Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Binding site analysis and identification of reciprocal targeting of Dom-A and Xbp1 in Drosophila melanogaster

ABSTRACT: The DOM-A complex regulates cell growth and proliferation in Drosophila. Analogous to the mammalian Tip60–p400 complex, DOM-A integrates two epigenetic effectors: a SWR1-type ATPase (Domino-A) that mediates histone exchange and the Tip60/KAT5 acetyltransferase. We identified Xbp1, a conserved transcriptional regulator of the unfolded protein response (UPR), as a tight interactor of the immunopurified DOM-A complex and explored the functional consequences of this association. Integrative analysis of Xbp1 and DOM-A occupancy in proliferating cells, together with reciprocal protein depletion, revealed two distinct modes of Xbp1 chromatin binding. In a sequence-specific mode, Xbp1 recruits DOM-A to motif-bearing promoters of UPR genes. In a second, motif-independent mode, Xbp1 localizes to hundreds of high-confidence DOM-A binding sites that lack Xbp1 recognition motifs; these interactions depend on DOM-A, consistent with a “reverse targeting” mechanism. Consistent with functional coupling, depletion of DOM-A reduces Xbp1 protein abundance without affecting Xbp1 mRNA levels, suggesting post-translational stabilization of Xbp1 upon association with DOM-A. Together, these findings indicate that the genome-wide interplay between Xbp1 and DOM-A may integrate UPR signaling with the broader, DOM-mediated regulation of cell growth and proliferation.

INSTRUMENT(S): NextSeq 2000

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Gizem Kars

PROVIDER: E-MTAB-15796 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

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Binding site analysis and identification of reciprocal targeting of Dom-A and Xbp1 in Drosophila melanogaster

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