Sudden infant death syndrome revisited: serotonin transporter gene, polymorphisms and promoter methylation

Background Based on findings in the brain stems of SIDS victims, the serotonin transporter (5-HTT) gene has been discussed to be associated with SIDS. Methods In the largest study to date, we investigated the promoter length (5-HTTLPR) and intron 2 VNTR polymorphisms in 274 cases and 264 controls and the Ile425Val polymorphism in 65 cases and 64 controls. Moreover, the methylation of the internal promoter region was investigated in 35 cases and 14 controls. Results For 5-HTTLPR, we observed a trend towards an association of allele L (58.8% vs. 53.4%) with SIDS and significant results were observed after stratifying for age, season at death, and prone position. Nevertheless, when pooling all published data, a significant association of allele L with SIDS is confirmed (p: 0.001). For the intron 2 VNTR polymorphism, no significant differences were observed. After pooling, a significant accumulation of the rare allele 9 was observed in SIDS (2.1% vs. 0.6%; p: 0.018). For the Ile425Val polymorphism, no differences were observed. Conclusion We conclude that genetic variation at this gene might be of some importance in SIDS. Epigenetic analysis of the internal promoter, however, revealed no influence on the relative risk to succumb to SIDS. Impact This is the largest study published up to now on 5-HTT gene polymorphisms and SIDS. Polymorphisms in the 5-HTT gene appear to contribute (although to a small degree) to the risk to die from SIDS. There is no evidence that a methylation of the promoter region is of impact for the etiology of SIDS.

The Regulation of Prototype Foamy Virus 5′Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors

<b><i>Background:</i></b> For foamy virus, the transactivator of spumaretrovirus (Tas) could bind directly to target DNA sequences termed as Tas responsive elements and trigger the viral internal promoter (IP) and long terminal repeat (LTR) promoters. The cellular endogenous factors also play an important role in viral gene expressions. We hypothesized that except the viral transcription factor Tas, the cellular endogenous factors also affect the viral gene expression. <b><i>Methods:</i></b> The full length of the prototype foamy virus (PFV) genome (U21247) was used to predict the potential binding sites of the transcription factors by online software JASPAR (http://jaspar.genereg.net) and Softberry (http://linux1.softberry.com/berry.phtml?topic=index&amp;group=programs&amp;subgroup=promoter). The Dual-Luciferase^® Reporter Assay System (Promega, USA) was used to confirm the relative luciferase activities of the test groups. The different representative activating agents or inhibitors of each canonical signal pathway were used to identify the impact of these pathways on PFV 5′LTR and IP promoters. <b><i>Results:</i></b> The results showed different cellular endogenous factors might have respective effects on PFV 5′LTR and IP. It is worth mentioning that activator protein-1 and BCL2-associated athanogene 3, 2 kinds of vital proteins associated with NF-κB and PKC pathways, could activate the basal activity of 5′LTR and IP promoters but inhibit the Tas-regulated activity of both promoters. Furthermore, PFV Tas was identified to trigger the transcription of the NF-κB promoter. <b><i>Conclusion:</i></b> NF-κB had a negative effect on PFV 5′LTR and IP promoter activities, the PKC pathway might upregulate 5′LTR and IP promoter activities, and the JNK and NF-AT signal pathway could increase the Tas-regulated promoter activity of PFV 5′LTR. This study sheds light on the interaction between PFV and the host cell and may help utilize the viral promoters in retroviral vectors designed for gene transfer experiments.

Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of theShigellaVirF Regulator

ABSTRACTVirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program ofShigellaspp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that thevirFmRNA is translated into two proteins, the major form, VirF30(30 kDa), and the shorter VirF21(21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30and VirF21and showed that the two different forms of VirF arise from differential translation. Interestingly,in vitroandin vivotranslation experiments showed that VirF21is also translated from a leaderless mRNA (llmRNA) whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30is responsible for activation of the virulence system, VirF21negatively autoregulatesvirFexpression itself. Since VirF21modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes invirFmRNA transcription and preferential translation of llmRNA.IMPORTANCEShigellaspp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that theShigella virFgene encodes two proteins of different sizes, VirF30and VirF21, that are functionally distinct. The major form, VirF30, activates the genes necessary for virulence, whereas the minor VirF21, which shares the C-terminal two-thirds of VirF30, negatively autoregulatesvirFexpression itself. VirF21is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of theShigellainvasive process and may help development of new therapies for shigellosis.