Dimitris Thanos

Virus Infection Induces NF-κB-Dependent Interchromosomal Associations Mediating Monoallelic IFN-β Gene Expression

Transcriptional activation of the IFN-beta gene by virus infection requires the cooperative assembly of an enhanceosome. We report that the stochastic and monoallelic expression of the IFN-beta gene depends on interchromosomal associations with three identified distinct genetic loci that could mediate binding of the limiting transcription factor NF-kappaB to the IFN-beta enhancer, thus triggering enhanceosome assembly and activation of transcription from this allele. The probability of a cell to express IFN-beta is dramatically increased when the cell is transfected with any of these loci. The secreted IFN-beta protein induces high-level expression of the enhanceosome factor IRF-7, which in turn promotes enhanceosome assembly and IFN-beta transcription from the remaining alleles and in other initially nonexpressing cells. Thus, the IFN-beta enhancer functions in a nonlinear fashion by working as a signal amplifier.

Transcription factors mediate long-range enhancer–promoter interactions

We examined how remote enhancers establish physical communication with target promoters to activate gene transcription in response to environmental signals. Although the natural IFN-β enhancer is located immediately upstream of the core promoter, it also can function as a classical enhancer element conferring virus infection-dependent activation of heterologous promoters, even when it is placed several kilobases away from these promoters. We demonstrated that the remote IFN-β enhancer “loops out” the intervening DNA to reach the target promoter. These chromatin loops depend on sequence-specific transcription factors bound to the enhancer and the promoter and thus can explain the specificity observed in enhancer–promoter interactions, especially in complex genetic loci. Transcription factor binding sites scattered between an enhancer and a promoter can work as decoys trapping the enhancer in nonproductive loops, thus resembling insulator elements. Finally, replacement of the transcription factor binding sites involved in DNA looping with those of a heterologous prokaryotic protein, the λ repressor, which is capable of loop formation, rescues enhancer function from a distance by re-establishing enhancer–promoter loop formation.

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.

S ‐glutathionylation of IRF3 regulates IRF3–CBP interaction and activation of the IFNβ pathway

Interferon regulatory factor 3 (IRF3) is an essential transcriptional regulator of the interferon genes. IRF3 is constitutively present in a latent conformation in the cell cytoplasm. In cells infected by Sendai virus, IRF3 becomes phosphorylated, homodimerizes, translocates to the nucleus, binds to target genes and activates transcription by interacting with CBP/p300 co‐activators. In this study, we report that in non‐infected cells IRF3 is post‐translationally modified by S‐glutathionylation. Upon viral‐infection, it undergoes a deglutathionylation step that is controlled by the cytoplasmic enzyme glutaredoxin‐1 (GRX‐1). In virus‐infected GRX‐1 knockdown cells, phosphorylation, homodimerization and nuclear translocation of IRF3 were not affected, but the transcriptional activity of IRF3 and the expression of interferon‐β (IFNβ), were severely reduced. We show that deglutathionylation of IRF3 is necessary for efficient interaction of IRF3 with CBP, an event essential for transcriptional activation of the interferon genes. Taken together, these findings reveal a crucial role for S‐glutathionylation and GRX‐1 in controlling the activation of IRF3 and IFNβ gene expression.

Gene regulatory networks modelling using a dynamic evolutionary hybrid.

BackgroundInference of gene regulatory networks is a key goal in the quest for understanding fundamental cellular processes and revealing underlying relations among genes. With the availability of gene expression data, computational methods aiming at regulatory networks reconstruction are facing challenges posed by the datas high dimensionality, temporal dynamics or measurement noise. We propose an approach based on a novel multi-layer evolutionary trained neuro-fuzzy recurrent network (ENFRN) that is able to select potential regulators of target genes and describe their regulation type.ResultsThe recurrent, self-organizing structure and evolutionary training of our network yield an optimized pool of regulatory relations, while its fuzzy nature avoids noise-related problems. Furthermore, we are able to assign scores for each regulation, highlighting the confidence in the retrieved relations. The approach was tested by applying it to several benchmark datasets of yeast, managing to acquire biologically validated relations among genes.ConclusionsThe results demonstrate the effectiveness of the ENFRN in retrieving biologically valid regulatory relations and providing meaningful insights for better understanding the dynamics of gene regulatory networks.The algorithms and methods described in this paper have been implemented in a Matlab toolbox and are available from: http://bioserver-1.bioacademy.gr/DataRepository/Project_ENFRN_GRN/.

Exploring and exploiting the systemic effects of deregulated replication licensing.

Maintenance and accurate propagation of the genetic material are key features for physiological development and wellbeing. The replication licensing machinery is crucial for replication precision as it ensures that replication takes place once per cell cycle. Thus, the expression status of the components comprising the replication licensing apparatus is tightly regulated to avoid re-replication; a form of replication stress that leads to genomic instability, a hallmark of cancer. In the present review we discuss the mechanistic basis of replication licensing deregulation, which leads to systemic effects, exemplified by its role in carcinogenesis and a variety of genetic syndromes. In addition, new insights demonstrate that above a particular threshold, the replication licensing factor Cdc6 acts as global transcriptional regulator, outlining new lines of exploration. The role of the putative replication licensing factor ChlR1/DDX11, mutated in the Warsaw Breakage Syndrome, in cancer is also considered. Finally, future perspectives focused on the potential therapeutic advantage by targeting replication licensing factors, and particularly Cdc6, are discussed.

Composite macroH2A/NRF-1 Nucleosomes Suppress Noise and Generate Robustness in Gene Expression.

The histone variant macroH2A (mH2A) has been implicated in transcriptional repression, but the molecular mechanisms that contribute to global mH2A-dependent genome regulation remain elusive. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with transcriptional profiling in mH2A knockdown cells, we demonstrate that singular mH2A nucleosomes occupy transcription start sites of subsets of both expressed and repressed genes, with opposing regulatory consequences. Specifically, mH2A nucleosomes mask repressor binding sites in expressed genes but activator binding sites in repressed genes, thus generating distinct chromatin landscapes that limit genetic or extracellular inductive signals. We show that composite nucleosomes containing mH2A and NRF-1 are stably positioned on gene regulatory regions and can buffer transcriptional noise associated with antiviral responses. In contrast, mH2A nucleosomes without NRF-1 bind promoters weakly and mark genes with noisier gene expression patterns. Thus, the strategic position and stabilization of mH2A nucleosomes in human promoters defines robust gene expression patterns.

A method for generating highly multiplexed ChIP-seq libraries

BackgroundThe barcoding of next generation sequencing libraries has become an essential part of the experimental design. Barcoding not only allows the sequencing of more than one sample per lane, but also reduces technical bias. However, current barcoding strategies impose significant limitations and/or technical barriers in their implementation for ChIP-sequencing.FindingsConverting Y-shaped sequencing adapters to double stranded DNA prior to agarose gel size selection reduces adapter dimer contamination and quantitating the number of cycles required for amplification of the library with qPCR prior to library amplification eliminates library over-amplification.ConclusionsWe describe an efficient and cost effective method for making barcoded ChIP-seq libraries for sequencing on the Illumina platform.

Time's up: Bursting out of Transcription

Many inducible genes are transcribed in bursts. In this issue, Degenhardt et al. (2009) report computational models that predict and validate patterns of stochastic gene expression.

Linking Differential Chromatin Loops to Transcriptional Decisions

GATA-1 and GATA-2 control proliferation and differentiation of hematopoietic progenitor cells via transcriptional regulation. In this issue of Molecular Cell, Jing et al. (2008) demonstrate that GATA factor exchange on the Kit locus directs a transcriptional switch by reconfiguring chromatin loops.