Computational identification and experimental characterization of preferred downstream positions in human core promoters

Author summary Transcription of genes by the RNA polymerase II enzyme initiates at a genomic region termed the core promoter. The core promoter is a regulatory region that may contain diverse short DNA sequence motifs/elements that confer specific properties to it. Interestingly, core promoter motifs can be located both upstream and downstream of the transcription start site. Variable compositions of core promoter elements were identified. The initiator (Inr) motif and the downstream core promoter element (DPE) is a combination of elements that has been identified and extensively characterized in fruit flies. Although a few Inr+DPE -containing human promoters were identified, the presence of transcriptionally important downstream core promoter positions within human promoters has been a matter of controversy in the literature. Here, using a newly-designed motif discovery strategy, we discovered preferred downstream positions in human promoters that resemble fruit fly DPE. Clustering of the corresponding sequence motifs in eight additional species indicated that such promoters could be common to multicellular non-plant organisms. Importantly, functional characterization of the newly discovered preferred downstream positions supports the existence of Inr+DPE-containing promoters in human genes. Metazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters

A Degenerate Type III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis

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

Engineered Promoters for Potent Transient Overexpression.

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

Identification of evolutionarily conserved downstream core promoter elements required for the transcriptional regulation of Fushi tarazu target genes.

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

Identification of evolutionarily conserved downstream core promoter elements required for the transcriptional regulation of Fushi tarazu target genes.

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

Live cell <i>EGFP</i> imaging of short-term expression of pRc/CMV-based constructs, in HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with either the pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were imaged once a day during 1–4 days post-transfection (P.T.). Each circle displays the whole well image constructed by stitching individual microscopic fields. (A) HeLa S3 cells. (B) SH-SY5Y cells. Data shown are representative of 3 independent experiments for each cell type.</p

Flow cytometric analysis of long-term fluorescence intensity and number of fluorescent HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were collected 4–8 days post-transfection (P.T.) for flow cytometric analysis. (A) Flow cytometric analysis of fluorescence intensity of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (B) Flow cytometric analysis of fluorescence intensity of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (C) Flow cytometric analysis of the number of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (D) Flow cytometric analysis of the number of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. Data shown are representative of 6 independent experiments using HeLa S3 cells and 5 independent experiments using SH-SY5Y cells. Statistical comparisons between the promoters were done using the Kruskal—Wallis test with pairwise comparisons. Significant p-values (p ≤0.05) are indicated in the results section.</p

Schematic representation of the engineered core promoters.

<p>The pRc/CMV vector (Life Technologies) contains the CMV enhancer and TATA box, but lacks any CMV sequences that are downstream of -16 relative to the +1 transcription start site (including the Inr element). Three variants of pRc/CMV were constructed in which the core promoter region (from -36 to +45) was replaced with either the natural CMV core promoter, which contains the CMV TATA and Inr elements, or with SCP2 or SCP3, which contains the CMV TATA and Inr, the <i>Tollo</i> MTE, and the <i>Calm2</i> DPE. Single nucleotide changes in SCP3 (relative to SCP2) are marked by red rectangles. Each of these pRc/CMV-based constructs contains the <i>EGFP</i> reporter gene.</p

Real-Time quantitative PCR of purified transiently transfected plasmid DNA in HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>, and harvested every other day during 8 days post-transfection (P.T.). Plasmid DNA was purified from cells and subjected to qPCR analysis with primers for the GAPDH, <i>EGFP</i> and Neomycin genes. Data shown are the averaged Ct values of 3 independent experiments (each performed in triplicates). (A) 2 days post-transfection. (B) 4 days post-transfection. (C) 6 days post-transfection. (D) 8 days post-transfection. Error bars represent SEM.</p

A comparison of the four core promoters DNA sequences.

<p>A comparison of the four core promoters DNA sequences.</p