USP13 interacts with cohesin and regulates its ubiquitination in human cells.

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

Genome-wide siRNA screen reveals coupling between mitotic apoptosis and adaptation.

The antimitotic anti-cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint-dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome-wide small interfering RNA (siRNA) screen in taxol-treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31(comet) actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro-apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.

The Cdc20-binding Phe box of the spindle checkpoint protein BubR1 maintains the mitotic checkpoint complex during mitosis.

The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.

Responsible and ethical conduct of research (RECR) diagnostic survey using case scenarios from biology course-based undergraduate research experiences (CUREs).

Course-based undergraduate research experiences (CUREs) are increasingly becoming the first, and perhaps only, research experience for many biology students. Responsible and ethical conduct of research (RECR) is crucial for the integrity of scientific research and essential for students to have an understanding of the scientific process at any academic level. However, there is a current lack of RECR education in biology CUREs. To understand the level of RECR knowledge and skills in undergraduate students, we created a diagnostic survey that uses case scenarios designed to illustrate RECR issues in the CURE classroom. Analysis of students' responses indicated that the overall percentage of students who are able to effectively use RECR terminology and identify the impact of RECR violations on science integrity and ultimately on society is low. Furthermore, some students equated RECR violations to academic dishonesty, indicating difficulties separating the research and academic aspects of CUREs. This diagnostic tool can aid instructors in identifying gaps in student RECR knowledge for the subsequent development of RECR educational interventions, particularly to ensure the integrity of the research performed in CURE settings.

Institution-Wide Analysis of Academic Outcomes Associated with Participation in UGR: Comparison of Different Research Modalities at a Hispanic-Serving Institution.

Most studies on the benefits of participation in undergraduate research (UGR) use data from student participants in undergraduate research programs (URPs), which offer a limited number of positions. In reality, however, the majority of UGR students participate in undergraduate research not in programs (URNPs). The authors conducted an institution-wide study at a Hispanic-serving institution to examine the relationship between academic success and participation in these two UGR modalities. Although there were some differences between URPs and URNPs, participation in research at this institution was largely equitable and inclusive, with UGR demographics that reflected those of the institution, and it was positively associated with increased benefits along multiple academic metrics, regardless of UGR modality. Importantly, these increases were observed for both first time in college and transfer students.

Current Approaches for Integrating Responsible and Ethical Conduct of Research (RECR) Education into Course-based Undergraduate Research Experiences: A National Assessment.

Course-based undergraduate research experiences (CUREs), which often engage students as early as freshman year, have become increasingly common in biology curricula. While many studies have highlighted the benefits of CUREs, little attention has been paid to responsible and ethical conduct of research (RECR) education in such contexts. Given this observation, we adopted a mixed methods approach to explore the extent to which RECR education is being implemented and assessed in biological sciences CUREs nationwide. Survey and semistructured interview data show a general awareness of the importance of incorporating RECR education into CUREs, with all respondents addressing at least one RECR topic in their courses. However, integration of RECR education within the CURE environment primarily focuses on the application of RECR during research practice, often takes the form of corrective measures, and appears to be rarely assessed. Participants reported lack of time and materials as the main barriers to purposeful inclusion of RECR education within their courses. These results underscore a need for the CURE community to develop resources and effective models to integrate RECR education into biology CUREs.

Recommendations for Effective Integration of Ethics and Responsible Conduct of Research (E/RCR) Education into Course-Based Undergraduate Research Experiences: A Meeting Report.

Advancement of the scientific enterprise relies on individuals conducting research in an ethical and responsible manner. Educating emergent scholars in the principles of ethics/responsible conduct of research (E/RCR) is therefore critical to ensuring such advancement. The recent impetus to include authentic research opportunities as part of the undergraduate curriculum, via course-based undergraduate research experiences (CUREs), has been shown to increase cognitive and noncognitive student outcomes. Because of these important benefits, CUREs are becoming more common and often constitute the first research experience for many students. However, despite the importance of E/RCR in the research process, we know of few efforts to incorporate E/RCR education into CUREs. The Ethics Network for Course-based Opportunities in Undergraduate Research (ENCOUR) was created to address this concern and promote the integration of E/RCR within CUREs in the biological sciences and related disciplines. During the inaugural ENCOUR meeting, a four-pronged approach was used to develop guidelines for the effective integration of E/RCR in CUREs. This approach included: 1) defining appropriate student learning objectives; 2) identifying relevant curriculum; 3) identifying relevant assessments; and 4) defining key aspects of professional development for CURE facilitators. Meeting outcomes, including the aforementioned E/RCR guidelines, are described herein.

UBL/UBA ubiquitin receptor proteins bind a common tetraubiquitin chain.

The ubiquitin-proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.

Integration of RCR and Ethics Education into Course-Based Undergraduate Research Experiences in the Biological Sciences: A Needed Discussion.

Course-based undergraduate research experiences (CUREs) have been identified as a promising vehicle to broaden novices' participation in authentic scientific opportunities. While recent studies in the bioeducation literature have focused on the influence of CUREs on cognitive and non-cognitive student outcomes (e.g., attitudes and motivation, science process skills development), few investigations have examined the extent to which the contextual features inherent in such experiences affect students' academic and professional growth. Central among these factors is that of ethics and the responsible conduct of research (RCR)-essential cornerstones of the scientific enterprise. In this article, we examine the intersectionality of ethics/RCR instruction within CURE contexts through a critical review of existing literature that details mechanisms for the integration of ethics/RCR education into undergraduate laboratory experiences in the science domains. Building upon this foundation, we propose a novel, evidence-based framework that seeks to illustrate posited interactions between core ethics/RCR principles and unique dimensions of CUREs. It is our intent that this framework will inform and encourage open dialogue around an often-overlooked aspect of CURE instruction-how to best prepare ethically responsible scholars for entrance into the global scientific workforce.

The complexity of life and death decisions in mitosis.

The anticancer drug taxol stabilizes microtubules and activates the spindle checkpoint, causing prolonged mitotic arrest in cancer cells. Our recent work suggests that the cellular decision to live or die following mitotic arrest is a complex process involving crosstalk between competing apoptotic and adaptation pathways.

Graded requirement for the spliceosome in cell cycle progression.

Genome stability is ensured by multiple surveillance mechanisms that monitor the duplication, segregation, and integrity of the genome throughout the cell cycle. Depletion of components of the spliceosome, a macromolecular machine essential for mRNA maturation and gene expression, has been associated with increased DNA damage and cell cycle defects. However, the specific role for the spliceosome in these processes has remained elusive, as different cell cycle defects have been reported depending on the specific spliceosome subunit depleted. Through a detailed cell cycle analysis after spliceosome depletion, we demonstrate that the spliceosome is required for progression through multiple phases of the cell cycle. Strikingly, the specific cell cycle phenotype observed after spliceosome depletion correlates with the extent of depletion. Partial depletion of a core spliceosome component results in defects at later stages of the cell cycle (G2 and mitosis), whereas a more complete depletion of the same component elicits an early cell cycle arrest in G1. We propose a quantitative model in which different functional dosages of the spliceosome are required for different cell cycle transitions.

Mutational inactivation of STAG2 causes aneuploidy in human cancer.

Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.

Determinants of Rad21 localization at the centrosome in human cells.

Cohesin proteins help maintain the physical associations between sister chromatids that arise in S-phase and are removed in anaphase. Recent studies found that cohesins also localize to the centrosomes, the organelles that organize the mitotic bipolar spindle. We find that the cohesin protein Rad21 localizes to centrosomes in a manner that is dependent upon known regulators of sister chromatid cohesion as well as regulators of centrosome function. These data suggest that Rad21 functions at the centrosome and that the regulators of Rad21 coordinate the centrosome and chromosomal functions of cohesin.

Cohesin is needed for bipolar mitosis in human cells.

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.

Rad21 is required for centrosome integrity in human cells independently of its role in chromosome cohesion.

Classically, chromosomal functions in DNA repair and sister chromatid association have been assigned to the cohesin proteins. More recent studies have provided evidence that cohesins also localize to the centrosomes, which organize the bipolar spindle during mitosis. Depletion of cohesin proteins is associated with multi-polar mitosis in which spindle pole integrity is compromised. However, the spindle pole defects after cohesin depletion could be an indirect consequence of a chromosomal cohesion defect which might impact centrosome integrity via alterations to the spindle microtubule network. Here we show that the cohesin Rad21 is required for centrosome integrity independently of its role as a chromosomal cohesin. Thus, Rad21 may promote accurate chromosome transmission not only by virtue of its function as a chromosomal cohesin, but also because it is required for centrosome function.

Cohesin: a multi-purpose chromatin glue.

Long thought to be the glue responsible for holding sister chromatids together, cohesin has been found to be stickier than previously thought. Recent discoveries point to cohesin having a role in transcription regulation by mediating long-distance intra-chromosomal interactions.

Chromosome cohesion and the spindle checkpoint.

Accurate chromosome segregation constitutes the basis of inheritance. Mistakes in chromosome segregation during mitosis lead to aneuploidy, a common feature of tumors. The accuracy of chromosome segregation is governed by a complex network of processes which ensure that each daughter cell receives the correct number of chromosomes. Herein we review recent developments in the understanding of chromosome segregation, focusing on the cohesion that holds the sister chromatids together and the spindle checkpoint which regulates anaphase onset.

Multiple anaphase-promoting complex/cyclosome degrons mediate the degradation of human Sgo1.

Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early mitosis and, thus, prevents premature sister-chromatid separation. The protein level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is down-regulated in G1/S. Here we show that Sgo1 is degraded during the exit from mitosis, and its degradation depends on the anaphase-promoting complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C bound to Cdh1 (APC/C(Cdh1)) in vitro. We have further identified two functional degradation motifs in Sgo1; that is, a KEN (Lys-Glu-Asn) box and a destruction box (D box). Although removal of either motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box deleted is stable in cells. Surprisingly, mitosis progresses normally in the presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not required for sister-chromatid separation or mitotic exit. Finally, we show that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1 steady-state protein levels in an APC/C-independent mechanism.