Cold Spring Harbor Perspectives in Biology, May 19, 2010
Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus t... more Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus through the activation or repression of RNA synthesis, processing, cytoplasmic export, and translation into protein. The major regulators of gene expression are chromatin remodeling and transcription machineries that are locally recruited to genes. However, enzymatic activities that act on genes are not ubiquitously distributed throughout the nucleoplasm, but limited to specific and spatially defined foci that promote preferred higher-order chromatin arrangements. The positioning of genes within the nuclear landscape relative to specific functional landmarks plays an important role in gene regulation and disease.
Journal of Histochemistry and Cytochemistry, Apr 1, 1999
The resolution of conventional light microscopy is limited to 002ف nm in the x-and y-axes and Ͼ... more The resolution of conventional light microscopy is limited to 002ف nm in the x-and y-axes and Ͼ500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100-200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of 04ف nm.
Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of develop... more Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders.
Zenodo (CERN European Organization for Nuclear Research), Apr 1, 2021
The brain is comprised of many different cell types with specialized functions which respond and ... more The brain is comprised of many different cell types with specialized functions which respond and adapt to the continuously changing environment, through tight spatiotemporal regulation of gene expression. The three-dimensional (3D) organisation of the genome is increasingly recognized as a major feature of gene regulation in brain cells, for the activation, repression and poising of gene expression, and in coupling transcription with RNA processing and transport. Here, we discuss the importance of dynamic chromatin organisation in the developmental patterning of the brain, and its role in fine tuning brain activity and plasticity. A better understanding of how disease-associated mutations interfere with chromatin organisation and long-range gene regulation will help reveal the molecular mechanisms underlying complex neurodevelopmental and neuropsychiatric disorders.
Nature Reviews Molecular Cell Biology, Aug 5, 2015
In the original article, the following sentence was incorrect: "Histone marks associated with enh... more In the original article, the following sentence was incorrect: "Histone marks associated with enhancers, such as histone 3 Lys4 monomethylation (H3K4me1), and with transcription repression, such as histone 3 Lys9 trimethylation (H3K9me3), were also enriched at TAD boundaries.²" The corrected sentence is as follows: "Histone marks associated with enhancers, such as histone 3 Lys4 monomethylation (H3K4me1), and with transcription repression, such as histone 3 Lys9 trimethylation (H3K9me3), were not found to be enriched at TAD boundaries." This has been corrected in the online version of the article.
Technology for measuring 3D genome topology is increasingly important for studying gene regulatio... more Technology for measuring 3D genome topology is increasingly important for studying gene regulation, for genome assembly and for mapping of genome rearrangements. Hi-C and other ligation-based methods have become routine but have specific biases. Here, we develop multiplex-GAM, a faster and more affordable version of genome architecture mapping (GAM), a ligation-free technique that maps chromatin contacts genome-wide. We perform a detailed comparison of multiplex-GAM and Hi-C using mouse embryonic stem cells. When examining the strongest contacts detected by either method, we find that only one-third of these are shared. The strongest contacts specifically found in GAM often involve ‘active’ regions, including many transcribed genes and super-enhancers, whereas in Hi-C they more often contain ‘inactive’ regions. Our work shows that active genomic regions are involved in extensive complex contacts that are currently underestimated in ligation-based approaches, and highlights the need ...
SummaryThe development of embryonic cell lineages is tightly controlled by transcription factors ... more SummaryThe development of embryonic cell lineages is tightly controlled by transcription factors that regulate gene expression and chromatin organisation. To investigate the specialisation of 3D genome structure in pluripotent or extra-embryonic endoderm lineages, we applied Genome Architecture Mapping (GAM) in embryonic stem (ES) cells, extra-embryonic endoderm (XEN) stem cells, and in theirin vivocounterparts, the epiblast (Epi) and primitive endoderm (PrE) cells, respectively. We discover extensive differences in 3D genome topology including the formation domain boundaries that differ between Epi and PrE lineages, bothin vivoandin vitro, at lineage commitment genes. In ES cells,Sox2contacts other active regions enriched for NANOG and SOX2 binding sites. PrE-specific genes, such asLama1andGata6, form repressive chromatin hubs in ES cells.Lama1activation in XEN or PrE cells coincides with its extensive decondensation. Putative binding sites for OCT4 and SNAIL, or GATA4/6, distingui...
DNA replication, similar to other cellular processes, occurs within dynamic macromolecular struct... more DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3Dstructured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3-to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.
Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, w... more Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single nucleotide polymorphism rs55705857 (A>G), which confers a 6-fold increased risk of IDH-mutant low-grade glioma (LGG) and is amongst the highest genetic associations with cancer. By fine-mapping the locus, we reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. To functionally test rs55705857, we generated an IDH1R132H-driven LGG mouse model and show that mutating the highly conserved, orthologous mouse rs55705857 locus dramatically accelerated tumor development from 463 to 172 days and increased penetrance from 30% to 75%. Overall, our work generates new LGG models and reveals mechanisms of the her...
Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in z... more Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos Graphical abstract Highlights d Single-cell DNA accessibility defines 17 cell types from the 24-hpf zebrafish embryo d Machine learning with ScregSeg classifies complex regulatory programs genome-wide d Integrating sc and bulk chromatin data reveals cross-modal regulatory principles d sci-ATAC profiling of npas4l mutants exposes unexpected changes in cell composition
Neurons and oligodendrocytes are terminally differentiated cells that sustain cascades of gene ac... more Neurons and oligodendrocytes are terminally differentiated cells that sustain cascades of gene activation and repression to execute highly specialized functions, while retaining homeostatic control. To study long-range chromatin folding without disturbing the native tissue environment, we developed Genome Architecture Mapping in combination with immunoselection (immunoGAM), and applied it to three cell types from the adult murine brain: dopaminergic neurons (DNs) from the midbrain, pyramidal glutamatergic neurons (PGNs) from the hippocampus, and oligodendroglia (OLGs) from the cortex. We find cell-type specific 3D chromatin structures that relate with patterns of gene expression at multiple genomic scales, including extensive reorganization of topological domains (TADs) and chromatin compartments. We discover the loss of TAD insulation, or ‘TAD melting’, at long genes (>400 kb) when they are highly transcribed. We find many neuron-specific contacts which contain accessible chroma...
Condensation is the hallmark of mitosis. Why, then, does the chromatin fibre not condense into th... more Condensation is the hallmark of mitosis. Why, then, does the chromatin fibre not condense into the most compact form, a sphere? Why are chromatids cylindrical, and not spherical? What are the basic principles that determine the cylindrical shape? Many models assume that chromosomes are constructed from a hierarchy of helices: the double helix first being coiled into a nucleosome (—11 nm dia.), nucleosomes into solenoids (—30 nm dia.), solenoids into chromatid fibres (225—250 nm dia.), and so on to give a cylinder. However, ...
The activity of locus control regions (LCR) has been correlated with chromatin decondensation, sp... more The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human b-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced.
The nuclear volumes occupied by each specific chromosome. Chromosomes tend to interact predominan... more The nuclear volumes occupied by each specific chromosome. Chromosomes tend to interact predominantly within themselves and occupy distinct regions within the interphase nucleus. Chromosomal compartments Chromosomes fold into distinct subcompartments, which correlate with transcriptional activity (A compartment) or repression (B compartment). The A and B compartments are defined by Hi-C contact frequencies.
Cold Spring Harbor Perspectives in Biology, May 19, 2010
Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus t... more Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus through the activation or repression of RNA synthesis, processing, cytoplasmic export, and translation into protein. The major regulators of gene expression are chromatin remodeling and transcription machineries that are locally recruited to genes. However, enzymatic activities that act on genes are not ubiquitously distributed throughout the nucleoplasm, but limited to specific and spatially defined foci that promote preferred higher-order chromatin arrangements. The positioning of genes within the nuclear landscape relative to specific functional landmarks plays an important role in gene regulation and disease.
Journal of Histochemistry and Cytochemistry, Apr 1, 1999
The resolution of conventional light microscopy is limited to 002ف nm in the x-and y-axes and Ͼ... more The resolution of conventional light microscopy is limited to 002ف nm in the x-and y-axes and Ͼ500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100-200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of 04ف nm.
Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of develop... more Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders.
Zenodo (CERN European Organization for Nuclear Research), Apr 1, 2021
The brain is comprised of many different cell types with specialized functions which respond and ... more The brain is comprised of many different cell types with specialized functions which respond and adapt to the continuously changing environment, through tight spatiotemporal regulation of gene expression. The three-dimensional (3D) organisation of the genome is increasingly recognized as a major feature of gene regulation in brain cells, for the activation, repression and poising of gene expression, and in coupling transcription with RNA processing and transport. Here, we discuss the importance of dynamic chromatin organisation in the developmental patterning of the brain, and its role in fine tuning brain activity and plasticity. A better understanding of how disease-associated mutations interfere with chromatin organisation and long-range gene regulation will help reveal the molecular mechanisms underlying complex neurodevelopmental and neuropsychiatric disorders.
Nature Reviews Molecular Cell Biology, Aug 5, 2015
In the original article, the following sentence was incorrect: "Histone marks associated with enh... more In the original article, the following sentence was incorrect: "Histone marks associated with enhancers, such as histone 3 Lys4 monomethylation (H3K4me1), and with transcription repression, such as histone 3 Lys9 trimethylation (H3K9me3), were also enriched at TAD boundaries.²" The corrected sentence is as follows: "Histone marks associated with enhancers, such as histone 3 Lys4 monomethylation (H3K4me1), and with transcription repression, such as histone 3 Lys9 trimethylation (H3K9me3), were not found to be enriched at TAD boundaries." This has been corrected in the online version of the article.
Technology for measuring 3D genome topology is increasingly important for studying gene regulatio... more Technology for measuring 3D genome topology is increasingly important for studying gene regulation, for genome assembly and for mapping of genome rearrangements. Hi-C and other ligation-based methods have become routine but have specific biases. Here, we develop multiplex-GAM, a faster and more affordable version of genome architecture mapping (GAM), a ligation-free technique that maps chromatin contacts genome-wide. We perform a detailed comparison of multiplex-GAM and Hi-C using mouse embryonic stem cells. When examining the strongest contacts detected by either method, we find that only one-third of these are shared. The strongest contacts specifically found in GAM often involve ‘active’ regions, including many transcribed genes and super-enhancers, whereas in Hi-C they more often contain ‘inactive’ regions. Our work shows that active genomic regions are involved in extensive complex contacts that are currently underestimated in ligation-based approaches, and highlights the need ...
SummaryThe development of embryonic cell lineages is tightly controlled by transcription factors ... more SummaryThe development of embryonic cell lineages is tightly controlled by transcription factors that regulate gene expression and chromatin organisation. To investigate the specialisation of 3D genome structure in pluripotent or extra-embryonic endoderm lineages, we applied Genome Architecture Mapping (GAM) in embryonic stem (ES) cells, extra-embryonic endoderm (XEN) stem cells, and in theirin vivocounterparts, the epiblast (Epi) and primitive endoderm (PrE) cells, respectively. We discover extensive differences in 3D genome topology including the formation domain boundaries that differ between Epi and PrE lineages, bothin vivoandin vitro, at lineage commitment genes. In ES cells,Sox2contacts other active regions enriched for NANOG and SOX2 binding sites. PrE-specific genes, such asLama1andGata6, form repressive chromatin hubs in ES cells.Lama1activation in XEN or PrE cells coincides with its extensive decondensation. Putative binding sites for OCT4 and SNAIL, or GATA4/6, distingui...
DNA replication, similar to other cellular processes, occurs within dynamic macromolecular struct... more DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3Dstructured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3-to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.
Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, w... more Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single nucleotide polymorphism rs55705857 (A>G), which confers a 6-fold increased risk of IDH-mutant low-grade glioma (LGG) and is amongst the highest genetic associations with cancer. By fine-mapping the locus, we reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. To functionally test rs55705857, we generated an IDH1R132H-driven LGG mouse model and show that mutating the highly conserved, orthologous mouse rs55705857 locus dramatically accelerated tumor development from 463 to 172 days and increased penetrance from 30% to 75%. Overall, our work generates new LGG models and reveals mechanisms of the her...
Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in z... more Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos Graphical abstract Highlights d Single-cell DNA accessibility defines 17 cell types from the 24-hpf zebrafish embryo d Machine learning with ScregSeg classifies complex regulatory programs genome-wide d Integrating sc and bulk chromatin data reveals cross-modal regulatory principles d sci-ATAC profiling of npas4l mutants exposes unexpected changes in cell composition
Neurons and oligodendrocytes are terminally differentiated cells that sustain cascades of gene ac... more Neurons and oligodendrocytes are terminally differentiated cells that sustain cascades of gene activation and repression to execute highly specialized functions, while retaining homeostatic control. To study long-range chromatin folding without disturbing the native tissue environment, we developed Genome Architecture Mapping in combination with immunoselection (immunoGAM), and applied it to three cell types from the adult murine brain: dopaminergic neurons (DNs) from the midbrain, pyramidal glutamatergic neurons (PGNs) from the hippocampus, and oligodendroglia (OLGs) from the cortex. We find cell-type specific 3D chromatin structures that relate with patterns of gene expression at multiple genomic scales, including extensive reorganization of topological domains (TADs) and chromatin compartments. We discover the loss of TAD insulation, or ‘TAD melting’, at long genes (>400 kb) when they are highly transcribed. We find many neuron-specific contacts which contain accessible chroma...
Condensation is the hallmark of mitosis. Why, then, does the chromatin fibre not condense into th... more Condensation is the hallmark of mitosis. Why, then, does the chromatin fibre not condense into the most compact form, a sphere? Why are chromatids cylindrical, and not spherical? What are the basic principles that determine the cylindrical shape? Many models assume that chromosomes are constructed from a hierarchy of helices: the double helix first being coiled into a nucleosome (—11 nm dia.), nucleosomes into solenoids (—30 nm dia.), solenoids into chromatid fibres (225—250 nm dia.), and so on to give a cylinder. However, ...
The activity of locus control regions (LCR) has been correlated with chromatin decondensation, sp... more The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human b-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced.
The nuclear volumes occupied by each specific chromosome. Chromosomes tend to interact predominan... more The nuclear volumes occupied by each specific chromosome. Chromosomes tend to interact predominantly within themselves and occupy distinct regions within the interphase nucleus. Chromosomal compartments Chromosomes fold into distinct subcompartments, which correlate with transcriptional activity (A compartment) or repression (B compartment). The A and B compartments are defined by Hi-C contact frequencies.
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Papers by Ana Pombo