Vikki M. Weake

Histone ubiquitination: triggering gene activity.

Recently, many of the enzymes responsible for the addition and removal of ubiquitin from the histones H2A and H2B have been identified and characterized. From these studies, it has become clear that H2A and H2B ubiquitination play critical roles in regulating many processes within the nucleus, including transcription initiation and elongation, silencing, and DNA repair. In this review, we present the enzymes involved in H2A and H2B ubiquitination and discuss new evidence that links histone ubiquitination to other chromatin modifications, which has provided a model for the role of H2B ubiquitination, in particular, in transcription initiation and elongation.

Inducible gene expression: diverse regulatory mechanisms

The rapid activation of gene expression in response to stimuli occurs largely through the regulation of RNA polymerase II-dependent transcription. In this Review, we discuss events that occur during the transcription cycle in eukaryotes that are important for the rapid and specific activation of gene expression in response to external stimuli. In addition to regulated recruitment of the transcription machinery to the promoter, it has now been shown that control steps can include chromatin remodelling and the release of paused polymerase. Recent work suggests that some components of signal transduction cascades also play an integral part in activating transcription at target genes.

SAGA‐mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system

Nonstop, which has previously been shown to have homology to ubiquitin proteases, is required for proper termination of axons R1–R6 in the optic lobe of the developing Drosophila eye. Herein, we establish that Nonstop actually functions as an ubiquitin protease to control the levels of ubiquitinated histone H2B in flies. We further establish that Nonstop is the functional homolog of yeast Ubp8, and can substitute for Ubp8 function in yeast cells. In yeast, Ubp8 activity requires Sgf11. We show that in Drosophila, loss of Sgf11 function causes similar photoreceptor axon‐targeting defects as loss of Nonstop. Ubp8 and Sgf11 are components of the yeast SAGA complex, suggesting that Nonstop function might be mediated through the Drosophila SAGA complex. Indeed, we find that Nonstop does associate with SAGA components in flies, and mutants in other SAGA subunits display nonstop phenotypes, indicating that SAGA complex is required for accurate axon guidance in the optic lobe. Candidate genes regulated by SAGA that may be required for correct axon targeting were identified by microarray analysis of gene expression in SAGA mutants.

SAGA function in tissue-specific gene expression

The Spt-Ada-Gcn5-acetyltransferase (SAGA) transcription coactivator plays multiple roles in regulating transcription because of the presence of functionally independent modules of subunits within the complex. We have recently identified a role for the ubiquitin protease activity of SAGA in regulating tissue-specific gene expression in Drosophila. Here, we discuss the modular nature of SAGA and the different mechanisms through which SAGA is recruited to target promoters. We propose that the genes sensitive to loss of the ubiquitin protease activity of SAGA share functional characteristics that require deubiquitination of monoubiquitinated histone H2B (ubH2B) for full activation. We hypothesize that deubiquitination of ubH2B by SAGA destabilizes promoter nucleosomes, thus enhancing recruitment of RNA polymerase II (Pol II) to weak promoters. In addition, SAGA-mediated deubiquitination of ubH2B may facilitate binding of factors that are important for the transition of paused Pol II into transcription elongation.

A novel histone fold domain-containing protein that replaces TAF6 in Drosophila SAGA is required for SAGA-dependent gene expression

The histone acetyltransferase complex SAGA is well characterized as a coactivator complex in yeast. In this study of Drosophila SAGA (dSAGA), we describe three novel components that include an ortholog of Spt20, a potential ortholog of Sgf73/ATXN7, and a novel histone fold protein, SAF6 (SAGA factor-like TAF6). SAF6, which binds directly to TAF9, functions analogously in dSAGA to TAF6/TAF6L in the yeast and human SAGA complexes, respectively. Moreover, TAF6 in flies is restricted to TFIID. Mutations in saf6 disrupt SAGA-regulated gene expression without disrupting acetylated or ubiquitinated histone levels. Thus, SAF6 is essential for SAGA coactivator function independent of the enzymatic activities of the complex.

The Essential Gene wda Encodes a WD40 Repeat Subunit of Drosophila SAGA Required for Histone H3 Acetylation

ABSTRACT Histone acetylation provides a switch between transcriptionally repressive and permissive chromatin. By regulating the chromatin structure at specific promoters, histone acetyltransferases (HATs) carry out important functions during differentiation and development of higher eukaryotes. HAT complexes are present in organisms as diverse as Saccharomyces cerevisiae, humans, and flies. For example, the well-studied yeast SAGA is related to three mammalian complexes. We previously identified Drosophila melanogaster orthologues of yeast SAGA components Ada2, Ada3, Spt3, and Tra1 and demonstrated that they associate with dGcn5 in a high-molecular-weight complex. To better understand the function of Drosophila SAGA (dSAGA), we sought to affinity purify and characterize this complex in more detail. A proteomic approach led to the identification of an orthologue of the yeast protein Ada1 and the novel protein encoded by CG4448, referred to as WDA (will decrease acetylation). Embryos lacking both alleles of the wda gene exhibited reduced levels of histone H3 acetylation and could not develop into adult flies. Our results point to a critical function of dSAGA and histone acetylation during Drosophila development.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Transcriptional Signatures of Aging

Genome-wide studies of aging have identified subsets of genes that show age-related changes in expression. Although the types of genes that are age regulated vary among different tissues and organisms, some patterns emerge from these large data sets. First, aging is associated with a broad induction of stress response pathways, although the specific genes and pathways involved differ depending on cell type and species. In contrast, a wide variety of functional classes of genes are downregulated with age, often including tissue-specific genes. Although the upregulation of age-regulated genes is likely to be governed by stress-responsive transcription factors, questions remain as to why particular genes are susceptible to age-related transcriptional decline. Here, we discuss recent findings showing that splicing is misregulated with age. While defects in splicing could lead to changes in protein isoform levels, they could also impact gene expression through nonsense-mediated decay of intron-retained transcripts. The discovery that splicing is misregulated with age suggests that other aspects of gene expression, such as transcription elongation, termination, and polyadenylation, must also be considered as potential mechanisms for age-related changes in transcript levels. Moreover, the considerable variation between genome-wide aging expression studies indicates that there is a critical need to analyze the transcriptional signatures of aging in single-cell types rather than whole tissues. Since age-associated decreases in gene expression could contribute to a progressive decline in cellular function, understanding the mechanisms that determine the aging transcriptome provides a potential target to extend healthy cellular lifespan.

Clearing the Way for Unpaused Polymerases

Heat shock loci in the polytene chromosomes of the fruit fly Drosophila undergo a characteristic change in appearance that coincides with the onset of gene expression. Petesch and Lis (2008) now show that nucleosomes are lost across the entire Hsp70 locus in an initial wave that precedes transcription by RNA polymerase II.

Cytochrome b5 protects photoreceptors from light stress-induced lipid peroxidation and retinal degeneration

Lipid peroxides are generated by oxidative stress in cells, and contribute to ageing and neurodegenerative disease. The eye is at special risk for lipid peroxidation because photoreceptors possess amplified sensory membranes rich in peroxidation-susceptible polyunsaturated fatty acids. Light-induced lipid peroxidation in the retina contributes to retinal degeneration, and lipid peroxidation has been implicated in the progression of age-associated ocular diseases such as age-related macular degeneration (AMD). Here, we show that exposing Drosophila melanogaster to strong blue light induces oxidative stress including lipid peroxidation that results in retinal degeneration. Surprisingly, very young flies are resilient to this acute light stress, suggesting they possess endogenous neuroprotective mechanisms. While lipophilic antioxidants partially suppressed blue light-induced retinal degeneration in older flies, we find that overexpression of cytochrome b5 (Cyt-b5) completely suppressed both blue light-induced lipid peroxidation and retinal degeneration. Our data identify Cyt-b5 as a neuroprotective factor that targets light-induced oxidative damage, particularly lipid peroxidation. Cyt-b5 may function via supporting antioxidant recycling, thereby providing a strategy to prevent oxidative stress in ageing photoreceptors that would be synergistic with dietary antioxidant supplementation.Neuroscience: Vision is stressful for old fliesParadoxically, light is essential for vision, yet it also induces stress that damages the sensitive cells in the eye. Vikki Weake and her team at Purdue University examined how exposure to blue light causes damage to the retina in fruit flies. Blue light causes death of photoreceptors, the light-sensing neurons. Surprisingly, very young flies are resistant to blue light. Increasing levels of a single protein, Cytochrome-b5, mimicked youthful resilience in older flies. Cytochrome-b5 is central to an ancient cellular defense system that protects membranes from oxidative damage. With expansive sensory membranes containing specialized lipids, photoreceptors are especially sensitive to membrane lipid peroxidation, an emerging final common pathway for cell death in aging and disease. Research into preventing lipid peroxidation might help to develop therapies for age-related diseases such as age-related macular degeneration.