InteractiveFly: GeneBrief

scrawny: Biological Overview | References

BIOLOGICAL OVERVIEW

Stem cells within diverse tissues share the need for a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. A Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, is required in germline, epithelial, and intestinal stem cells. Like its yeast relative UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of ubiquitinylated H2B and trimethylated H3K4. These findings suggest that inhibiting H2B ubiquitylation through scny represents a common mechanism within stem cells that is used to repress the premature expression of key differentiation genes, including Notch target genes (Buszczak, 2009).

Stem cells are maintained in an undifferentiated state by signals they receive within the niche and are subsequently guided toward particular fates upon niche exit. Within ES cells and during differentiation, cell state changes are controlled at the level of chromatin by alterations involving higher order nucleosome packaging and histone tail modifications. Polycomb group (PcG) and Trithorax group (trxG) genes influence key histone methylation events at the promoters of target genes, including H3K27 and H3K4 modifications associated with gene repression and activation, respectively, but few other genes with a specific role in stem cells are known (Buszczak, 2009).

Histone H2A and H2B mono-ubiquitylation play fundamental roles in chromatin regulation, and H2A ubiquitylation has been linked to PcG-mediated gene repression and stem cell maintenance. The mammalian Polycomb repressive complex 1 (PRC1) component RING1B is a H2A ubiquitin ligase that is required to block the elongation of poised RNA polymerase II on bivalent genes in ES cells. Mutations in the PRC1 component, BMI-1, the ortholog of Psc in the mammalian PRC1, complexes with RING1B, and causes multiple types of adult stem cells to be prematurely lost. The role of H2B ubiquitylation in stem cells is unclear, however. In yeast, ubiquitylation of Histone H2B by the RAD6 and BRE1 ligases controls H3K4 methylation (H3K4me3), a process that requires the polymerase accessory factor PAF1. Conversely, H2B deubiquitylation by the ubiquitin-specific protease (USP) family member UBP10 is required for silencing telomeres, rDNA and other loci (Gardner, 2005). The Drosophila homolog of BRE1, dBRE1, also is needed for H3K4 methylation, suggesting that this pathway is conserved. Furthermore, the Drosophila ubiquitin-specific protease USP7 is part of a complex that selectively deubiquitylates H2B and genetically interacts with PcG mutations (van der Knaap, 2005). Mutations in another USP family member, Nonstop, increase H2B ubiquitylation and cause axon targeting defects in the eye (Buszczak, 2009).

In order to gain further insight into the role of H2B ubiquitylation in stem cells, a novel Drosophila gene, scrawny (scny) (CG5505), was identified, whose encoded USP family protein shares homology with human USP36 and among yeast USPs closely matches UBP10 within the core protease domain. Strains bearing scny insertions, except for a viable GFP protein trap (CA06690), were female sterile or lethal, and proved to be allelic. Transposon excision or expression of a scny-RB cDNA reverts the phenotype of tested alleles. An anti-SCNY antibody raised against a domain common to all SCNY isoforms recognizes wild type and SCNY-GFP on a Western blot. SCNY protein levels in homozygous third instar larvae are greatly reduced in lethal mutants, and SCNY expression is also lower in stem cell-enriched ovarian tissue from adults homozygous for the sterile d06513 allele. Consistent with a role in gene silencing, several scny mutations act as dominant suppressors of position effect variegation (Buszczak, 2009).

Further studies strongly suggested that SCNY functions in vivo as an H2B-ubiquitin protease. Recombinant full-length SCNY protein, but not a version bearing a point mutation in the protease domain, efficiently deubiquitylates histone H2B in vitro. scny^f01742 homozygous tissue contains levels of Ub-H2B that are elevated at least twofold compared to wild type. As expected if Ub-H2B is required for H3K4 methylation, clones of homozygous scny^e00340 mutant cells stain more strongly for H3K4me3 than heterozygous cells. Consistent with a direct rather than an indirect action on Ub-H2B levels, anti-SCNY antibodies co-immunoprecipitate H2B from Drosophila embryonic nuclear extracts. Moreover, epitope-tagged SCNY co-immunoprecipitates Drosophila PAF1, but not Cyclin T (or several other tested chromatin proteins) when co-expressed in S2 tissue culture cells. Together, these data support the view that SCNY participates in a conserved pathway of chromatin regulation linking H2B ubiquitylation with H3K4me3 methylation. Because the effects of scny mutation on Ub-H2B and H3K4me3 are opposite to those of dBre1 mutation, SCNY likely opposes dBRE1 action on H2B, just as UBP10 opposes BRE1 action on H2B in yeast (Buszczak, 2009).

Drosophila male and female gonads contain well characterized germline stem cells (GSCs) that allow the effects of genes on stem cell maintenance to be quantitatively analyzed. High levels of scny expression were observed in female and male GSCs using SCNY-GFP and identical staining was observed using anti-SCNY immunofluoresence. SCNY protein resides in cell nuclei and is enriched in nucleoli. In sterile or semi-fertile scny mutant adults, the numbers of germline stem cells surrounding the testis hub and within germaria were clearly reduced. The half-lives of female GSCs bearing clones of three different scny alleles were all sharply reduced. Later follicular development was also abnormal suggesting that scny continues to function after the stem cell stage. However, previous studies indicate that accelerated GSC loss is a specific phenotype, and hence that scny has a preferential requirement in GSCs (Buszczak, 2009).

A known mechanism of increased GSC loss is the premature activation of differentiation genes. Staining germaria with an antibody specific for multiple sites of histone H3 acetylation (H3-Ac) suggested that scny mutation affects the global chromatin organization of GSCs. Wild type GSCs contain lower levels of H3-Ac than slightly older germ cells within cysts. Presumptive GSCs located in the GSC niche in scny mutants frequently stained more strongly, suggesting that they have begun to upregulate general transcription. Some scny GSC-like cells also expressed bag-of-marbles (bam), a key cystoblast differentiation gene, and GSC-like cells in scny^d06513; bam^Δ86 mutant females persist in the germarium. However, it could not be completely ruled out that the observed increases in H3-Ac levels and bam expression were a result rather than a cause of the premature differentiation and loss of scny GSCs (Buszczak, 2009).

To determine if scny is also required in a very different type of stem cell, the epithelial follicle stem cell (FSC), the persistence of individual scny mutant FSCs was quantitatively. The half-life of FSCs mutant for scny^l(3)02331 was reduced more than 10-fold, while the scny^f01742 mutation also caused a sharp decline. However, mutant follicle cells continued to develop normally at later stages. Thus, scny is preferentially required to maintain FSCs as well as GSCs (Buszczak, 2009).

The largest population of Drosophila stem cells are the hundreds of multipotent intestinal stem cells (ISCs) that maintain the adult posterior midgut. ISCs signal to their daughters via Delta-Notch signaling to specify enterocyte vs. enteroendocrine cell fate, but the pathway must remain inactive in the ISCs themselves to avoid differentiation. Most ISCs (those about to produce enterocytes) express high levels of the Notch ligand Delta, allowing them to be specifically distinguished from other diploid gut cells. This study found that SCNY-GFP is expressed in ISCs suggesting that SCNY plays a role in these stem cells as well. While 7-day old normal adult midguts contain a high density of ISCs, as revealed by Delta staining, it was found that corresponding tissue from 7-day-old scny^f01742 or scny^f01742/scny^l(3)02331 escaper adults possess very few Delta-positive cells. ISCs are present in near normal numbers at eclosion, but are rapidly lost in the mutant adults, indicating that scny is required for ISC maintenance (Buszczak, 2009).

It is suspected that inappropriate Notch pathway activation was responsible for the premature ISC loss in scny mutants. dBre1 mutations strongly reduce Notch signaling, suggesting that Notch target genes are particularly dependent on H2B mono-ubiquitylation and H3K4 methylation. Consequently, scny mutations, which have the opposite effects on Ub-H2B and H3K4me3 levels, might upregulate Notch target genes, stimulating ISCs to differentiate prematurely. This idea was tested by supplementing the food of newly eclosed scny^f01742/scny^l(3)02331 adults with 8 mM DAPT, a gamma-secretase inhibitor that blocks Notch signaling and phenocopies Notch mutation when fed to wild type animals. scny^f01742/scny^l(3)02331 DAPT-treated adults remained healthy and the guts of 7-day old animals still contained many ISCs, although not as many as wild type. Tumors like those produced in wild type animals fed DAPT were not observed. Thus, in these animals endogenous stem cell loss can be slowed by drug treatment (Buszczak, 2009).

These experiments provide strong evidence that a pathway involving the ubiquitin protease Scrawny and the ubiquitin ligase dBRE1 controls the levels of Ub-H2B, and H3K4me3 at multiple target sites in the Drosophila genome. Although, other ubiquitin proteases also act on Ub-H2B in Drosophila, the direct interaction between SCNY and H2B, and the strong effects of scny mutations argue that it plays an essential, direct role in silencing genomic regions critical for cellular differentiation, including Notch target genes. SCNY interacts with the RNA polymerase accessory factor complex component, PAF1. Upregulation of H2B ubiquitinylation and H3 methylation in yeast is mediated by the PAF1 complex and is associated with elongating RNA Pol II. Drosophila PAF1 is required for normal levels of H3K4me3 at the hsp70 gene, and another PAF1 complex member, RTF1, is needed for H3K4 methylation and Notch target gene expression. Indeed, the pathway connecting Ub-H2B, H3K4me3 and gene silencing appears to be conserved in organisms as distant as Arabidopsis. A human protein closely related to SCNY, USP36, is overexpressed in ovarian cancer cells (Li, 2008), and the results suggest it may act as an oncogene by suppressing differentiation (Buszczak, 2009).

Above all, these experiments indicate that SCNY-mediated H2B deubiquitylation is required to maintain multiple Drosophila stem cells, including progenitors of germline, epithelial and endodermal lineages. In ES cells and presumably in adult stem cells, many differentiation genes contain promoter-bound, arrested RNA Pol II and are associated with Polycomb group proteins. It is envisioned that in the niche environment SCNY activity overrides that of dBRE1, keeping levels of Ub-H2B (and hence H3K4me3) low at key differentiation genes. Upon exit from the niche, the balance of signals shifts to favor H2B ubiquitylation, H3K4 trimethylation, and target gene activation. Thus, the control of H2B ubiquitylation, like H2A ubiquitylation, plays a fundamental interactive role in maintaining the chromatin environment of the stem cell state (Buszczak, 2009).

The deubiquitinase emperor's thumb is a regulator of apoptosis in Drosophila

The gene emperor's thumb (et) is required for the regulation of apoptosis in Drosophila. Loss-of-function mutations in et result in apoptosis associated with a decrease in the concentration of DIAP1. Overexpression of one form of et inhibits apoptosis, consistent with et having an anti-apoptotic function; however, overexpression of a second form of et induces apoptosis, indicating that the two forms of et may have competing functions. et, also known as scrawny, encodes a protein deubiquitinase, suggesting it regulates apoptosis by controlling the stability of apoptotic regulatory proteins (Ribaya, 2009).

The core mediators of apoptosis are a family of highly conserved cystein proteases called caspases (cystein aspartic acid proteases). Two classes of caspases, 'initiator' and 'effector', exist within the apoptotic pathway. Upon receiving death stimuli, initiator caspases activate the downstream effector caspases whose protease activity is directed toward the deconstruction of cellular machinery, resulting in a controlled cell death. Caspase activation must be strongly regulated in cells not scheduled to die because activated caspases can initiate a cascade of proteolysis. The only known cellular caspase inhibitors are members of the 'inhibitor of apoptotic proteins' (IAP) family. IAPs were first identified in baculovirus as cell death inhibitors. These proteins contain several 70-amino acid repeat motifs known as the baculovirus IAP repeats (BIRs) and a C-terminal RING finger domain. The BIR domains are important for protein-protein interactions and are required to inhibit caspase function. DIAP1 in flies, for instance, binds and inhibits the caspase Drice and Dronc via its BIR domains. These same BIR domains are also required for the deregulation of IAPs in cells fated to die. In flies, the proapoptotic BIR-interacting proteins Reaper, Hid, and Grim (RHG proteins), can bind competitively with caspases (Drice and Dronc) for DIAP1's BIR domains, which displaces the caspases from DIAP1 inhibition. The RING finger domain confers E3 ubiquitin ligase activity that allows IAPs to ubiquitinate themselves (autoubiquitination) or bound substrate proteins, such as the caspases and RHG proteins. A number of proteins that contain one or more BIR repeats have now been identified in vertebrates. Studies on several of these, including XIAP, have shown that they act as cell death inhibitors (Ribaya, 2009 and references therein).

Many of the proteins involved in the regulation of apoptosis, including DIAP1, the RHG proteins and Dronc, are regulated by ubiquitination. Ubiquitin (Ub) is a 76-amino-acid polypeptide that can be linked covalently to other proteins via an isopeptide bond between the terminal glycine residue of Ub and an internal lysine on the substrate protein. Polyubiquitination marks proteins for degradation by the 26S proteasome, a multisubunit proteolytic complex. Once thought to be a mechanism only for disposing of damaged proteins, it is now well established that Ub-mediated proteolysis is widely used to modulate the levels of critical regulatory proteins (Ribaya, 2009 and references therein).

Deubiquitinating enzymes (DUBs) are a large group of proteins that cleave Ub-protein bonds and play an important role in the ubiquitin pathway by reversing the effects of ubiquitination. Where polyubiquitination will mark a protein for degradation, deubiquitination will remove the ubiquitin and stabilize the protein. This paper describes et, a DUB required for the proper regulation of apoptosis in Drosophila. Loss-of-function mutations in et result in apoptosis associated with a decrease in the concentration of DIAP1, indicating an antiapoptotic function for et. Consistent with this notion, overexpression of one form of et inhibits apoptosis in the developing retina and apoptosis caused by Reaper and Grim induction; however, overexpression of a second form of et induces apoptosis, suggesting that et also has a proapoptotic function. Many of the proteins involved in the regulation of apoptosis are ubiquitinated, suggesting a mechanism for the deubiquitinase activity of Et. Although specific Et targets are not known, proposed targets for Et include the ubiquitinated proteins Reaper, Grim, Diap1, and Dronc (Ribaya, 2009).

Mutations in et result in apoptosis coupled with a decrease in the concentration of DIAP1. Overexpression of long et inhibits apoptosis, whereas overexpression of short et promotes apoptosis. In addition, et has been shown to be induced 71-fold during the programmed cell death of larval salivary glands. Together, these data make a compelling argument that et is required for the proper regulation of apoptosis in Drosophila (Ribaya, 2009).

Many Drosophila proteins involved in apoptosis are regulated by ubiquitination, including the E3 ubiquitin ligase DIAP1, Dronc and the RHG proteins (see Drosophila apoptotic pathway). In unstressed cells, DIAP1 and the caspases are kept at low concentrations by ubiquitination and proteosomal degradation. et may function by deubiquitinating one or more of these proteins. A mechanism comparable to this is seen with the mammalian deubiquitinase HAUSP, a regulator of p53. p53 is primarily regulated by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 and keeps it at low concentrations in unstressed cells. To maintain p53 at low levels, a cell must maintain Mdm2 at optimal levels. It does this through HAUSP, which is capable of deubiquitinating and thereby stabilizing both p53 and Mdm2 by creating a feedback loop between the three proteins that keeps them at the required concentrations. Et belongs to the same deubiquitinase family of proteins as HAUSP and could regulate DIAP1 and the caspases in a manner similar to that of HAUSP (Ribaya, 2009).

Loss-of-function mutations in et induced apoptosis in the ovary and developing eye, therefore et has an antiapoptotic function in these tissues. Consistent with these results was the finding that overexpression of long et also inhibits apoptosis. It was surprising to find that overexpression of short et induces cell death. The simplest explanation for this finding is that the long and short Et proteins have competing functions, this is supported by the finding that long et overexpression can partially suppress apoptosis induced by short et. RT-PCR analysis showed that both short and long et transcripts are present in larval eye discs. It is therefore proposed that there is a balance between short and long Et proteins in the eye. A minimal amount of long Et is necessary to prevent apoptosis, but that long Et activity is inhibited by short Et. Loss of both proteins would result in apoptosis, but overexpression of either long or short Et would throw the balance towards life or death. The results also reveal a strong association between et and DIAP1, including a strong genetic interaction and a reduction in DIAP1 concentration in cells mutant for et. Also pertinent is the finding that overexpression of long et suppresses apoptosis induced by Reaper and Grim, but not Hid. It has been shown in one study that overexpression of Reaper and Grim causes a reduction in the concentration of DIAP1, but overexpression of Hid does not affect DIAP1 levels. This suggests that Reaper and Grim induce apoptosis by reducing DIAP1 concentrations, possibly by promoting Ub-mediated DIAP1 degradation, whereas Hid must induce apoptosis by a different mechanism. The data therefore suggests that long et may inhibit the ability of Reaper and Grim to promote the degradation of DIAP1 (Ribaya, 2009).

These results put forward the possibility that the human homologues of et also function as regulators of apoptosis. The biology of USP36 has not been studied in detail, except for determining that it has deubiquitinase activity and that it is polyubiquitinated in tissue culture cells. USP42 has also not been studied in detail; however, a manuscript reported that a seven-year-old boy with acute myeloid leukemia (AML) had a novel cryptic translocation in the cancer cells that made a hybrid gene between RUNX1 and USP42, and the hybrid RUNX1/USP42 fusion protein was expressed at high levels in blood cells. It is possible that the overexpression of the USP42 ubiquitin protease activity has a role in the AML phenotype. The DUB/USP17 family of genes are located on the RS447 mega-satellite DNA, which has a 4.7 kb repetitive unit that contains the DUB/USP17 gene, with the number of repeats varying from 20 to 103 per person. Work on mouse and human DUB/USP17 genes show that their expression is induced by a number of different cytokines and that overexpression in tissue culture cells can block cell proliferation and induce apoptosis (Burrows, 2004; Zhu, 1997). Further analysis will be required to determine whether Et and its vertebrate homologues have conserved functions (Ribaya, 2009).

The germline specificity of et^roo is unique as all other et alleles affect both germline and soma. RT-PCR shows that et^roo makes a hybrid transcript containing both et and roo sequences. This hybrid transcript is assumed to make a truncated, but still active Et protein, which would explain the lack of a somatic phenotype. There are many possible explanations for why this truncated protein functions in the soma but not in the germline. One possibility is that the truncated Et protein is fully functional in the somatic tissue, but the protein coding region downstream of the roo insertion (which is conserved in USP42) has a germline-specific function. A second possibility is that an RNAi mechanism is involved. The expression of retrotransposons has been shown to be inhibited by rasiRNA (repeat associated small interfering RNA), and that rasiRNAs work preferentially in the germline. It is possible that the hybrid et-roo transcript is repressed in the germline by rasiRNAs (Ribaya, 2009).

Search PubMed for articles about Drosophila Scrawny

Burrows, J. F., et al. (2004). DUB-3, a cytokine-inducible deubiquitinating enzyme that blocks proliferation. J. Biol. Chem. 279: 13993-14000. PubMed ID: 14699124

Buszczak, M., Paterno, S. and Spradling, A. C. (2009). Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny. Science 323: 248-251. PubMed ID: 19039105

Gardner, R. G., Nelson, Z. W. and Gottschling, D. E. (2005). Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin. Mol. Cell Biol. 25(14): 6123-39. PubMed ID: 15988024

Li, J., et al. (2008). Differential display identifies overexpression of the USP36 gene, encoding a deubiquitinating enzyme, in ovarian cancer. Int. J. Med. Sci. 5(3): 133-42. PubMed ID: 18566677

Ribaya, J. P., et al. (2009). The deubiquitinase emperor's thumb is a regulator of apoptosis in Drosophila. Dev. Biol. 329(1): 25-35. PubMed ID: 19217892

van der Knaap, J. A., et al. (2005). GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 17(5): 695-707. PubMed ID: 15749019

Zhu, Y., et al. (1997). DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes. J. Biol. Chem. 272: 51-57. PubMed ID: 8995226

date revised: 12 January 2011

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