Identification of molecular sub-networks associated with cell survival in a chronically SIVmac-infected human CD4+ T cell line.

BACKGROUND: The deciphering of cellular networks to determine susceptibility to infection by HIV or the related simian immunodeficiency virus (SIV) is a major challenge in infection biology. RESULTS: Here, we have compared gene expression profiles of a human CD4+ T cell line at 24 h after infection with a cell line of the same origin permanently releasing SIVmac. A new knowledge-based-network approach (Inter-Chain-Finder, ICF) has been used to identify sub-networks associated with cell survival of a chronically SIV-infected T cell line. Notably, the method can identify not only differentially expressed key hub genes but also non-differentially expressed, critical, 'hidden' regulators. Six out of the 13 predicted major hidden key regulators were among the landscape of proteins known to interact with HIV. Several sub-networks were dysregulated upon chronic infection with SIV. Most prominently, factors reported to be engaged in early stages of acute viral infection were affected, e.g. entry, integration and provirus transcription and other cellular responses such as apoptosis and proliferation were modulated. For experimental validation of the gene expression analyses and computational predictions, individual pathways/sub-networks and significantly altered key regulators were investigated further. We showed that the expression of caveolin-1 (Cav-1), the top hub in the affected protein-protein interaction network, was significantly upregulated in chronically SIV-infected CD4+ T cells. Cav-1 is the main determinant of caveolae and a central component of several signal transduction pathways. Furthermore, CD4 downregulation and modulation of the expression of alternate and co-receptors as well as pathways associated with viral integration into the genome were also observed in these cells. Putatively, these modifications interfere with re-infection and the early replication cycle and inhibit cell death provoked by syncytia formation and bystander apoptosis. CONCLUSIONS: Thus, by using the novel approach for network analysis, ICF, we predict that in the T cell line chronically infected with SIV, cellular processes that are known to be crucial for early phases of HIV/SIV replication are altered and cellular responses that result in cell death are modulated. These modifications presumably contribute to cell survival despite chronic infection.

The positive aspects of stress: strain initiates domain decondensation (SIDD).

The conventional string-based bioinformatic methods of genomic sequence analysis are often insufficient to identify DNA regulatory elements, since many of these do not have a recognizable motif. Even in case a sequence pattern is known to be associated with an element it may only partially mediate its function. This suggests that properties not correlated with the details of base sequence contribute to regulation. One of these attributes is the DNA strand-separation potential, known as SIDD (stress-induced duplex destabilization) which facilitates the access of tracking proteins and the formation of local secondary structures. Using the type 1 interferon gene cluster as a paradigm, we demonstrate that the imprints in a SIDD profile coincide with chromatin domain borders and with DNAse I hypersensitive sites to which regulatory potential could be assigned. The approach permits the computer-guided identification of yet unknown, mostly remote sites and the design of artificial elements with predictable properties for multiple applications.

Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy.

Scaffold or matrix-attachment regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.

Co-localization of PARP-1 and lamin B in the nuclear architecture: a halo-fluorescence- and confocal-microscopy study.

A functional interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and lamin B has recently been proposed by nuclear fractionation, crosslinking, and immunoprecipitation experiments. Here we use fluorescence microscopy to verify and extend these findings. We analyze nuclear halo preparations by fluorescence in situ immuno staining (FISIS), which shares attributes with traditional nuclear fractionation techniques, and by confocal laser scanning microscopy (CLSM). The results agree in that a major part of the enzyme co-localizes with lamin B under physiological conditions, where PARP-1 only has basal activity. After DNA damage and the associated activation of PARP-1, and during the subsequent entry into apoptosis, dramatic changes occur: a gradual release of the enzyme from the lamina, accompanied by its accumulation in nucleoli. Our observations are in line with biochemical evidence for lamin B-PARP-1 interactions under physiological conditions and suggest ways by which these interactions are modified to support PARP-functions in damage and its fate in apoptosis.

Performance of genomic bordering elements at predefined genomic loci.

Eukaryotic DNA is organized into chromatin domains that regulate gene expression and chromosome behavior. Insulators and/or scaffold-matrix attachment regions (S/MARs) mark the boundaries of these chromatin domains where they delimit enhancing and silencing effects from the outside. By recombinase-mediated cassette exchange (RMCE), we were able to compare these two types of bordering elements at a number of predefined genomic loci. Flanking an expression vector with either S/MARs or two copies of the non-S/MAR chicken hypersensitive site 4 insulator demonstrates that while these borders confer related expression characteristics at most loci, their effect on chromatin organization is clearly distinct. Our results suggest that the activity of bordering elements is most pronounced for the abundant class of loci with a low but negligible expression potential in the case of highly expressed sites. By the RMCE procedure, we demonstrate that expression parameters are not due to a potential targeting action of bordering elements, in the sense that a linked transgene is directed into a special class of loci. Instead, we can relate the observed transcriptional augmentation phenomena to their function as genomic insulators.