Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells.

TCR signaling pathways cooperate to activate the inducible transcription factors NF-κB, NFAT, and AP-1. In this study, using the calcium ionophore ionomycin and/or PMA on Jurkat T cells, we show that the gene expression program associated with activation of TCR signaling is closely related to specific chromatin landscapes. We find that calcium and kinase signaling cooperate to induce chromatin remodeling at ∼2100 chromatin regions, which demonstrate enriched binding motifs for inducible factors and correlate with target gene expression. We found that these regions typically function as inducible enhancers. Many of these elements contain composite NFAT/AP-1 sites, which typically support cooperative binding, thus further reinforcing the need for cooperation between calcium and kinase signaling in the activation of genes in T cells. In contrast, treatment with PMA or ionomycin alone induces chromatin remodeling at far fewer regions (∼600 and ∼350, respectively), which mostly represent a subset of those induced by costimulation. This suggests that the integration of TCR signaling largely occurs at the level of chromatin, which we propose plays a crucial role in regulating T cell activation.

Physical activity in older age: perspectives for healthy ageing and frailty.

Regular physical activity helps to improve physical and mental functions as well as reverse some effects of chronic disease to keep older people mobile and independent. Despite the highly publicised benefits of physical activity, the overwhelming majority of older people in the United Kingdom do not meet the minimum physical activity levels needed to maintain health. The sedentary lifestyles that predominate in older age results in premature onset of ill health, disease and frailty. Local authorities have a responsibility to promote physical activity amongst older people, but knowing how to stimulate regular activity at the population-level is challenging. The physiological rationale for physical activity, risks of adverse events, societal and psychological factors are discussed with a view to inform public health initiatives for the relatively healthy older person as well as those with physical frailty. The evidence shows that regular physical activity is safe for healthy and for frail older people and the risks of developing major cardiovascular and metabolic diseases, obesity, falls, cognitive impairments, osteoporosis and muscular weakness are decreased by regularly completing activities ranging from low intensity walking through to more vigorous sports and resistance exercises. Yet, participation in physical activities remains low amongst older adults, particularly those living in less affluent areas. Older people may be encouraged to increase their activities if influenced by clinicians, family or friends, keeping costs low and enjoyment high, facilitating group-based activities and raising self-efficacy for exercise.

How dormant origins promote complete genome replication.

Many replication origins that are licensed by loading MCM2-7 complexes in G1 are not normally used. Activation of these dormant origins during S phase provides a first line of defence for the genome if replication is inhibited. When replication forks fail, dormant origins are activated within regions of the genome currently engaged in replication. At the same time, DNA damage-response kinases activated by the stalled forks preferentially suppress the assembly of new replication factories, thereby ensuring that chromosomal regions experiencing replicative stress complete synthesis before new regions of the genome are replicated. Mice expressing reduced levels of MCM2-7 have fewer dormant origins, are cancer-prone and are genetically unstable, demonstrating the importance of dormant origins for preserving genome integrity. We review the function of dormant origins, the molecular mechanism of their regulation and their physiological implications.

Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription.

Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease.

Cell-surface Notch1 expression identifies a primitive phenotype within CD34+ CD38- haematopoietic cells.

OBJECTIVE: Notch signalling has been implicated in haematopoietic stem cell self-renewal. Although several studies have tested the effect of activating or inhibiting the Notch signalling pathway in stem cells, no study has yet determined the functional differences associated with expressing Notch1. The aims of this study were to characterise the expression of human cell-surface Notch1 in cord blood (CB) CD34(+) cells and to study the function of Notch in CD34(+) cells in vitro. METHODS: A monoclonal antibody against the extracellular domain of Notch1 was developed, and Notch1 expression in CB CD34(+) cells was assessed by flow cytometry. CB CD34(+) cells were sorted on the basis of their Notch1 expression and cultured in serum-free media. Single sorted CD34(+) CD38(-) Notch1(+) /(-) cells were cultured for 8 wks on murine stroma monolayers and assayed for stem cell activity and lineage potential using a cobblestone area-forming cell (CAFC) assay. RESULTS: Cell-surface Notch1 expression was characterised in various primitive CD34(+) cell compartments including a small subpopulation of CD34(+) CD38(-) cells. We found the CD34(+) CD38(-) Notch1(+) population to be enriched for stem cell activity. Moreover, CD34(+) CD38(-) Notch1(+) , but not Notch1(-) cells, demonstrated multilineage potential. CONCLUSIONS: These data show that Notch1 is expressed on a functionally distinct subpopulation of CD34(+) cells that is highly enriched for stem cell activity and multilineage potential and could suggest that Notch1 could be used as a novel stem cell marker.

EdU induces DNA damage response and cell death in mESC in culture.

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

Visualising chromosomal replication sites and replicons in mammalian cells.

The precise regulation of DNA replication is fundamental to the preservation of intact genomes during cell proliferation. Our understanding of this process has been based traditionally on a combination of techniques including biochemistry, molecular biology and cell biology. In this report we describe how the analysis of the S phase in mammalian cells using classical cell biology techniques has contributed to our understanding of the replication process. We describe traditional and state-of-the-art protocols for imaging sites of DNA synthesis in nuclei and the organisation of active replicons along DNA, as visualised on individual DNA fibres. We evaluate how the different approaches inform our understanding of the replication process, placing particular emphasis on ways in which the higher order chromatin structures and the spatial architecture of replication sites contribute to the orderly activation of defined regions of the genome at precise times of S phase.

S phase progression in human cells is dictated by the genetic continuity of DNA foci.

DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.

Overexpression of IκB⍺ modulates NF-κB activation of inflammatory target gene expression.

Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-κB (NF-κB) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm in some cell types. We investigate the relationship between p65 and inhibitor-κB⍺ (IκBα) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of IκB⍺-eGFP protein overexpression in a pseudo-native genomic context. We find that cells with high levels of the negative regulator IκBα remain responsive to inflammatory stimuli and maintain dynamics for both p65 and IκBα. In contrast, canonical target gene expression is dramatically reduced by overexpression of IκBα, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of IκB⍺ also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IκB⍺ accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immunoprecipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both IκB⍺ and p65. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.

B-Myb is critical for proper DNA duplication during an unperturbed S phase in mouse embryonic stem cells.

A common feature of early embryo cells from the inner cell mass (ICM) and of ESCs is an absolute dependence on an atypical cell cycle in which the G1 phase is shortened to preserve their self-renewing and pluripotent nature. The transcription factor B-Myb has been attributed a role in proliferation, in particular during the G2/M phases of the cell cycle. Intriguingly, B-Myb levels in ICM/ESCs are greater than 100 times compared with those in normal proliferating cells, suggesting a particularly important function for this transcription factor in pluripotent stem cells. B-Myb is essential for embryo development beyond the preimplantation stage, but its role in ICM/ESCs remains unclear. Using a combination of mouse genetics, single DNA fiber analyses and high-resolution three-dimensional (3D) imaging, we demonstrate that B-Myb has no influence on the expression of pluripotency factors, but instead B-Myb ablation leads to stalling of replication forks and superactivation of replication factories that result in disorganization of the replication program and an increase in double-strand breaks. These effects are partly due to aberrant transcriptional regulation of cell cycle proliferation factors, namely c-Myc and FoxM1, which dictate normal S phase progression. We conclude that B-Myb acts crucially during the S phase in ESCs by facilitating proper progression of replication, thereby protecting the cells from genomic damage. Our findings have particular relevance in the light of the potential therapeutic application of ESCs and the need to maintain their genomic integrity.

The anatomy of transcription sites.

Patterns of gene expression in multicellular eukaryotes are regulated by numerous extremely sophisticated mechanisms. Over the past year, developments in our ability to monitor the organisation and dynamic properties of the components involved in gene expression have emphasised how both global nuclear architecture and chromosome structure can influence this fundamental process.

Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress.

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Inheriting nuclear organization: can nuclear lamins impart spatial memory during post-mitotic nuclear assembly?

Cell type and tissue architecture correlate with genome organization in higher eukaryotes, and structural nuclear landmarks are faithfully transmitted from one cell generation to the next. However, how nuclear components find their place in the nucleus after mitosis is still a matter of debate. As the major structural proteins within nuclei, the nuclear lamins are good candidates to re-establish nuclear compartments following mitosis. Human cells with reduced expression of the major B-type lamin protein, lamin B1, were generated using RNA interference. Mitotic and nuclear assembly phenotypes were then visualized in both fixed and living cells. Mitotic defects in lamin B1-depleted cells correlated with a general deterioration in nuclear compartmentalization and chromatin structure, frequent failure of chromosome segregation, and profound disorganization of centromeres. Examination of cells with normal lamin B1 expression indicated that small lamin B1 foci remain associated with major nuclear compartments--chromatin, nucleoli, and nuclear speckles--during an unperturbed mitosis. Our experiments show that normal lamin B1 expression is required for successful cell division and provide preliminary evidence that lamin B1-containing remnants of the interphase nucleoskeleton persist throughout mitosis. We suggest that these residual structures provide landmarks that are targeted during nuclear reassembly to allow key features of nuclear organization to be inherited from one cell cycle to the next.

S-phase progression in mammalian cells: modelling the influence of nuclear organization.

The control of DNA replication is of fundamental importance as cell proliferation demands that identical copies of the genetic material are passed to the two daughter cells that form during mitosis. These genetic copies are generated in the preceding S phase, where the entire DNA complement of the mother cell must be copied exactly once. As part of this process, it is known that different regions of mammalian genomes are replicated at specific times of a temporally defined replication programme. The key feature of this programme is that active genes in euchromatin are replicated before inactive ones in heterochromatin. This separation of S phase into periods where different classes of chromatin are duplicated is important in maintaining changes in gene expression that define individual cell types. Recent attempts to understand the structure of the S-phase timing programme have focused on the use of genome-wide strategies that inevitably use DNA isolated from large cell populations for analysis. However, this approach provides a composite view of events that occur within a population without knowledge of the cell-to-cell variability across the population. In this review, we attempt to combine information generated using genome-wide and single cell strategies in order to develop a coherent molecular understanding of S-phase progression. During this integration, we have explored how available information can be introduced into a modelling environment that best describes S-phase progression in mammalian cells.

Stable S/MAR-based episomal vectors are regulated at the chromatin level.

Episomal vectors assembled from defined genetic components are a promising alternative to traditional gene therapy vectors that integrate in the host genome and may cause insertional mutations. The vector pEPI-eGFP is stably retained in the episomal state in cultured mammalian cells at low copy number for many generations without integration into the host genome. Although pEPI-eGFP is a fully engineered vector, little is known about how it interacts with the host genome and about the molecular mechanisms that are responsible for its transcriptional activity. We have analyzed the expression of the episomal reporter gene eGFP under conditions that affect the chromatin state of the genome. We have also constructed pEPI derivatives carrying a tandem array of lac operator sequences, which allows in vivo visualization and manipulation of the chromatin state of the episome. We show that changes in chromatin state of both the host and pEPI-eGFP induces changes in episomal gene activity and influences the episome's nuclear distributions. We conclude that episomal genes are subject to control systems of the host, similarly to their counterparts in the host genome.

Gene-Specific Linear Trends Constrain Transcriptional Variability of the Toll-like Receptor Signaling.

Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1ß involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.

Interaction with checkpoint kinase 1 modulates the recruitment of nucleophosmin to chromatin.

The Checkpoint kinase 1 (Chk1) plays a central role in the cellular response to DNA damage and also contributes to the efficacy of DNA replication in the absence of genomic stress. However, we have only limited knowledge regarding the molecular mechanisms that regulate differential Chk1 function in the absence and presence of DNA damage. To address this, we used vertebrate cells with compromised Chk1 function to analyze how altered Chk1 activity influences protein interactions in chromatin. Avian and mammalian cells with compromised Chk1 activity were used in combination with genomic stress, induced by UV, and DNA-associated proteomes were analyzed using 2-DE/MS proteomics and Western-blot analysis. Only one protein, the histone chaperone nucelophosmin, was altered consistently in line with changes in chromatin-associated Chk1 and increased in response to DNA damage. Purified Chk1 and NPM were shown to interact in vitro and strong in vivo interactions were implied from immunoprecipitation analysis of chromatin extracts. During chromatin immunoprecipitation, coassociation of the major cell cycle regulator proteins p53 and CDC25A with both Chk1 and NPM suggests that these proteins are components of complex interaction networks that operate to regulate cell proliferation and apoptosis in vertebrate cells.

Reduced expression of lamin A/C results in modified cell signaling and metabolism coupled with changes in expression of structural proteins.

Nuclear lamins are intermediate filament proteins that define the shape and stability of nuclei in mammalian cells. In addition to this dominant structural role, recent studies have suggested that the lamin proteins also regulate fundamental aspects of nuclear function. In order to understand different roles played by lamin proteins, we used RNA interference to generate a series of HeLa cell lines to study loss-of-function phenotypes associated with depletion of lamin protein expression. In this study, we used genome-wide proteomic approaches to monitor global changes in protein expression in cells with <10% of normal lamin A/C expression. Of approximately 2000 protein spots analyzed by two-dimensional electrophoresis, only 38 showed significantly altered expression in lamin A/C depleted cells. Of these, 4 protein spots were up-regulated, and 34 were down-regulated. Significant changes were seen to involve the general reduction in expression of cytoskeletal proteins, consistent with altered functionality of the structural cellular networks. At the same time, alterations in expression of proteins involved in cellular metabolism correlated with altered patterns of metabolic activity. In order to link these two features, we used antibody microarrays to perform a focused analysis of expression of cell cycle regulatory proteins. This confirmed a general reduction in expression of proteins regulating cell cycle progression and alteration in signaling pathways that regulate the metabolic activity of cells. The cross-talk between signal transduction and the cytoskeleton emphasizes how structural and kinase-based networks are integrated in mammalian cells to fine-tune metabolic responses.

Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase.

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1(-/-) chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1(-/-) cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3(-/-) DT40 cells. Rather, we observed an increased number of replication fibers in Chk1(-/-) cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1(-/-) cells are associated with the accumulation of aberrant replication fork structures.