Transcriptional Regulation

Common motifs shared by conserved enhancers of Drosophila midline glial genes

Coding sequences are usually the most highly conserved sectors of DNA, but genomic regions controlling the expression pattern of certain genes can also be conserved across diverse species. In this study, five enhancers were identified capable of activating transcription in the midline glia of Drosophila melanogaster and each contains sequences conserved across at least 11 Drosophila species. In addition, the conserved sequences contain reiterated motifs for binding sites of the known midline transcriptional activators, Single-minded, Tango, Dichaete, and Pointed. To understand the molecular basis for the highly conserved genomic subregions within enhancers of the midline genes, the ability of various motifs to affect midline expression, both individually and in combination, were tested within synthetic reporter constructs. Multiple copies of the binding site for the midline regulators Single-minded and Tango can drive expression in midline cells; however, small changes to the sequences flanking this transcription factor binding site can inactivate expression in midline cells and activate expression in tracheal cells instead. For the midline genes described in this study, the highly conserved sequences appear to juxtapose positive and negative regulatory factors in a configuration that activates genes specifically in the midline glia, while maintaining them inactive in other tissues, including midline neurons and tracheal cells (Fulkerson, 2010).

The results described in this study indicate that the four genes expressed in the midline glia contain enhancers with subregions conserved in 11 or 12 of the sequenced Drosophila genomes. These conserved subregions contain one or more of the four motifs previously identified in the wrapper regulatory region, are highly A/T rich, and needed for robust expression in the midline. These results confirm the importance of several transcription factor-binding sites for midline glial activation. One of these sites, the CME, binds both Sim/Tgo and Trh/Tgo heterodimers and, when multimerized, can drive reporter gene expression in both midline and tracheal cells. Two lines of evidence indicate that the context of the CME determines whether or not it can be utilized to drive expression in these two tissues. (1) The sequences flanking the CMEs are highly conserved in the four genes discussed in this study, Glec, oatp26f, liprinγ and wrapper, suggesting that the location and sequence of other transcription factor-binding sites are constrained. (2) Changing the sequences flanking the CME in the synthetic multimers can eliminate expression in the midline, trachea, or both tissues (Fulkerson, 2010).

A multimerized CME in the context of the 4Toll:GFP reporter was expressed in both the midline and trachea and quite sensitive to slight modifications in flanking sequences. Changing 5-7 nucleotides on either side of the CME within this multimerized construct either substantially elevated expression in the trachea and eliminated it in the midline (T rich:GFP) or eliminated expression in both tissues (Sox:GFP). Additional combinations between the CME and one of the other midline glial motifs restricted expression to the midline (Pnt:GFP) or the trachea (POU:GFP). These results indicate that testing binding sites for two different factors next to one another can disrupt the endogenous ordering and spacing of the sites within the enhancers. Significantly, the Toll:GFP and Pnt:GFP reporters, unlike the intact enhancers described in this study, drive GFP expression in both midline neurons and glia. This midline expression pattern suggests that the synthetic multimers may lack repressor-binding sites that restrict expression to midline glia. Taken together, these results demonstrate the sensitivity of CME function to flanking sequences within the midline enhancers (Fulkerson, 2010).

Existing experimental evidence suggests that unlike most transcription factors, Sim/Tgo heterodimers (as well as Trh/Tgo heterodimers) preferentially binds one sequence over all others: ACGTG, the CME. Within the enhancers described in this study, sites flanking the CME have remained unchanged over evolutionary time due, in part, to similarities between binding sites for Sim and Trh and the molecular consequences of changing nucleotides adjacent to the CME. This conservation may ensure transcription is restricted to the midline glia and repressed in tracheal cells. In addition to the midline enhancers reported in this study, regions conserved among Drosophila species were found within the known midline enhancers. For instance, a 1.0-kb enhancer present in the first intron of slit drives expression in the midline glia and it contains a single CME and a 32-bp sequence conserved in 11 Drosophila species. It is important to note that the number of midline enhancers described in this study is limited and not all the midline glial enhancers are likely to exhibit such a high degree of conservation. For instance, a midline enhancer of the ectoderm3 gene, was identified that exhibits much less conservation among Drosophila species and presently, the basis for the observed variation among enhancers is unknown (Fulkerson, 2010).

The Ets transcriptional activator, pnt, a downstream effector of EGFR signaling, and Drifter, a POU domain protein, are expressed in both embryonic midline glia and tracheal cells. Previous studies have shown that deleting a POU domain-binding site within an enhancer of rhomboid eliminated expression in tracheal cells, but did not affect its midline glial expression. The results described in this study confirm and extend these results and suggest that the location of a POU domain-binding site relative to the CME can play a role in determining if a gene is expressed in the midline glia, the trachea or both. Moreover, swapping the PAS domains between Sim and Trh proteins indicated that additional, midline or tracheal specific cofactors bind to the PAS domains of the individual proteins and likely to determine which genes are expressed in the two different cell types. This may be the reason sequences adjacent to the CME play such a critical and sensitive role in determining which tissues express the various reporter genes described here. To activate the midline genes, Sim may interact with Drifter and Pnt and bind to sequences flanked by different binding sites compared with sequences bound by Trh, Drifter, and Pnt needed to activate tracheal genes. The simplicity of the multimers studied in this paper raise the possibility that different PAS heterodimers may specifically interact with other factors, such as Drifter and Pnt, in a manner that depends on the relative location and/or distance between each binding site, as has been described for nuclear hormone receptor complexes (Fulkerson, 2010).

The results confirm those of Swanson (2010), who found binding sites can be juxtaposed in different ways within enhancers to favor particular short-range interactions, and, in this way, various combinations of transcription factor binding sites (inputs) can result in more than one output. Similarly, the motifs described in this study can be combined in different ways that result in either midline or tracheal expression. The results indicate the proximity of the CME to activators, one another and/or to repressors could contribute to the level of expression observed in the trachea and midline. This study focused on activator sites, but repressor sites are also likely present and restrict expression to certain cell types. Previous studies in Drosophila embryos have revealed the complexity of the transcriptional regulatory 'grammar' and have shown that the transcriptional output from various genes can be determined by the stoichiometry, affinity, spacing, arrangement, and distance between activator and repressor sites (Fulkerson, 2010).

The high degree of conservation within the midline enhancer subregions examined in this study here belies known properties of transcription factors and their recognition sequences, as well as observations made for many early developmental regulators of Drosophila development. Most transcription factors can vary considerably in the sequences they recognize and tend to bind to related sites with different affinities. This property would suggest that enhancers need not be strictly conserved to function, in contrast to what is reported here. The pattern of conserved sequences within these identified enhancers suggests that the transcription factors that bind these regions do so in a conserved order and spacing pattern. These results suggest that Sim and Trh may interact with other proteins to form an 'enhanceosome'-like complex, similar to that observed in the regulation of the interferon-β gene, in which activators and HMG proteins interact to form a specific multiprotein complex, with a defined structure. This model contrasts with the 'information display/billboard' model of enhancer function. In that model, enhancers are bound by a group of independent factors or group of factors that work together to promote or repress transcription in particular cell types. An important distinction between the two models is the arrangement of binding sites within an enhancer. Within an enhanceosome, the arrangement of binding sites relative to each other is constrained, whereas within a billboard enhancer, the relative arrangement of binding sites is rather flexible as long as a sufficient number of binding sites work together, in many possible configurations, to recruit factors for transcriptional activation (Fulkerson, 2010).

Results obtained with the midline glial genes examined in this study suggest that midline enhancers may consist of a nucleating enhanceosome-like region that combines with an 'information display/billboard' constellation of additional binding sites. This is supported by results obtained with the 70-bp conserved region of wrapper. When tested alone, it only marginally drives midline expression, whereas in the context of the 166-bp enhancer, it works quite well. Moreover, the 166-bp region of virilis cannot function on its own, but drives high levels of expression in the midline glia of melanogaster in the context of the larger, 476 bp region. That the 166-bp region from virilis cannot work efficiently in the midline suggests the transcription complex that binds to this region may be slightly different in virilis compared with melanogaster. For each enhancer described in this study, the presence of the conserved region is required to obtain expression in the midline glia (Fulkerson, 2010).

After comparing vertebrate genomes and generating reporter constructs with highly conserved noncoding sequences, Bailey (2006) noticed that many of these direct expression to regions of the CNS. It is possible that enhancers of CNS genes are more conserved compared with other gene sets, such as early developmental regulators of Drosophila that have been studied in detail. This may be due to the highly conserved nature of the transcription factors that regulate gene expression in this tissue, many of which have analogous functions in flies and mammals). Sox-binding sites are present throughout conserved regions of CNS genes and one of the similarities between these conserved CNS genes, the extensively characterized interferon-β enhanceosome and midline glial genes is the importance of HMG proteins. These proteins may bend the DNA, facilitating binding to highly structured, multiprotein complexes. The enhancers described here likely bind PAS and Sox proteins together with other conserved CNS regulators and it may be this combination of transcription factors that contributes to the similarly conserved arrangement of binding sites (Fulkerson, 2010).

Numerous combinations of transcription factor binding sites can be used to drive expression in many tissue types. Despite the conservation found in this study, binding sites for transcription factors do vary considerably, making it, at times, difficult to identify enhancers based on sequence conservation. In certain cases, changes within enhancers can generate diverse phenotypes between Drosophila populations. The continuing challenge is to understand both the forces constraining the enhancer sequences between Drosophila species, as well as how changes in these regions lead to significant modifications in the expression pattern of a gene, which over the long term, leads to variation among Drosophila populations and eventually, Drosophila species. For the midline genes described in this study, selection has stabilized the constellation of binding sites found within enhancers, resulting in their conservation among Drosophila species over approximately 40 million years of evolution (Fulkerson, 2010).

Protein Interactions

Three fusion proteins were constructed, N, C and NC, made up of the N-terminal, C-terminal, and both N- and C-terminal portions of the protein. Only the N fusion protein demonstrates binding to glycolipids. Binding was detected to the NZ fraction (extracted lipids that run through an anion exchange column, that is, neutral and zwitterionic species) and to the A fraction (extracted acidic components). Binding to the NZ fraction is restricted to a subset of its glycolipids (Tiemeyer, 1996).

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