromatin decondensation. This process does not appear to involve common remodeling complexes, including SWI/SNF and histone acetyltransferases, and is independent of histone H3 lysine 9 acetylation. However, UBF recruits the pol I-specific, TATA box-binding protein containing complex SL1 and pol I subunits. Our results suggest a working hypothesis in which the dynamic association of UBF with ribosomal DNA clusters recruits the pol I transcription machinery and maintains these loci in a transcriptionally competent configuration. These studies also provide an in vivo model simulating ribosomal DNA transactivation outside the nucleolus, allowing temporal and spatial analyses of chromatin remodeling and assembly of the pol I transcription machinery.

Acetylation↗
PubMed2004

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure.

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."

Adenosine Triphosphatases↗
PubMed2004

Differential large-scale chromatin compaction and intranuclear positioning of transcribed versus non-transcribed transgene arrays containing beta-globin regulatory sequences.

Previous work has demonstrated a more decondensed large-scale chromatin structure and a more internal nuclear position for gene-rich versus gene-poor chromosome regions. Here, we show that large-scale chromatin opening and changes in intranuclear positioning of chromosome regions can be induced by normal levels of endogenous transcription factors acting on mammalian regulatory sequences. We transfected mouse erythroleukemia cells with a 15 kbp plasmid containing a lac operator repeat plus beta-globin regulatory sequences driving a beta-galactosidase reporter gene. After green-fluorescent-protein/lac-repressor fusion-protein binding or after fluorescence in situ hybridization, the volume and location of the transgene array signal were measured. With both detection methods, we found that the volume was severalfold larger when transcription was on. While silent transgene arrays were located close to the nuclear membrane, we observed a significantly more internal position for the transcriptionally active state. Our results indicate that both large-scale chromatin decondensation and changes in nuclear positioning as observed for large, complex gene-rich chromosome regions can be reproduced by endogenous regulatory sequences acting within simple repetitive transgene arrays.

Animals↗
PubMed2004

Automated microscopy identifies estrogen receptor subdomains with large-scale chromatin structure unfolding activity.

BACKGROUND: Recently, several transcription factors were found to possess large-scale chromatin unfolding activity; these include the VP16 acidic activation domain, BRCA1, E2F1, p53, and the glucocorticoid and estrogen steroid receptors. In these studies, proteins were fluorescently labeled and targeted to a multimerized array of DNA sequences in mammalian cultured cells, and changes in the appearance and/or size of the array were observed. This type of experiment is impeded by the low throughput of traditional microscopy. METHODS: We report the application of automated microscopy to provide unattended, rapid, quantitative measurements of fluorescently labeled chromosome regions. RESULTS: The automated image collection routine produced results comparable to results previously obtained by manual methods and was significantly faster. Using this approach, we identified two subdomains within the E domain of estrogen receptor alpha capable of inducing large-scale chromatin decondensation. CONCLUSIONS: This work confirms that, like BRCA1, the activation function 2 region of the estrogen receptor has more than one distinct chromatin unfolding domain. In addition, we demonstrate the feasibility of using automated microscopy as a high-throughput screen for identifying modulators of large-scale chromatin folding. The Supplementary Material referred to in this article can be found at the CYTO Part A website (http://www.interscience.wiley.com/jpages/0196-4763/suppmat/v58A.html)

Animals↗
PubMed2003

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Animals↗
PubMed2003

Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16.

Eukaryotic transcription initiation requires the complex dynamics of hundreds of proteins, many of which are found in large multisubunit complexes. Recent experiments have suggested stepwise recruitment of preassembled complexes, including chromatin remodeling, general transcription factor, mediator, and polymerase complexes, in which the actual order of recruitment may vary for different promoters. How do these complexes access target sequences contained within tightly condensed chromatin? While chromatin remodeling activities may facilitate the accessibility of large transcription and polymerase complexes to promoters, it is not known how they themselves are targeted within condensed chromatin. Gene activation in the context of condensed chromatin does occur. A yeast acidic activator, Gal4, can overcome heterochromatin gene silencing in Drosophila, and the addition of LCRs (locus control regions) to transgenes overcomes position effect silencing, even within centromeric chromatin. Here, we directly visualize the recruitment of HAT and SWI/SNF components after tethering the VP16 acidic activation domain within condensed chromatin. A recruitment delay of tens to hundreds of minutes for catalytic HAT subunits and SWI/SNF subunits, relative to other HAT and SWI/SNF components, suggests sequential recruitment/assembly of chromatin remodeling complexes within condensed chromatin.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Effects of tethering HP1 to euchromatic regions of the Drosophila genome.

Heterochromatin protein 1 (HP1) is a conserved non-histone chromosomal protein enriched in heterochromatin. On Drosophila polytene chromosomes, HP1 localizes to centric and telomeric regions, along the fourth chromosome, and to specific sites within euchromatin. HP1 associates with centric regions through an interaction with methylated lysine nine of histone H3, a modification generated by the histone methyltransferase SU(VAR)3-9. This association correlates with a closed chromatin configuration and silencing of euchromatic genes positioned near heterochromatin. To determine whether HP1 is sufficient to nucleate the formation of silent chromatin at non-centric locations, HP1 was tethered to sites within euchromatic regions of Drosophila chromosomes. At 25 out of 26 sites tested, tethered HP1 caused silencing of a nearby reporter gene. The site that did not support silencing was upstream of an active gene, suggesting that the local chromatin environment did not support the formation of silent chromatin. Silencing correlated with the formation of ectopic fibers between the site of tethered HP1 and other chromosomal sites, some containing HP1. The ability of HP1 to bring distant chromosomal sites into proximity with each other suggests a mechanism for chromatin packaging. Silencing was not dependent on SU(VAR)3-9 dosage, suggesting a bypass of the requirement for histone methylation.

Animals↗
PubMed2002

The dynamics of homologous chromosome pairing during male Drosophila meiosis.

BACKGROUND: Meiotic pairing is essential for the proper orientation of chromosomes at the metaphase plate and their subsequent disjunction during anaphase I. In male Drosophila melanogaster, meiosis occurs in the absence of recombination or a recognizable synaptonemal complex (SC). Due to limitations in available cytological techniques, the early stages of homologous chromosome pairing in male Drosophila have not been observed, and the mechanisms involved are poorly understood. RESULTS: Chromosome tagging with GFP-Lac repressor protein allowed us to track, for the first time, the behavior of meiotic chromosomes at high resolution, live, at all stages of male Drosophila meiosis. Homologous chromosomes pair throughout the euchromatic regions in spermatogonia and during the early phases of spermatocyte development. Extensive separation of homologs and sister chromatids along the chromosome arms occurs in mid-G2, several hours before the first meiotic division, and before the G2/M transition. Centromeres, on the other hand, show complex association patterns, with specific homolog pairing taking place in mid-G2. These changes in chromosome pairing parallel changes in large-scale chromosome organization. CONCLUSIONS: Our results suggest that widespread interactions along the euchromatin are required for the initiation, but not the maintenance, of meiotic pairing of autosomes in male Drosophila. We propose that heterochromatic associations, or chromatid entanglement, may be responsible for the maintenance of homolog association during late G2. Our data also suggest that the formation of chromosome territories in the spermatocyte nucleus may play an active role in ensuring the specificity of meiotic pairing in late prophase by disrupting interactions between nonhomologous chromosomes.

Anaphase↗