David A. Wassarman

Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death

Apoptotic cell death is a mechanism by which organisms eliminate superfluous or harmful cells. Expression of the cell death regulatory protein REAPER (RPR) in the developing Drosophila eye results in a small eye owing to excess cell death. We show that mutations in thread (th) are dominant enhancers of RPR-induced cell death and that th encodes a protein homologous to baculovirus inhibitors of apoptosis (IAPs), which we call Drosophila IAP1 (DIAP1). Overexpression of DIAP1 or a related protein, DIAP2, in the eye suppresses normally occurring cell death as well as death due to overexpression of rpr or head involution defective. IAP death-preventing activity localizes to the N-terminal baculovirus IAP repeats, a motif found in both viral and cellular proteins associated with death prevention.

KSR, a novel protein kinase required for RAS signal transduction

We have identified and characterized two genes in Drosophila whose products are required for activated RAS to signal with normal efficiency, but do not appear to effect signaling by activated RAF. One encodes the beta subunit of type I geranylgeranyl transferase, a prenylation enzyme essential for targeting RAS to the plasma membrane. The other encodes a protein kinase that we have named kinase suppressor of ras (ksr). By genetic criteria, we show that KSR functions in multiple receptor tyrosine kinase pathways. We have isolated mammalian homologs of KSR that, together with the Drosophila gene, define a novel class of kinases. Our results suggest that KSR is a general and evolutionarily conserved component of the RAS signaling pathway that acts between RAS and RAF.

The Ras signaling pathway in Drosophila.

During Drosophila eye development, a Ras cascade mediates the decision between neuronal and non-neuronal differentiation of the R7 photoreceptor precursor. Recent genetic and molecular studies have identified a set of protein kinases as components of the Ras cascade and nuclear targets of the cascade, including Yan, Pointed, Jun, and Phyllopod. The Ras cascade functions in other Drosophila signal transduction pathways, eliciting a distinct response in each case, presumably through phosphorylation of specific transcription factors.

Altered expression of a novel adaptin leads to defective pigment granule biogenesis in the Drosophila eye color mutant garnet.

Drosophila eye pigmentation defects have thus far been attributed to mutations in genes encoding enzymes required for biosynthesis of pigments and to ABC‐type membrane transporters for pigments or their precursors. We report here that a defect in a gene encoding a putative coat adaptor protein leads to the eye color defect of garnet mutants. We first identified a human cDNA encoding δ‐adaptin, a structural homolog of the α‐ and γ‐adaptin subunits of the clathrin coat adaptors AP‐1 and AP‐2, respectively. Biochemical analyses demonstrated that δ‐adaptin is a component of the adaptor‐like complex AP‐3 in human cells. We then isolated a full‐length cDNA encoding the Drosophila ortholog of δ‐adaptin and found that transcripts specified by this cDNA are altered in garnet mutant flies. Examination by light and electron microscopy indicated that these mutant flies have reduced numbers of eye pigment granules, which correlates with decreased levels of both pteridine (red) and ommachrome (brown) pigments. Thus, the eye pigmentation defect in the Drosophila garnet mutant may be attributed to compromised function of a coat protein involved in intracellular transport processes required for biogenesis or function of pigment granules.

phyllopod functions in the fate determination of a subset of photoreceptors in drosophila

phyllopod (phyl) encodes a novel protein required for fate determination of photoreceptors R1, R6, and R7, the last three photoreceptors to be recruited into the ommatidia of the developing Drosophila eye. Genetic data suggests that phyl acts downstream of Ras1, raf, and yan to promote neuronal differentiation in this subset of photoreceptors. Ectopic expression of phyl in the cone cell precursors mimics the effect of ectopic activation of Ras1, suggesting that phyl expression is regulated by Ras1. phyl is also required for embryonic nervous system and sensory bristle development.

Ubiquilin Modifies TDP-43 Toxicity in a Drosophila Model of Amyotrophic Lateral Sclerosis (ALS)

TDP-43 (43-kDa TAR DNA-binding protein) is a major constituent of ubiquitin-positive cytosolic aggregates present in neurons of patients with amyotrophic lateral sclerosis (ALS) and ubiquitin-positive fronto-temporal lobar degeneration (FTLD-U). Inherited mutations in TDP-43 have been linked to familial forms of ALS, indicating a key role for TDP-43 in disease pathogenesis. Here, we describe a Drosophila melanogaster model of TDP-43 proteinopathy. Expression of wild-type human TDP-43 protein in Drosophila motor neurons led to motor dysfunction and dramatic reduction of life span. Interestingly, coexpression of ubiquilin 1, a previously identified TDP-43-interacting protein with suspected functions in autophagy and proteasome targeting, reduced steady-state TDP-43 expression but enhanced the severity of TDP-43 phenotypes. Finally, ectopically expressed TDP-43 was largely localized to motor neuron nuclei, suggesting that expression of wild-type TDP-43 alone is detrimental even in the absence of cytosolic aggregation. Our findings demonstrate that TDP-43 exerts cell-autonomous neurotoxicity in Drosophila and further imply that dose-dependent alterations of TDP-43 nuclear function may underlie motor neuron death in ALS.

Chromosomal localization links the SIN3–RPD3 complex to the regulation of chromatin condensation, histone acetylation and gene expression

Acetylation of core histone N‐terminal tails influences chromatin condensation and transcription. To examine how the SIN3–RPD3 deacetylase complex contributes to these events in vivo, we examined binding of SIN3 and RPD3 to Drosophila salivary gland polytene chromosomes. The binding patterns of SIN3 and RPD3 were highly coincident, suggesting that the SIN3–RPD3 complex is the most abundant chromatin‐bound RPD3 complex in salivary gland cells. SIN3– RPD3 binding was restricted to less condensed, hypoacetylated euchromatic interbands and was absent from moderately condensed, hyperacetylated euchromatic bands and highly condensed, differentially acetylated centric heterochromatin. Consistent with its demonstrated role in transcriptional repression, SIN3–RPD3 did not co‐localize with RNA polymer ase II. Chromatin binding of the complex, mediated by SMRTER, decreased upon ecdysone‐induced transcriptional activation but was restored when transcription was reduced. These results implicate SIN3–RPD3 in maintaining histone acetylation levels or patterns within less condensed chromatin domains and suggest that SIN3–RPD3 activity is required, in the absence of an activation signal, to repress transcription of particular genes within transcriptionally active chromatin domains.

The SIN3/RPD3 Deacetylase Complex Is Essential for G2 Phase Cell Cycle Progression and Regulation of SMRTER Corepressor Levels

ABSTRACT The SIN3 corepressor and RPD3 histone deacetylase are components of the evolutionarily conserved SIN3/RPD3 transcriptional repression complex. Here we show that the SIN3/RPD3 complex and the corepressor SMRTER are required for Drosophila G2 phase cell cycle progression. Loss of the SIN3, but not the p55, SAP18, or SAP30, component of the SIN3/RPD3 complex by RNA interference (RNAi) causes a cell cycle delay prior to initiation of mitosis. Loss of RPD3 reduces the growth rate of cells but does not cause a distinct cell cycle defect, suggesting that cells are delayed in multiple phases of the cell cycle, including G2. Thus, the role of the SIN3/RPD3 complex in G2 phase progression appears to be independent of p55, SAP18, and SAP30. SMRTER protein levels are reduced in SIN3 and RPD3 RNAi cells, and loss of SMRTER by RNAi is sufficient to cause a G2 phase delay, demonstrating that regulation of SMRTER protein levels by the SIN3/RPD3 complex is a vital component of the transcriptional repression mechanism. Loss of SIN3 does not affect global acetylation of histones H3 and H4, suggesting that the G2 phase delay is due not to global changes in genome integrity but rather to derepression of SIN3 target genes.

The SIN3 Deacetylase Complex Represses Genes Encoding Mitochondrial Proteins IMPLICATIONS FOR THE REGULATION OF ENERGY METABOLISM

Deacetylation of histones by the SIN3 complex is a major mechanism utilized in eukaryotic organisms to repress transcription. Presumably, developmental and cellular phenotypes resulting from mutations in SIN3 are a consequence of altered transcription of SIN3 target genes. Therefore, to understand the molecular mechanisms underlying SIN3 mutant phenotypes in Drosophila, we used full-genome oligonucleotide microarrays to compare gene expression levels in wild type Drosophila tissue culture cells versus SIN3-deficient cells generated by RNA interference. Of the 13,137 genes tested, 364 were induced and 35 were repressed by loss of SIN3. The ∼10-fold difference between the number of induced and repressed genes suggests that SIN3 plays a direct role in regulating these genes. The identified genes are distributed throughout euchromatic regions but are preferentially excluded from heterochromatic regions of Drosophila chromosomes suggesting that the SIN3 complex can only access particular chromatin structures. A number of cell cycle regulators were repressed by loss of SIN3, and functional studies indicate that repression of string, encoding the Drosophila homologue of the yeast CDC25 phosphatase, contributes to the G2 cell cycle delay of SIN3-deficient cells. Unexpectedly, a substantial fraction of genes induced by loss of SIN3 is involved in cytosolic and mitochondrial energy-generating pathways and other genes encode components of the mitochondrial translation machinery. Increased expression of mitochondrial proteins in SIN3-deficient cells is manifested in an increase in mitochondrial mass. Thus, SIN3 may play an important role in regulating mitochondrial respiratory activity.

Promoting developmental transcription

Animal growth and development depend on the precise control of gene expression at the level of transcription. A central role in the regulation of developmental transcription is attributed to transcription factors that bind DNA enhancer elements, which are often located far from gene transcription start sites. Here, we review recent studies that have uncovered significant regulatory functions in developmental transcription for the TFIID basal transcription factors and for the DNA core promoter elements that are located close to transcription start sites.