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The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2 sepsis , which, although degenerate, contributes to pathogenesis. ETT2 sepsis has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.Type III secretion systems (T3SSs) are utilized by gramnegative pathogenic bacteria to deliver target-specific effector proteins into eukaryotic cells (14). This energy-requiring system is composed of more than 20 largely conserved proteins, which form a secretion apparatus for direct transfer of effector proteins from bacterial cytoplasm into the host cell or the medium. These effectors interfere with specific cellular processes and facilitate bacterial survival and replication. Typical features of the T3SS are the lack of an obvious secretion signal in the delivered proteins and the requirement for binding of a chaperone to the substrate before secretion (11,16).Previously characterized T3SSs in members of the family Enterobacteriaceae are composed of connected inner and outer membrane rings and a needle, through which the effector proteins are most likely passed into the host cytosol. Proteins translocated by this system promote critical processes such as bacterial internalization by mammalian cells in Salmonella and Shigella spp. (34, 36), induction of macrophage apoptosis in Salmonella, Shigella, and Yersinia spp. (24,27), and creation of attachment and effacement lesions in enteropathogenic and enterohemorrhagic Escherichia coli (18).In enteropathogenic and enterohemorrhagic E. coli, a chromosomal pathogenicity island (PAI) called the locus of enterocyte effacement contains genes encoding the components of T3SS which are involved in the formation of attaching and effacing lesions (18).The published sequences of the enterohemorrhagic E. coli O157:H7 genome revealed the presence of an additional putative T3SS (13, 30), which has not been previously described. This type III secretion system was termed ETT2, for E. coli type III secretion system 2 (13), to distinguish it from the locus of e...

Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, we used a biochemical complementation approach and identified the Drosophila TBP (TATA-boxbinding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. We demonstrate that the short TRF2 isoform preferentially activates DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 up-regulated genes. Using primer extension analysis and reporter assays, we show the importance of the DPE in transcriptional regulation of TRF2 target genes. It was previously shown that, unlike TBP, TRF2 fails to bind DNA containing TATA-boxes. Using microfluidic affinity analysis, we discovered that short TRF2-bound DNA oligos are enriched for Inr and DPE motifs. Taken together, our findings highlight the role of short TRF2 as a preferential core promoter regulator.

Long noncoding RNAs (lncRNAs) are major regulators of many cellular processes including cell cycle progression and tumorigenesis. In this study, we identify a novel lncRNA, MA-linc1, and reveal its effects on cell cycle progression and cancer growth. Inhibition of MA-linc1 expression alters cell cycle distribution, leading to a decrease in the number of G1 cells and a concomitant increase in all other stages of the cell cycle, and in particular G2/M, suggesting its involvement in the regulation of M phase. Accordingly, knock down of MA-linc1 inhibits M phase exit upon release from a mitotic block. We further demonstrate that MA-linc1 predominantly functions in cis to repress expression of its neighboring gene, Purα, which is often deleted in human cancers and whose ectopic expression inhibits cell cycle progression. Knock down of Purα partially rescues the MA-linc1 dependent inhibition of M phase exit. In agreement with its suggested role in M phase, inhibition of MA-linc1 enhances apoptotic cell death induced by the antimitotic drug, Paclitaxel and this enhancement of apoptosis is rescued by Purα knockdown. Furthermore, high levels of MA-linc1 are associated with reduced survival in human breast and lung cancer patients.Taken together, our data identify MA-linc1 as a novel lncRNA regulator of cell cycle and demonstrate its potential role in cancer progression and treatment.

These authors contributed equally to this work.Keywords: core promoter elements/motifs, DPE, embryonic development, histone gene cluster, RNAP II transcription, ribosomal protein genes, spermiogenesis, TBP-related factors, TRF2, TCT Transcriptional regulation is pivotal for development and differentiation of organisms. Transcription of eukaryotic protein-coding genes by RNA polymerase II (RNAP II) initiates at the core promoter. Core promoters, which encompass the transcription start site, may contain functional core promoter elements, such as the TATA box, initiator, TCT and downstream core promoter element. TRF2 (TATA-box-binding protein-related factor 2) does not bind TATA box-containing promoters. Rather, it is recruited to core promoters via sequences other than the TATA box. We review the recent findings implicating TRF2 as a basal transcription factor in the regulation of diverse biological processes and specialized transcriptional programs.

The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3 (SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as gene-therapy-related applications that require potent expression for extended time periods.

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The regulation of transcription initiation is critical for developmental and cellular processes. RNA polymerase II (Pol II) is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. We had previously shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the engrailed locus. Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. Here, we examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. Our analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. We further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination (Inr, Bridge and DPE) of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.