Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

Abstract

The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 59 end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression.

Affinity-based Isolation of Tagged Nuclei from <em>Drosophila</em> Tissues for Gene Expression Analysis

“…Thus, cell-specific effects of recessive lethal alleles can be examined in the embryonic or larval stages, prior to the stage of lethality. This genetic technique has been previously used to positively label cells in embryonic muscle that were homozygous for mutations in the SAGA subunit, sgf11 1 .…”

“…Methods to isolate cells from intact tissues include laser microdissection, and fluorescence-activated cell sorting (FACS) of whole cells. While FACS has been used to isolate cells and nuclei from Drosophila embryos and from Caenorhabditis elegans for gene expression and chromatin profiling [1][2][3] , FACS and laser microdissection can be difficult to perform successfully in tissues that contain highly intermixed cell types or that contain cells with irregular morphology, such as neurons. To overcome this difficulty, nuclei rather than cells can be isolated from specific cell types and used for subsequent gene expression profiling.…”

(Ubi)quitin’ the h2bit: recent insights into the roles of H2B ubiquitylation in DNA replication and transcription

“…Genome-wide location studies of SAGA complex subunits in different organisms reached various conclusions as to the extent of SAGA binding; while some reported only a few hundred binding sites for SAGA subunits, mainly at a subset of promoters, others observed more wide-spread occupancy. 22,23,24,25,26 Bonnet et al recently confirmed that SAGA deubiquitylates H2B in the coding region of all expressed genes in yeast and human cells-whereas earlier studies reached their conclusions by inspecting the occupancy of SAGA subunits, Bonnet et al examined deubiquitylase activity by measuring H2Bub distribution in the coding regions of active genes upon disruption of SAGA. 8 Earlier studies that concluded that SAGA is associated with a subset of active genes may have detected only the most stable interactions of the examined SAGA subunit with DNA.…”

Drosophila models reveal novel insights into mechanisms underlying neurodegeneration

“…8 Increasingly, however, evidence has indicated that regulation of SAGA in multicellular organisms involves more sophisticated mechanisms than in their unicellular counterparts. 9,10 In order to gain more insight into SAGA function in multicellular eukaryotes, we turned to Drosophila. This model provided powerful genetic and biochemical tools in which to explore principles underlying SAGA-mediated gene regulation.…”