Participation in a High-Structure General Chemistry Course Increases Student Sense of Belonging and Persistence to Organic Chemistry.

A parallel series of general chemistry courses for Life Science Majors was created in an effort to support students and improve general chemistry outcomes. We created a two-quarter enhanced general chemistry course series that is not remedial, but instead implements several evidence-based teaching practices including Process Oriented Guided Inquiry Learning (POGIL), Peer-Led Team Learning (PLTL), and the Learning Assistant (LA) model. We found that students who took enhanced general chemistry had higher persistence to the subsequent first organic chemistry course, and performed equally well in the organic course compared to their peers who took standard general chemistry. Students in the first enhanced general chemistry course also reported significantly higher belonging, although we were unable to determine if increased belonging was associated with the increased persistence to organic chemistry. Rather we found that the positive association between taking the enhanced general chemistry course and persistence to organic chemistry was mediated by higher grades received in the enhanced general chemistry course. Our findings highlight the responsibility we have as educators to carefully consider the pedagogical practices we use, in addition to how we assign student grades.

SUMO Interacting Motifs: Structure and Function.

Small ubiquitin-related modifier (SUMO) is a member of the ubiquitin-related protein family. SUMO modulates protein function through covalent conjugation to lysine residues in a large number of proteins. Once covalently conjugated to a protein, SUMO often regulates that protein's function by recruiting other cellular proteins. Recruitment frequently involves a non-covalent interaction between SUMO and a SUMO-interacting motif (SIM) in the interacting protein. SIMs generally consist of a four-residue-long hydrophobic stretch of amino acids with aliphatic non-polar side chains flanked on one side by negatively charged amino acid residues. The SIM assumes an extended ß-strand-like conformation and binds to a conserved hydrophobic groove in SUMO. In addition to hydrophobic interactions between the SIM non-polar core and hydrophobic residues in the groove, the negatively charged residues in the SIM make favorable electrostatic contacts with positively charged residues in and around the groove. The SIM/SUMO interaction can be regulated by the phosphorylation of residues adjacent to the SIM hydrophobic core, which provide additional negative charges for favorable electrostatic interaction with SUMO. The SUMO interactome consists of hundreds or perhaps thousands of SIM-containing proteins, but we do not fully understand how each SUMOylated protein selects the set of SIM-containing proteins appropriate to its function. SIM/SUMO interactions have critical functions in a large number of essential cellular processes including the formation of membraneless organelles by liquid-liquid phase separation, epigenetic regulation of transcription through histone modification, DNA repair, and a variety of host-pathogen interactions.

SUMOylation in development and neurodegeneration.

In essentially all eukaryotes, proteins can be modified by the attachment of small ubiquitin-related modifier (SUMO) proteins to lysine side chains to produce branched proteins. This process of 'SUMOylation' plays essential roles in plant and animal development by altering protein function in spatially and temporally controlled ways. In this Primer, we explain the process of SUMOylation and summarize how SUMOylation regulates a number of signal transduction pathways. Next, we discuss multiple roles of SUMOylation in the epigenetic control of transcription. In addition, we evaluate the role of SUMOylation in the etiology of neurodegenerative disorders, focusing on Parkinson's disease and cerebral ischemia. Finally, we discuss the possibility that SUMOylation may stimulate survival and neurogenesis of neuronal stem cells.

Mechanisms of Groucho-mediated repression revealed by genome-wide analysis of Groucho binding and activity.

BACKGROUND: The transcriptional corepressor Groucho (Gro) is required for the function of many developmentally regulated DNA binding repressors, thus helping to define the gene expression profile of each cell during development. The ability of Gro to repress transcription at a distance together with its ability to oligomerize and bind to histones has led to the suggestion that Gro may spread along chromatin. However, much is unknown about the mechanism of Gro-mediated repression and about the dynamics of Gro targeting. RESULTS: Our chromatin immunoprecipitation sequencing analysis of temporally staged Drosophila embryos shows that Gro binds in a highly dynamic manner primarily to clusters of discrete (<1 kb) segments. Consistent with the idea that Gro may facilitate communication between silencers and promoters, Gro binding is enriched at both cis-regulatory modules, as well as within the promotors of potential target genes. While this Gro-recruitment is required for repression, our data show that it is not sufficient for repression. Integration of Gro binding data with transcriptomic analysis suggests that, contrary to what has been observed for another Gro family member, Drosophila Gro is probably a dedicated repressor. This analysis also allows us to define a set of high confidence Gro repression targets. Using publically available data regarding the physical and genetic interactions between these targets, we are able to place them in the regulatory network controlling development. Through analysis of chromatin associated pre-mRNA levels at these targets, we find that genes regulated by Gro in the embryo are enriched for characteristics of promoter proximal paused RNA polymerase II. CONCLUSIONS: Our findings are inconsistent with a one-dimensional spreading model for long-range repression and suggest that Gro-mediated repression must be regulated at a post-recruitment step. They also show that Gro is likely a dedicated repressor that sits at a prominent highly interconnected regulatory hub in the developmental network. Furthermore, our findings suggest a role for RNA polymerase II pausing in Gro-mediated repression.

SUMO in Drosophila Development.

The ubiquitin -like protein SUMO is conjugated covalently to hundreds of target proteins in organisms throughout the eukaryotic domain. Genetic and biochemical studies using the model organism Drosophila melanogaster are beginning to reveal many essential functions for SUMO in cell biology and development. For example, SUMO regulates multiple signaling pathways such as the Ras/MAPK, Dpp, and JNK pathways. In addition, SUMO regulates transcription through conjugation to many transcriptional regulatory proteins, including Bicoid, Spalt , Scm, and Groucho. In some cases, conjugation of SUMO to a target protein inhibits its normal activity, while in other cases SUMO conjugation stimulates target protein activity. SUMO often modulates a biological process by altering the subcellular localization of a target protein. The ability of SUMO and other ubiquitin-like proteins to diversify protein function may be critical to the evolution of developmental complexity.

The Central Region of the Drosophila Co-repressor Groucho as a Regulatory Hub.

Groucho (Gro) is a Drosophila co-repressor that regulates the expression of a large number of genes, many of which are involved in developmental control. Previous studies have shown that its central region is essential for function even though its three domains are poorly conserved and intrinsically disordered. Using these disordered domains as affinity reagents, we have now identified multiple embryonic Gro-interacting proteins. The interactors include protein complexes involved in chromosome organization, mRNA processing, and signaling. Further investigation of the interacting proteins using a reporter assay showed that many of them modulate Gro-mediated repression either positively or negatively. The positive regulators include components of the spliceosomal subcomplex U1 small nuclear ribonucleoprotein (U1 snRNP). A co-immunoprecipitation experiment confirms this finding and suggests that a sizable fraction of nuclear U1 snRNP is associated with Gro. The use of RNA-seq to analyze the gene expression profile of cells subjected to knockdown of Gro or snRNP-U1-C (a component of U1 snRNP) showed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data with Gro and RNA polymerase II ChIP data led to a number of insights, including the finding that Gro-repressed genes are enriched for promoter-proximal RNA polymerase II. We conclude that the Gro central domains mediate multiple interactions required for repression, thus functioning as a regulatory hub. Furthermore, interactions with the spliceosome may contribute to repression by Gro.

SUMO as a solubility tag and in vivo cleavage of SUMO fusion proteins with Ulp1.

Expression of proteins in E. coli is often plagued by insolubility of the protein of interest. A solution to this problem is the expression of proteins as fusions to solubility tags such as the SUMO protein. SUMO fusion proteins can be cleaved to remove the SUMO moiety using SUMO-specific proteases such as Ulp1. Here, we describe the use of vectors for the expression of recombinant proteins in E. coli as fusions to the Drosophila SUMO protein. This includes a vector that encodes not only the SUMO tagged protein of interest but also SUMO-tagged Ulp1. Coexpression of these two proteins results in the in vivo cleavage of the protein of interest from the SUMO tag, while still leaving the protein of interest in a form that can be purified from a soluble cell lysate by nickel affinity chromatography.

Groucho: a corepressor with instructive roles in development.

Drosophila Groucho (Gro) is the founding member of a family of metazoan corepressors. Gro mediates repression through interactions with a myriad of DNA-binding repressor proteins to direct the silencing of genes involved in many developmental processes, including neurogenesis and patterning of the main body axis, as well as receptor tyrosine kinase/Ras/MAPK, Notch, Wingless (Wg)/Wnt, and Decapentaplegic (Dpp) signaling. Gro mediates repression by multiple molecular mechanisms, depending on the regulatory context. Because Gro is a broadly expressed nuclear factor, whereas its repressor partners display restricted temporal and spatial distribution, it was presumed that this corepressor played permissive rather than instructive roles in development. However, a wide range of studies demonstrates that this is not the case. Gro can sense and integrate many cellular inputs to modulate the expression of variety of genes, making it a versatile corepressor with crucial instructive roles in development and signaling.

The unconserved groucho central region is essential for viability and modulates target gene specificity.

Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding repressors, many of which are distributed in gradients and provide positional information during development. Gro contains well-conserved domains at its N- and C-termini, and a poorly conserved central region that includes the GP, CcN, and SP domains. All lethal point mutations in gro map to the conserved regions, leading to speculation that the unconserved central domains are dispensable. However, our sequence analysis suggests that the central domains are disordered leading us to suspect that the lack of lethal mutations in this region reflects a lack of order rather than an absence of essential functions. In support of this conclusion, genomic rescue experiments with Gro deletion variants demonstrate that the GP and CcN domains are required for viability. Misexpression assays using these same deletion variants show that the SP domain prevents unrestrained and promiscuous repression by Gro, while the GP and CcN domains are indispensable for repression. Deletion of the GP domain leads to loss of nuclear import, while deletion of the CcN domain leads to complete loss of repression. Changes in Gro activity levels reset the threshold concentrations at which graded repressors silence target gene expression. We conclude that co-regulators such as Gro are not simply permissive components of the repression machinery, but cooperate with graded DNA-binding factors in setting borders of gene expression. We suspect that disorder in the Gro central domains may provide the flexibility that allows this region to mediate multiple interactions required for repression.

SUMOylation in Drosophila Development.

Small ubiquitin-related modifier (SUMO), an ~90 amino acid ubiquitin-like protein, is highly conserved throughout the eukaryotic domain. Like ubiquitin, SUMO is covalently attached to lysine side chains in a large number of target proteins. In contrast to ubiquitin, SUMO does not have a direct role in targeting proteins for proteasomal degradation. However, like ubiquitin, SUMO does modulate protein function in a variety of other ways. This includes effects on protein conformation, subcellular localization, and protein-protein interactions. Significant insight into the in vivo role of SUMOylation has been provided by studies in Drosophila that combine genetic manipulation, proteomic, and biochemical analysis. Such studies have revealed that the SUMO conjugation pathway regulates a wide variety of critical cellular and developmental processes, including chromatin/chromosome function, eggshell patterning, embryonic pattern formation, metamorphosis, larval and pupal development, neurogenesis, development of the innate immune system, and apoptosis. This review discusses our current understanding of the diverse roles for SUMO in Drosophila development.

Small ubiquitin-like modifier (SUMO) conjugation impedes transcriptional silencing by the polycomb group repressor Sex Comb on Midleg.

The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. Recent findings have identified PcG proteins both as targets for modification by the small ubiquitin-like modifier (SUMO) protein and as catalytic components of the SUMO conjugation pathway. We have found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of sumoylation augments binding of Scm to the PRE. This is likely to be a direct effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm enhance its recruitment to the PRE, whereas translational fusion of SUMO to the Scm N terminus interferes with this recruitment. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced, we observe decreased expression of Ubx and partial haltere-to-wing transformation phenotypes. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE.

Capicua DNA-binding sites are general response elements for RTK signaling in Drosophila.

RTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMG-box factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.

A SUMO-Groucho Q domain fusion protein: characterization and in vivo Ulp1-mediated cleavage.

We describe here a system for the expression and purification of small ubiquitin-related modifier (SUMO) fusion proteins, which often exhibit dramatically increased solubility and stability during expression in bacteria relative to unfused proteins. The vector described here allows expression of a His-tagged protein of interest fused at its N-terminus to SUMO. Using this vector, we have produced a polypeptide consisting of SUMO fused to the Q domain of Drosophila Groucho in a concentrated soluble form. Hydrodynamic analysis shows that, consistent with previous studies on full-length Groucho, the fusion protein forms an elongated tetramer, as well as higher order oligomers. After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, we have constructed a dual expression vector encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO-Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage.

Groucho-mediated repression may result from a histone deacetylase-dependent increase in nucleosome density.

Groucho (Gro) is a Drosophila melanogaster transcriptional corepressor that directly interacts with the histone deacetylase Rpd3. Although previous studies suggest that this interaction is required for repression of Gro-responsive reporters in cultured cells, the in vivo significance of this interaction and the mechanism by which it leads to repression remain largely unexplored. In this study, we show that Gro is partially dependent on Rpd3 for repression, supporting the idea that Rpd3-mediated repression is one mode of Gro-mediated repression. We demonstrate that Gro colocalizes with Rpd3 to the chromatin of a target gene and that this is accompanied by the deacetylation of specific lysines within the N-terminal tails of histones H3 and H4. Gro overexpression leads to wing patterning defects and ectopic repression in the wing disc of transcription directed by the vestigial quadrant enhancer. These effects are reversed by the histone deacetylase inhibitors TSA and HC-Toxin and by the reduction of Rpd3 gene dosage. Furthermore, repression of the vestigial quadrant enhancer is accompanied by a Gro-mediated increase in nucleosome density, an effect that is reversed by histone deacetylase inhibitors. We propose a model in which Gro-mediated histone deacetylation results in increased nucleosome density leading to transcriptional repression.

Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation.

SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

Non-cell-autonomous inhibition of photoreceptor development by Dip3.

We show here that the Drosophila MADF/BESS domain transcription factor Dip3, which is expressed in differentiating photoreceptors, regulates neuronal differentiation in the compound eye. Loss of Dip3 activity in photoreceptors leads to an extra photoreceptor in many ommatidia, while ectopic expression of Dip3 in non-neuronal cells results in photoreceptor loss. These findings are consistent with the idea that Dip3 is required non-cell autonomously to block extra photoreceptor formation. Dip3 may mediate the spatially restricted potentiation of Notch (N) signaling since the Dip3 misexpression phenotype is suppressed by reducing N signaling and misexpression of Dip3 leads to ectopic activity of a N-responsive enhancer. Analysis of mosaic ommatidia suggests that no specific photoreceptor must be mutant to generate the mutant phenotype. Remarkably, however, mosaic pupal ommatidia with three or fewer Dip3(+) photoreceptors always differentiate an extra photoreceptor, while those with four or more Dip3(+) photoreceptors never differentiate an extra photoreceptor. These findings are consistent with the notion that Dip3 in photoreceptors activates a heretofore unsuspected diffusible ligand that may work in conjunction with the N pathway to prevent a subpopulation of undifferentiated cells from choosing a neuronal fate.

Dorsal interacting protein 3 potentiates activation by Drosophila Rel homology domain proteins.

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal Rel homology domain. We show here that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters.

Transformation of eye to antenna by misexpression of a single gene.

In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance our understanding of the mechanisms that restrict stem cell fate. We show here that Dip3, a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and have taken advantage of this observation to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, our results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae.

Uncoupling dorsal-mediated activation from dorsal-mediated repression in the Drosophila embryo.

The Rel family transcription factor Dorsal patterns the dorsoventral axis of the Drosophila embryo by activating genes such as twist and snail and repressing genes such as decapentaplegic and zerknüllt. Dorsal represses transcription by recruiting the co-repressor Groucho. However, repression occurs only when Dorsal-binding sites are close to binding sites for other factors that also bind Groucho. The need for additional factors to assist Dorsal in repression may result from the intrinsically weak interaction between Dorsal and Groucho. To test this idea, we generated a Dorsal variant containing a high-affinity Groucho recruitment motif at its C terminus. As predicted, this variant functions as a dedicated repressor, silencing decapentaplegic and zerknüllt while failing to activate twist and snail. We also converted Dorsal into a dedicated activator by replacing its weak Groucho-recruitment motif with heterologous activation domains. Although the dedicated activator alleles fail to repress decapentaplegic and zerknüllt in the syncytial blastoderm embryo, they are able to pattern the dorsoventral axis. This indicates that dorsoventral patterning is not dependent upon Dorsal-mediated repression, reflecting the existence of redundant mechanisms to block Decapentaplegic signaling.