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1.  A biophysical model for transcription factories 
BMC Biophysics  2013;6:2.
Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene. Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active and inactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in the chromatin fibre. The result of this change in flexibility is that chromatin could behave like a multi-block copolymer with repetitions of stiff-flexible (active-inactive chromatin) components. Copolymers with such structure self-organize through spontaneous phase separation into microdomains. Consistent with such model H4K16ac chromatin form foci that associates with nascent transcripts. We propose that transcription factories are the result of the spontaneous concentration of H4K16ac chromatin that are in proximity, mainly in cis.
PMCID: PMC3740778  PMID: 23394119
Epigenetics; Biophysics; H4K16Ac; BrUTP; Transcription Factories; RNA pol II; Nuclear organization
2.  Mitochondrial Variability as a Source of Extrinsic Cellular Noise 
PLoS Computational Biology  2012;8(3):e1002416.
We present a study investigating the role of mitochondrial variability in generating noise in eukaryotic cells. Noise in cellular physiology plays an important role in many fundamental cellular processes, including transcription, translation, stem cell differentiation and response to medication, but the specific random influences that affect these processes have yet to be clearly elucidated. Here we present a mechanism by which variability in mitochondrial volume and functionality, along with cell cycle dynamics, is linked to variability in transcription rate and hence has a profound effect on downstream cellular processes. Our model mechanism is supported by an appreciable volume of recent experimental evidence, and we present the results of several new experiments with which our model is also consistent. We find that noise due to mitochondrial variability can sometimes dominate over other extrinsic noise sources (such as cell cycle asynchronicity) and can significantly affect large-scale observable properties such as cell cycle length and gene expression levels. We also explore two recent regulatory network-based models for stem cell differentiation, and find that extrinsic noise in transcription rate causes appreciable variability in the behaviour of these model systems. These results suggest that mitochondrial and transcriptional variability may be an important mechanism influencing a large variety of cellular processes and properties.
Author Summary
Cellular variability has been found to play a major role in diverse and important phenomena, including stem cell differentiation and drug resistance, but the sources of this variability have yet to be satisfactorily explained. We propose a mechanism, supported by a substantial number of recent and new experiments, by which cell-to-cell differences in both the number and functionality of mitochondria – the organelles responsible for energy production in eukaryotes – leads to variability in transcription rate between cells and may hence be a significant source of cellular noise in many downstream processes. We illustrate the downstream effect of mitochondrial variability through simulated studies of protein expression and stem cell differentiation, and suggest possible experimental approaches to further elucidate this mechanism.
PMCID: PMC3297557  PMID: 22412363
3.  Connecting Variability in Global Transcription Rate to Mitochondrial Variability 
PLoS Biology  2010;8(12):e1000560.
The authors demonstrate a connection between variability in the rate of transcription and differences in cellular mitochondrial content.
Populations of genetically identical eukaryotic cells show significant cell-to-cell variability in gene expression. However, we lack a good understanding of the origins of this variation. We have found marked cell-to-cell variability in average cellular rates of transcription. We also found marked cell-to-cell variability in the amount of cellular mitochondrial mass. We undertook fusion studies that suggested that variability in transcription rate depends on small diffusible factors. Following this, in vitro studies showed that transcription rate has a sensitive dependence on [ATP] but not on the concentration of other nucleotide triphosphates (NTPs). Further experiments that perturbed populations by changing nutrient levels and available [ATP] suggested this connection holds in vivo. We found evidence that cells with higher mitochondrial mass, or higher total membrane potential, have a faster rate of transcription per unit volume of nuclear material. We also found evidence that transcription rate variability is substantially modulated by the presence of anti- or prooxidants. Daughter studies showed that a cause of variability in mitochondrial content is apparently stochastic segregation of mitochondria at division. We conclude by noting that daughters that stochastically inherit a lower mitochondrial mass than their sisters have relatively longer cell cycles. Our findings reveal a link between variability in energy metabolism and variability in transcription rate.
Author Summary
Though pairs of cells may have identical genes, they still show behavioural differences. These cell-to-cell differences may arise from variations in how genes are transcribed and translated by the cellular machinery. Identifying the origins of this variation is important as it helps us understand why genetically identical cells can show a range of responses to the environment. In this work, we measured the rate at which the genes yield transcripts in cultured human cells. We found marked cell-to-cell variability in average rates of transcription. This variability is related to mitochondrial content. Cells with a higher mitochondrial mass have a faster rate of transcription, and we show that part of this variability is due to the unequal distribution of mitochondria to daughter cells when cells divide. Additionally, we find that cells that inherit more mitochondria divide earlier. These findings make a connection between variability in transcript production and variability in cellular mitochondrial content.
PMCID: PMC3001896  PMID: 21179497
4.  Association between active genes occurs at nuclear speckles and is modulated by chromatin environment 
The Journal of Cell Biology  2008;182(6):1083-1097.
Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse α-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human α-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.
PMCID: PMC2542471  PMID: 18809724
5.  A novel histone exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A–H2B 
The Journal of Cell Biology  2006;175(3):389-400.
In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.
PMCID: PMC2064517  PMID: 17074886
6.  Can visco-elastic phase separation, macromolecular crowding and colloidal physics explain nuclear organisation? 
The cell nucleus is highly compartmentalized with well-defined domains, it is not well understood how this nuclear order is maintained. Many scientists are fascinated by the different set of structures observed in the nucleus to attribute functions to them. In order to distinguish functional compartments from non-functional aggregates, I believe is important to investigate the biophysical nature of nuclear organisation.
The various nuclear compartments can be divided broadly as chromatin or protein and/or RNA based, and they have very different dynamic properties. The chromatin compartment displays a slow, constrained diffusional motion. On the other hand, the protein/RNA compartment is very dynamic. Physical systems with dynamical asymmetry go to viscoelastic phase separation. This phase separation phenomenon leads to the formation of a long-lived interaction network of slow components (chromatin) scattered within domains rich in fast components (protein/RNA). Moreover, the nucleus is packed with macromolecules in the order of 300 mg/ml. This high concentration of macromolecules produces volume exclusion effects that enhance attractive interactions between macromolecules, known as macromolecular crowding, which favours the formation of compartments. In this paper I hypothesise that nuclear compartmentalization can be explained by viscoelastic phase separation of the dynamically different nuclear components, in combination with macromolecular crowding and the properties of colloidal particles.
I demonstrate that nuclear structure can satisfy the predictions of this hypothesis. I discuss the functional implications of this phenomenon.
PMCID: PMC1853075  PMID: 17430588
7.  Coregulated human globin genes are frequently in spatial proximity when active 
The Journal of Cell Biology  2006;172(2):177-187.
The organization of genes within the nucleus may influence transcription. We have analyzed the nuclear positioning of the coordinately regulated α- and β-globin genes and show that the gene-dense chromatin surrounding the human α-globin genes is frequently decondensed, independent of transcription. Against this background, we show the frequent juxtaposition of active α- and β-globin genes and of homologous α-globin loci that occurs at nuclear speckles and correlates with transcription. However, we did not see increased colocalization of signals, which would be expected with direct physical interaction. The same degree of proximity does not occur between human β-globin genes or between murine globin genes, which are more constrained to their chromosome territories. Our findings suggest that the distribution of globin genes within erythroblast nuclei is the result of a self-organizing process, involving transcriptional status, diffusional ability of chromatin, and physical interactions with nuclear proteins, rather than a directed form of higher-order control.
PMCID: PMC2063548  PMID: 16418531
8.  The functional organization of mitochondrial genomes in human cells 
BMC Biology  2004;2:9.
We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with the yellow fluorescent protein.
Mitochondrial DNA is organized in ~475 discrete foci containing 6–10 genomes. These foci (nucleoids) are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 × 10-3 μm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine) reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time ~43 min) long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins – that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).
The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.
PMCID: PMC425603  PMID: 15157274
9.  Numbers and Organization of RNA Polymerases, Nascent Transcripts, and Transcription Units in HeLa Nuclei 
Molecular Biology of the Cell  1998;9(6):1523-1536.
Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15,000 transcripts were elongated by polymerase I, and ∼75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in ∼2400 sites with diameters of ∼80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a “cloud” of loops around a site; each site, or transcription “factory,” would contain ∼30 active polymerases and associated transcripts.
PMCID: PMC25378  PMID: 9614191

Results 1-9 (9)