Subunits of the PBAP Chromatin Remodeler Are Capable of Mediating Enhancer-Driven Transcription in Drosophila

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.

PBAP chromatin remodeler mediates enhancer-driven transcription in Drosophila

Chromatin remodeler SWI/SNF is an important participant of gene activation acting predominantly by opening chromatin structure on promoters and enhancers. Here we describe its novel mode of action by mediating targeted action of enhancer. We studied functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered onto transgene reporter carrying hsp70 core promoter. Tethered subunits mediate transcription of reporter in a pattern prescribed by nearby enhancer in multiple loci throughout the genome, where the reporter construct was located. Both tethered SAYP and BAP170 recruit the whole PBAP complex onto reporter promoter. Studying difference between these subunits, we found that BAP170-dependent transcription is more resistant to depletion of other PBAP subunits, what may imply the principal role of BAP170 in establishing enhancer-dependent transcription.Author SummaryChromatin remodelers are key molecular machines that are responsible for local changes in chromatin structure in the nucleus. However, their functions in gene expression regulation seem to be broader. We describe the involvement of the SWI/SNF family of remodelers in establishing enhancer-promoter communication, which is apparently independent of its local remodeling activity.Using an artificial tethering of a remodeler on a promoter, we demonstrated that promoters of a certain type become responsive to activation by a nearby enhancer only in the presence of the remodeler, while a remodeler tethering itself is insufficient for gene activation. Thus, our approach helps to uncover novel aspects of molecular interplay on regulatory elements during the gene activation process.

Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits

Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter gene activities ensure control of the cell with synthetic genetic circuits, as achieved using a combination of in silico and in vivo experiments. However, unexpected effects of downstream reporter genes on upstream regulatory genes may interfere with in vivo observations. This phenomenon is termed as retroactivity. Using in silico and in vivo experiments, we found that a different copy number of regulatory protein-binding sites in a downstream gene altered the upstream dynamics, suggesting retroactivity of reporters in this synthetic genetic oscillator. Furthermore, by separating the two sources of retroactivity (titration of the component and competition for degradation), we showed that, in the dual-feedback oscillator, the level of the fluorescent protein reporter competing for degradation with the circuits’ components is important for the stability of the oscillations. Altogether, our results indicate that the selection of reporter promoters using a combination of in silico and in vivo experiments is essential for the advanced design of genetic circuits.

Two regulatory elements required for enhancing ospA expression in Borrelia burgdorferi grown in vitro but repressing its expression during mammalian infection

During cycling between the tick vector and a mammal, the Lyme disease spirochaete Borrelia burgdorferi must coordinate expression of outer-surface proteins (Osps) A and B to quickly respond to environmental changes. The pathogen abundantly produces OspA/B in the tick, but represses their expression during mammalian infection. This paper reports a regulatory structure, consisting of two sequences flanking the ospAB promoter, that is required for enhancing ospA expression in B. burgdorferi grown in vitro, but repressing its expression during murine infection. Deletion or replacement of either the upstream or downstream sequence of the ospAB promoter caused a significant decrease in ospA expression in vitro, but a dramatic increase during murine infection. Fusion of either sequence with the flaB reporter promoter led to increased expression of an ospA reporter gene in vitro, but a decrease in the murine host. Furthermore, simultaneous fusion of both sequences with the reporter promoter showed a synergistic effect in enhancing expression of the ospA reporter in vitro, but repressing its expression during murine infection. Taken together, the results demonstrate that the regulatory structure functions oppositely in the two different environments and potentially provides B. burgdorferi with a molecular mechanism to quickly adapt to the distinct environments during its enzootic life cycle.

Inhibition of Interferon Signaling by the Kaposi's Sarcoma-Associated Herpesvirus Full-Length Viral Interferon Regulatory Factor 2 Protein

ABSTRACT Interferon (IFN) signal transduction involves interferon regulatory factors (IRF). Kaposi's sarcoma-associated herpesvirus (KSHV) encodes four IRF homologues: viral IRF 1 (vIRF-1) to vIRF-4. Previous functional studies revealed that the first exon of vIRF-2 inhibited alpha/beta interferon (IFN-α/β) signaling. We now show that full-length vIRF-2 protein, translated from two spliced exons, inhibited both IFN-α- and IFN-λ-driven transactivation of a reporter promoter containing the interferon stimulated response element (ISRE). Transactivation of the ISRE promoter by IRF-1 was negatively regulated by vIRF-2 protein as well. Transactivation of a full-length IFN-β reporter promoter by either IRF-3 or IRF-1, but not IRF-7, was also inhibited by vIRF-2 protein. Thus, vIRF-2 protein is an interferon induction antagonist that acts pleiotropically, presumably facilitating KSHV infection and dissemination in vivo.