Proteome plasticity in response to persistent environmental change.

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

Enzymatic modules of the SAGA chromatin-modifying complex play distinct roles in Drosophila gene expression and development.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and whether they have any SAGA-independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Loss of the histone acetyltransferase (HAT) activity blocks oogenesis, while loss of the H2B deubiquitinase (DUB) activity does not. However, the DUB module regulates a subset of genes in early embryogenesis, and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the HAT module. Our results suggest that the DUB module has functions within SAGA and independent functions.

The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters.

The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. We show that depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. Our data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.

37/67-laminin receptor facilitates neural crest cell migration during enteric nervous system development.

Enteric nervous system (ENS) development is governed by interactions between neural crest cells (NCC) and the extracellular matrix (ECM). Hirschsprung disease (HSCR) results from incomplete NCC migration and failure to form an appropriate ENS. Prior studies implicate abnormal ECM in NCC migration failure. We performed a comparative microarray of the embryonic distal hindgut of wild-type and EdnrBNCC-/- mice that model HSCR and identified laminin-ß1 as upregulated in EdnrBNCC-/- colon. We identified decreased expression of 37/67 kDa laminin receptor (LAMR), which binds laminin-ß1, in human HSCR myenteric plexus and EdnrBNCC-/- NCC. Using a combination of in vitro gut slice cultures and ex vivo organ cultures, we determined the mechanistic role of LAMR in NCC migration. We found that enteric NCC express LAMR, which is downregulated in human and murine HSCR. Binding of LAMR by the laminin-ß1 analog YIGSR promotes NCC migration. Silencing of LAMR abrogated these effects. Finally, applying YIGSR to E13.5 EdnrBNCC-/- colon explants resulted in 80%-100% colonization of the hindgut. This study adds LAMR to the large list of receptors through which NCC interact with their environment during ENS development. These results should be used to inform ongoing integrative, regenerative medicine approaches to HSCR.

Characterization of a metazoan ADA acetyltransferase complex.

The Gcn5 acetyltransferase functions in multiple acetyltransferase complexes in yeast and metazoans. Yeast Gcn5 is part of the large SAGA (Spt-Ada-Gcn5 acetyltransferase) complex and a smaller ADA acetyltransferase complex. In flies and mammals, Gcn5 (and its homolog pCAF) is part of various versions of the SAGA complex and another large acetyltransferase complex, ATAC (Ada2A containing acetyltransferase complex). However, a complex analogous to the small ADA complex in yeast has never been described in metazoans. Previous studies in Drosophila hinted at the existence of a small complex which contains Ada2b, a partner of Gcn5 in the SAGA complex. Here we have purified and characterized the composition of this complex and show that it is composed of Gcn5, Ada2b, Ada3 and Sgf29. Hence, we have named it the metazoan 'ADA complex'. We demonstrate that the fly ADA complex has histone acetylation activity on histones and nucleosome substrates. Moreover, ChIP-Sequencing experiments identified Ada2b peaks that overlap with another SAGA subunit, Spt3, as well as Ada2b peaks that do not overlap with Spt3 suggesting that the ADA complex binds chromosomal sites independent of the larger SAGA complex.

Schizosaccharomyces pombe Pol II transcription elongation factor ELL functions as part of a rudimentary super elongation complex.

ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.

Codependency of H2B monoubiquitination and nucleosome reassembly on Chd1.

Monoubiquitination of histone H2B on Lys 123 (H2BK123ub) is a transient histone modification considered to be essential for establishing H3K4 and H3K79 trimethylation by Set1/COMPASS and Dot1, respectively. Here, we identified Chd1 as a factor that is required for the maintenance of high levels of H2B monoubiquitination, but not for H3K4 and H3K79 trimethylation. Loss of Chd1 results in a substantial loss of H2BK123ub levels with little to no effect on the genome-wide pattern of H3K4 and H3K79 trimethylation. Our data show that nucleosomal occupancy is reduced in gene bodies in both chd1Δ and, as has been shown, K123A mutant backgrounds. We also demonstrated that Chd1's function in maintaining H2BK123ub levels is conserved from yeast to humans. Our study provides evidence that only small levels of H2BK123ub are necessary for full levels of H3K4 and H3K79 trimethylation in vivo and points to a possible role for Chd1 in positively regulating gene expression through promoting nucleosome reassembly coupled with H2B monoubiquitination.

The little elongation complex regulates small nuclear RNA transcription.

Eleven-nineteen lysine-rich leukemia (ELL) participates in the super elongation complex (SEC) with the RNA polymerase II (Pol II) CTD kinase P-TEFb. SEC is a key regulator in the expression of HOX genes in mixed lineage leukemia (MLL)-based hematological malignancies, in the control of induced gene expression early in development, and in immediate early gene transcription. Here, we identify an SEC-like complex in Drosophila, as well as a distinct ELL-containing complex that lacks P-TEFb and other components of SEC named the "little elongation complex" (LEC). LEC subunits are highly enriched at RNA Pol II-transcribed small nuclear RNA (snRNA) genes, and the loss of LEC results in decreased snRNA expression in both flies and mammals. The specialization of the SEC and LEC complexes for mRNA and snRNA-containing genes, respectively, suggests the presence of specific classes of elongation factors for each class of genes transcribed by RNA polymerase II.

Dynamic transcriptional events in embryonic stem cells mediated by the super elongation complex (SEC).

Transcriptional regulation of developmentally controlled genes is at the heart of differentiation and organogenesis. In this study, we performed global genomic analyses in murine embryonic stem (ES) cells and in human cells in response to activation signals. We identified an essential role for the ELL (eleven-nineteen lysine-rich leukemia gene)/P-TEFb (positive transcription elongation factor)-containing super elongation complex (SEC) in the regulation of gene expression, including several genes bearing paused RNA polymerase II (Pol II). Paused Pol II has been proposed to be associated with loci that respond rapidly to environmental stimuli. However, our studies in ES cells also identified a requirement for SEC at genes without paused Pol II, which also respond dynamically to differentiation signals. Our findings suggest that SEC is a major class of active P-TEFb-containing complexes required for transcriptional activation in response to environmental cues such as differentiation signals.

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

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 5' 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.

Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells.

The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line.

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/ß-catenin signaling following hair cell death. We propose that Wnt/ß-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.

Mechanisms of tentacle morphogenesis in the sea anemone Nematostella vectensis.

Evolution of the capacity to form secondary outgrowths from the principal embryonic axes was a crucial innovation that potentiated the diversification of animal body plans. Precisely how such outgrowths develop in early-branching metazoan species remains poorly understood. Here we demonstrate that three fundamental processes contribute to embryonic tentacle development in the cnidarian Nematostella vectensis. First, a pseudostratified ectodermal placode forms at the oral pole of developing larvae and is transcriptionally patterned into four tentacle buds. Subsequently, Notch signaling-dependent changes in apicobasal epithelial thickness drive elongation of these primordia. In parallel, oriented cell rearrangements revealed by clonal analysis correlate with shaping of the elongating tentacles. Taken together, our results define the mechanism of embryonic appendage development in an early-branching metazoan, and thereby provide a novel foundation for understanding the diversification of body plans during animal evolution.

Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye.

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to cross-talk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.

Molecular evolution of the Yap/Yorkie proto-oncogene and elucidation of its core transcriptional program.

Throughout Metazoa, developmental processes are controlled by a surprisingly limited number of conserved signaling pathways. Precisely how these signaling cassettes were assembled in early animal evolution remains poorly understood, as do the molecular transitions that potentiated the acquisition of their myriad developmental functions. Here we analyze the molecular evolution of the proto-oncogene yes-associated protein (Yap)/Yorkie, a key effector of the Hippo signaling pathway that controls organ size in both Drosophila and mammals. Based on heterologous functional analysis of evolutionarily distant Yap/Yorkie orthologs, we demonstrate that a structurally distinct interaction interface between Yap/Yorkie and its partner TEAD/Scalloped became fixed in the eumetazoan common ancestor. We then combine transcriptional profiling of tissues expressing phylogenetically diverse forms of Yap/Yorkie with ChIP-seq validation to identify a common downstream gene expression program underlying the control of tissue growth in Drosophila. Intriguingly, a subset of the newly identified Yorkie target genes are also induced by Yap in mammalian tissues, thus revealing a conserved Yap-dependent gene expression signature likely to mediate organ size control throughout bilaterian animals. Combined, these experiments provide new mechanistic insights while revealing the ancient evolutionary history of Hippo signaling.

Antagonism of PDGF-BB suppresses subretinal neovascularization and enhances the effects of blocking VEGF-A.

Hypoxia-inducible factor-1 (HIF-1) plays an important role in retinal and subretinal neovascularization (NV). Increased levels of HIF-1 cause increased expression of vascular endothelial growth factor (VEGF-A) and current therapies for ocular NV focus on neutralizing VEGF-A, but there is mounting evidence that other HIF-1-responsive gene products may also participate. In this study, we tested the effect of a designed ankyrin repeat protein (DARPin) that selectively binds and antagonizes the hypoxia-regulated gene product PDGF-BB in three models of subretinal NV (relevant to neovascular age-related macular degeneration) and compared its effects to a DARPin that selectively antagonizes VEGF-A. Daily intraperitoneal injections of 10 mg/kg of the anti-PDGF-BB DARPin or 1 mg/kg of the anti-VEGF DARPin significantly suppressed subretinal NV from laser-induced rupture of Bruch's membrane. Injections of 1 mg/kg/day of the anti-PDGF-BB DARPin had no significant effect, but when combined with 1 mg/kg/day of the anti-VEGF-A DARPin there was greater suppression than injection of the anti-VEGF-A DARPin alone. In Vldlr (-/-) mice which spontaneously develop subretinal NV, intraocular injection of 1.85 µg of anti-PDGF-BB or anti-VEGF-A DARPin caused significant suppression of NV and when combined there was greater suppression than with either alone. The two DARPins also showed an additive effect in Tet/opsin/VEGF double transgenic mice, a particularly severe model of subretinal NV and exudative retinal detachment. In addition, intraocular injection of 1.85 µg of anti-PDGF-BB DARPin strongly suppressed ischemia-induced retinal NV, which is relevant to proliferative diabetic retinopathy and retinopathy of prematurity. These data demonstrate that PDGF-BB is another hypoxia-regulated gene product that along with VEGF-A contributes to ocular NV and suppression of both provides an additive effect.

Nuclear cGMP-dependent kinase regulates gene expression via activity-dependent recruitment of a conserved histone deacetylase complex.

Elevation of the second messenger cGMP by nitric oxide (NO) activates the cGMP-dependent protein kinase PKG, which is key in regulating cardiovascular, intestinal, and neuronal functions in mammals. The NO-cGMP-PKG signaling pathway is also a major therapeutic target for cardiovascular and male reproductive diseases. Despite widespread effects of PKG activation, few molecular targets of PKG are known. We study how EGL-4, the Caenorhabditis elegans PKG ortholog, modulates foraging behavior and egg-laying and seeks the downstream effectors of EGL-4 activity. Using a combination of unbiased forward genetic screen and proteomic analysis, we have identified a conserved SAEG-1/SAEG-2/HDA-2 histone deacetylase complex that is specifically recruited by activated nuclear EGL-4. Gene expression profiling by microarrays revealed >40 genes that are sensitive to EGL-4 activity in a SAEG-1-dependent manner. We present evidence that EGL-4 controls egg laying via one of these genes, Y45F10C.2, which encodes a novel protein that is expressed exclusively in the uterine epithelium. Our results indicate that, in addition to cytoplasmic functions, active EGL-4/PKG acts in the nucleus via a conserved Class I histone deacetylase complex to regulate gene expression pertinent to behavioral and physiological responses to cGMP. We also identify transcriptional targets of EGL-4 that carry out discrete components of the physiological response.

Suppression of GLUT1; a new strategy to prevent diabetic complications.

High blood glucose results in high glucose levels in retina, because GLUT1, the sole glucose transporter between blood and retina, transports more glucose when blood glucose is high. This is the ultimate cause of diabetic retinopathy. Knockdown of GLUT1 by intraocular injections of a pool of siRNAs directed against SLC2A1 mRNA which codes for GLUT1 significantly reduced mean retinal glucose levels in diabetic mice. Systemic treatment of diabetic mice with forskolin or genistein, which bind GLUT1 and inhibit glucose transport, significantly reduced retinal glucose to the same levels seen in non-diabetics. 1,9-Dideoxyforskolin, which binds GLUT1 but does not stimulate adenylate cyclase had an equivalent effect to that of forskolin regarding lowering retinal glucose in diabetics indicating that cyclic AMP is noncontributory. GLUT1 inhibitors also reduced glucose and glycohemoglobin levels in red blood cells providing a peripheral biomarker for the effect. In contrast, brain glucose levels were not increased in diabetics and not reduced by forskolin. Treatment of diabetics with forskolin prevented early biomarkers of diabetic retinopathy, including elevation of superoxide radicals, increased expression of the chaperone protein ß2 crystallin, and increased expression of vascular endothelial growth factor (VEGF). These data identify GLUT1 as a promising therapeutic target for prevention of diabetic retinopathy.

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.

Delayed correlation of mRNA and protein expression in rapamycin-treated cells and a role for Ggc1 in cellular sensitivity to rapamycin.

To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Deltaggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression under TOR control.