The regulated production of a mature mRNA in eukaryotes requires highly coordinated molecular interactions and biochemical processes that involve the participation of hundreds of polypeptides. Understanding these underlying processes is important in creating novel strategies for intervening in abnormal regulation caused by mutations, epigenetic abnormalities, or infectious agents. The heat shock (HS) genes are a highly regulated set of genes particularly well suited to investigate fundamental features of inducible mRNA production. Here, both well-established and newly developed technologies will be used to discern critical features of promoter architecture and mechanisms of regulation. A critical feature of the promoter regions of heat shock genes and many important highly regulated genes is promoter-proximal RNA Polymerase II pausing (Paused Pol II).
In Aim 1 of this application, the establishment and properties of Paused Pol II will be investigated using a variation of our newly developed Global nuclear Run-On and massively-parallel sequencing protocol (GRO- seq) that is designed to provide near-nucleotide resolution mapping of Paused Pol II on a genome-wide scale in Drosophila cells. The precise positioning of all pauses coupled with bioinformatic approaches should greatly aid the identification of general elements and factors used in Paused Pol II regulation. The role of DNA elements and their spatial relationships to each other will be tested using targeted mutagenesis of existing model genes like Hsp70, and new genes uncovered by GRO-seq analyses. We will also define important mechanistic and dynamic properties of Paused Pol II using both our newly developed live cell imaging method and classical pulse-labeling experiments coupled with sensitive and high-resolution biochemical assays of paused RNAs.
In Aim 2, we investigate the mechanism of activating promoter-proximal paused Pol II by performing a comprehensive biochemical search for factors that interact with master regulator, HSF, as well as analysis of factors affecting Hsp70 expression that we obtained from directed and genome-wide screens. Existing and newly identified transcription factors will be examined to determine when and where they participate in the activation process in vivo by ChIP assays as well as two-photon microscopy to track in real time the recruitment and dynamics of factors at specific activated loci.
In Aim 3, we investigate the mechanisms that bring about rapid large scale loss of nucleosomes over an entire activated locus. Upon HS, nucleosomes are rapidly lost from heat shock loci in a transcription-independent manner. The region of nucleosome loss extends beyond the gene and up to, but not beyond, the locus insulators, scs and scs'. This locus-wide loss of nucleosomes depends on HSF and PARP. The proposed analyses will probe the spatial relationship of HSF and PARP during gene activation and nucleosome loss, testing for interactions between HSF and PARP, and identifying the critical barrier to nucleosome loss in the scs and scs'insulator regions.
The development, health, and viability of an organism depend on a plethora of intra- and extracellular signals that produce a highly orchestrated and regulated gene expression, with much of this regulation occurring at the level of transcription. Some of the approaches in this proposal and the technology being developed are providing direct insights to these molecular mechanisms in the complex but relevant milieu of living cells, while others provide unprecedented genome-wide views of promoter and gene architecture and expression. This information will provide the necessary background for understanding normal and disease states at the level of gene regulation and expression, and for designing strategies for intervening with abnormal expression of genes associated with cancer or disorders originating from mutation or disruption of transcription factor functions.
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