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Ana Pombo

Publications and source records attributed to Ana Pombo.

15 recordsLinked to original sources
PubMed2007

Chromosome organization: new facts, new models.

The study of nuclear organization has radically changed the way we envision gene regulation, imposing a paradigm shift from a seemingly featureless nucleus to a highly compartmentalized and complex organelle. The positioning of genes, regulatory sequences and transcription factors in relation to each other and to landmarks in the nucleus, such as nuclear bodies and the lamina, is important in determining which genes are transcribed at any one time. Investigating chromatin organization during interphase is therefore essential to the understanding of gene expression. The recent discovery of interactions between distal chromatin segments that occur within the same chromosome or across different chromosomes, and that have a role in transcription regulation, suggests a re-evaluation of current models of chromosome organization and the development of new ones.

Animals↗
PubMed2006

Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations.

After mitosis, mammalian chromosomes partially decondense to occupy distinct territories in the cell nucleus. Current models propose that territories are separated by an interchromatin domain, rich in soluble nuclear machinery, where only rare interchromosomal interactions can occur via extended chromatin loops. In contrast, recent evidence for chromatin mobility and high frequency of chromosome translocations are consistent with significant levels of chromosome intermingling, with important consequences for genome function and stability. Here we use a novel high-resolution in situ hybridization procedure that preserves chromatin nanostructure to show that chromosome territories intermingle significantly in the nucleus of human cells. The degree of intermingling between specific chromosome pairs in human lymphocytes correlates with the frequency of chromosome translocations in the same cell type, implying that double-strand breaks formed within areas of intermingling are more likely to participate in interchromosomal rearrangements. The presence of transcription factories in regions of intermingling and the effect of transcription impairment on the interactions between chromosomes shows that transcription-dependent interchromosomal associations shape chromosome organization in mammalian cells. These findings suggest that local chromatin conformation and gene transcription influence the extent with which chromosomes interact and affect their overall properties, with direct consequences for cell-type specific genome stability.

Cells, Cultured↗
PubMed2006

Splicing speckles are not reservoirs of RNA polymerase II, but contain an inactive form, phosphorylated on serine2 residues of the C-terminal domain.

"Splicing speckles" are major nuclear domains rich in components of the splicing machinery and polyA(+) RNA. Although speckles contain little detectable transcriptional activity, they are found preferentially associated with specific mRNA-coding genes and gene-rich R bands, and they accumulate some unspliced pre-mRNAs. RNA polymerase II transcribes mRNAs and is required for splicing, with some reports suggesting that the inactive complexes are stored in splicing speckles. Using ultrathin cryosections to improve optical resolution and preserve nuclear structure, we find that all forms of polymerase II are present, but not enriched, within speckles. Inhibition of polymerase activity shows that speckles do not act as major storage sites for inactive polymerase II complexes but that they contain a stable pool of polymerase II phosphorylated on serine(2) residues of the C-terminal domain, which is transcriptionally inactive and may have roles in spliceosome assembly or posttranscriptional splicing of pre-mRNAs. Paraspeckle domains lie adjacent to speckles, but little is known about their protein content or putative roles in the expression of the speckle-associated genes. We find that paraspeckles are transcriptionally inactive but contain polymerase II, which remains stably associated upon transcriptional inhibition, when paraspeckles reorganize around nucleoli in the form of caps.

Antibodies↗
PubMed2005

Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: an ultrastructural study.

The mammalian nucleus is a highly organised organelle that contains many subcompartments with roles in DNA replication and repair, gene expression and RNA processing. Cajal and promyelocytic leukaemia (PML) bodies are discrete nuclear structures with specific molecular signatures. RNA polymerase II and many transcription factors have been identified within these compartments by immunofluorescence microscopy, suggesting a role in polymerase II assembly or transcriptional activity. Here, we have examined the presence of different phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-resolution imaging of ultrathin cryosections (approximately 120 nm thick) with fluorescence and electron microscopes. We show that Cajal bodies contain polymerase II phosphorylated on Ser5, and not the Ser2-phosphorylated (active) form or newly made RNA. The presence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in polymerase assembly or transport, but not in gene transcription. PML bodies contain no detectable polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances>25 nm. These results support the view that although PML bodies are present in transcriptionally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.

Antibodies↗
PubMed2005

Variant histone H3.3 marks promoters of transcriptionally active genes during mammalian cell division.

Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role.

Animals↗
PubMed2005

Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G.

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2 vif-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1 vif-minus nonpermissive cell line H9 does not show a similar phenotype for vif-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.

APOBEC-3G Deaminase↗
PubMed2004

Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells.

RNA polymerase II (pol II) transcribes the most varied group of genes and is present in hypo- and hyperphosphorylated forms, with residues Ser(2) and Ser(5) of the C-terminal domain (CTD) of the largest subunit as main targets of phosphorylation. The elongating (active) form is phosphorylated on Ser(2) and can be specifically recognized with the H5 antibody. It has been found in different nuclear distributions: in discrete sites throughout the nucleoplasm, consistent with a role in transcription, and/or concentrated in "splicing speckles", a nuclear compartment mostly devoid of transcriptional activity. Here, we assess the effects of cell fixation and permeabilization on the distribution of polymerase II and correlate its distribution with the preservation of cellular ultrastructure. We show that phospho-Ser(2) polymerase II can redistribute to, or be differentially retained in, "speckles" in conditions that do not preserve cellular ultrastructure. The fixation protocols that disrupt polymerase II distribution also cause partial or total loss of TATA-binding protein, Sm antigen and PML staining in PML bodies, and have no noticeable effect in the labeling of SC35 in "splicing speckles" or coilin in Cajal bodies. When nuclear ultrastructure is preserved, phospho-Ser(2) polymerase II is found in discrete sites throughout the nucleoplasm, without visible enrichment within splicing speckles. A minor proportion of the total amount of the phospho-Ser(2) form is present in these domains.

Antibodies, Monoclonal↗
PubMed2004

Measuring the size of biological nanostructures with spatially modulated illumination microscopy.

Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads ( approximately 40-100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.

Cell Nucleus↗
PubMed2004

CD8 alpha is an activation marker for a subset of peripheral CD4 T cells.

Rat CD4 T lymphocytes express CD8 alpha upon activation. Here, we show that double-positive cells express CD8 alpha alpha homodimers, and we study their phenotype and function. Most activated CD4(+) lymphocytes expressing CD8 alpha are recent thymic emigrants. Accordingly, most activated CD4 single-positive thymocytes express CD8 alpha, and thymectomy and aging decrease the frequency of CD4(+)CD8 alpha(+) lymphocytes. However, CD8 induction is not restricted to CD4(+) recent thymic emigrants. CD4(+)CD8 alpha(+) and CD4(+)CD8 alpha(-)cells were generated in vitro from naive or from primed donors and, to study their function, were transferred to normal rats. Both cell types helped primary humoral responses, but only CD4(+)CD8 alpha(-) cells promoted secondary responses. Thus, memory CD4 T cells mediating antibody responses and some naive CD4(+) lymphocytes do not express CD8 alpha. In addition, CD4(+)CD8 alpha(+) cells produce mainly Th1 cytokines while CD4(+)CD8 alpha(-) cells produce IL-10 and showed a sustained proliferative response. Hence, CD8 alpha expression after activation distinguishes two distinct CD4 T cell subsets.

Animals↗
PubMed2003

Interaction of proteins with promoter elements of the human U2 snRNA genes in vivo.

The multicopy human U2 small nuclear (sn)RNA genes are transcribed by RNA polymerase (pol) II and contain two major promoter elements upstream of the transcription start site: an essential proximal sequence element (PSE) at around -55 and a distal sequence element (DSE) at around -220. We have carried out an in vivo footprinting analysis on these genes, and the results suggest that most, if not all, of the U2 gene promoters are bound by factors in interphase. Both the DSE and the PSE are protected from digestion, and the pattern of methylation protection over the DSE is virtually identical to that obtained in vitro using nuclear extract. Our results also indicate that the DNA between the PSE and the transcription start site is distorted and that proteins interact with the promoter between -20 and -33. Mutation of this sequence affects both the accuracy of initiation and polymerase specificity, underlining the importance of this region in U2 gene expression. We have also analysed the pattern of protection over the DSE and PSE of the U2 genes in mitotic cells. The degree of protection over all promoter elements is drastically reduced, suggesting that loss of DNA binding factors from the promoter plays a role in the shutdown of U2 gene transcription in mitosis.

Base Sequence↗
PubMed2003

Genome function and nuclear architecture: from gene expression to nanoscience.

Biophysical, chemical, and nanoscience approaches to the study of nuclear structure and activity have been developing recently and hold considerable promise. A selection of fundamental problems in genome organization and function are reviewed and discussed in the context of these new perspectives and approaches. Advancing these concepts will require coordinated networks of physicists, chemists, and materials scientists collaborating with cell, developmental, and genome biologists.

Animals↗
PubMed2003

Cellular genomics: which genes are transcribed, when and where?

Gene expression involves a cascade of events, from transcription factor transactivation and RNA polymerase binding to RNA processing and maturation. mRNA profiling has yielded a vast amount of information about the total RNA content in different tissues physiological states. However, it has been unclear how the variable amounts of mRNA might correlate with transcriptional events. Recent advances in single-cell imaging offer a platform for 'cellular genomics', the high throughput analysis of transcriptional activity in single cells.

Animals↗
PubMed2003

Transcription factories: quantitative studies of nanostructures in the mammalian nucleus.

Transcription by the three nuclear RNA polymerases is carried out in transcription factories. This conclusion has been drawn from estimates of the total number of nascent transcripts or active polymerase molecules and the number of transcription sites within a cell. Here we summarise the variety of methods used to determine these parameters, discuss their associated problems and outline future prospects.

Cell Nucleus↗