Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.

USP22 controls multiple signaling pathways that are essential for vasculature formation in the mouse placenta.

USP22, a component of the SAGA complex, is overexpressed in highly aggressive cancers, but the normal functions of this deubiquitinase are not well defined. We determined that loss of USP22 in mice results in embryonic lethality due to defects in extra-embryonic placental tissues and failure to establish proper vascular interactions with the maternal circulatory system. These phenotypes arise from abnormal gene expression patterns that reflect defective kinase signaling, including TGFß and several receptor tyrosine kinase pathways. USP22 deletion in endothelial cells and pericytes that are induced from embryonic stem cells also hinders these signaling cascades, with detrimental effects on cell survival and differentiation as well as on the ability to form vessels. Our findings provide new insights into the functions of USP22 during development that may offer clues to its role in disease states.

Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies.

Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs.

Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration.

During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR) as well as HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. Pif1 has a role in DNA synthesis across hard-to-replicate sites and in lagging-strand synthesis with polymerase δ (Polδ). Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

Stabilization of the promoter nucleosomes in nucleosome-free regions by the yeast Cyc8-Tup1 corepressor.

The yeast Cyc8 (also known as Ssn6)-Tup1 complex regulates gene expression through a variety of mechanisms, including positioning of nucleosomes over promoters of some target genes to limit accessibility to the transcription machinery. To further define the functions of Cyc8-Tup1 in gene regulation and chromatin remodeling, we performed genome-wide profiling of changes in nucleosome organization and gene expression that occur upon loss of CYC8 or TUP1 and observed extensive nucleosome alterations in both promoters and gene bodies of derepressed genes. Our improved nucleosome profiling and analysis approaches revealed low-occupancy promoter nucleosomes (P nucleosomes) at locations previously defined as nucleosome-free regions. In the absence of CYC8 or TUP1, this P nucleosome is frequently lost, whereas nucleosomes are gained at -1 and +1 positions, accompanying up-regulation of downstream genes. Our analysis of public ChIP-seq data revealed that Cyc8 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of CYC8 or TUP1. These results suggest that stabilization of the P nucleosome on TATA-containing promoters may be a central feature of the repressive chromatin architecture created by the Cyc8-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.

The impact of holistic review on correlations between doctoral student outcomes, and GPA and GRE scores in the biomedical sciences.

Graduate admissions committees throughout the United States examine both quantitative and qualitative data from applicants to make admissions determinations. A number of recent studies have examined the ability of commonly used quantitative metrics such as the GRE and undergraduate GPA to predict the likelihood of applicant success in graduate programs. We examined whether an admissions committee could predict applicant success at The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences based on quantitative metrics. We analyzed the predictive validity of admissions scores, undergraduate GPA, and the GRE for student success. We observed nuanced differences based on gender, ethnicity, race, and citizenship status. The scores assigned to applicants by the admissions committee could not predict time to degree in PhD students regardless of demographic group. Undergraduate GPA was correlated with time to degree in some instances. Interestingly, while GRE scores could predict time to degree, GRE percentile scores could predict both time to degree and PhD candidacy examination results. These findings suggest that there is a level of nuance that is required for interpretation of these quantitative metrics by admissions committees.

Diverse aberrancies target yeast mRNAs to cytoplasmic mRNA surveillance pathways.

Eukaryotic gene expression is a complex, multistep process that needs to be executed with high fidelity and two general methods help achieve the overall accuracy of this process. Maximizing accuracy in each step in gene expression increases the fraction of correct mRNAs made. Fidelity is further improved by mRNA surveillance mechanisms that degrade incorrect or aberrant mRNAs that are made when a step is not perfectly executed. Here, we review how cytoplasmic mRNA surveillance mechanisms selectively recognize and degrade a surprisingly wide variety of aberrant mRNAs that are exported from the nucleus into the cytoplasm.

A Model for Holistic Review in Graduate Admissions That Decouples the GRE from Race, Ethnicity, and Gender.

Graduate schools around the United States are working to improve access to science, technology, engineering, and mathematics (STEM) in a manner that reflects local and national demographics. The admissions process has been the focus of examination, as it is a potential bottleneck for entry into STEM. Standardized tests are widely used as part of the decision-making process; thus, we examined the Graduate Record Examination (GRE) in two models of applicant review: metrics-based applicant review and holistic applicant review to understand whether it affected applicant demographics at The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences. We measured the relationship between GRE scores of doctoral applicants and admissions committee scores. Metrics-based review of applicants excluded twice the number of applicants who identified as a historically underrepresented minority compared with their peers. Efforts to implement holistic applicant review resulted in an unexpected result: the GRE could be used as a tool in a manner that did not reflect its reported bias. Applicant assessments in our holistic review process were independent of gender, racial, and citizenship status. Importantly, our recommendations provide a blueprint for institutions that want to implement a data-driven approach to assess applicants in a manner that uses the GRE as part of the review process.

A genomic screen in yeast reveals novel aspects of nonstop mRNA metabolism.

Nonstop mRNA decay, a specific mRNA surveillance pathway, rapidly degrades transcripts that lack in-frame stop codons. The cytoplasmic exosome, a complex of 3'-5' exoribonucleases involved in RNA degradation and processing events, degrades nonstop transcripts. To further understand how nonstop mRNAs are recognized and degraded, we performed a genomewide screen for nonessential genes that are required for nonstop mRNA decay. We identified 16 genes that affect the expression of two different nonstop reporters. Most of these genes affected the stability of a nonstop mRNA reporter. Additionally, three mutations that affected nonstop gene expression without stabilizing nonstop mRNA levels implicated the proteasome. This finding not only suggested that the proteasome may degrade proteins encoded by nonstop mRNAs, but also supported previous observations that rapid decay of nonstop mRNAs cannot fully explain the lack of the encoded proteins. Further, we show that the proteasome and Ski7p affected expression of nonstop reporter genes independently of each other. In addition, our results implicate inositol 1,3,4,5,6-pentakisphosphate as an inhibitor of nonstop mRNA decay.

Institutional Interventions That Remove Barriers to Recruit and Retain Diverse Biomedical PhD Students.

The faculty and student populations in academia are not representative of the diversity in the U.S. POPULATION: Thus, research institutions and funding agencies invest significant funds and effort into recruitment and retention programs that focus on increasing the flow of historically underrepresented minorities (URMs) into the science, technology, engineering, and mathematics (STEM) pipeline. Here, we outline challenges, interventions, and assessments by the University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences (GSBS) that increased the diversity of the student body independently of grade point averages and Graduate Record Examination scores. Additionally, we show these efforts progressively decreased the attrition rates of URM students over time while eliminating attrition in the latest cohort. Further, the majority of URM students who graduate from the GSBS are likely to remain in the STEM pipeline beyond the postdoctoral training period. We also provide specific recommendations based on the data presented to identify and remove barriers that prevent entry, participation, and inclusion of the underrepresented and underserved in the STEM pipeline.

DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.

Most spontaneous DNA double-strand breaks (DSBs) result from replication-fork breakage. Break-induced replication (BIR), a genome rearrangement-prone repair mechanism that requires the Pol32/POLD3 subunit of eukaryotic DNA Polδ, was proposed to repair broken forks, but how genome destabilization is avoided was unknown. We show that broken fork repair initially uses error-prone Pol32-dependent synthesis, but that mutagenic synthesis is limited to within a few kilobases from the break by Mus81 endonuclease and a converging fork. Mus81 suppresses template switches between both homologous sequences and diverged human Alu repetitive elements, highlighting its importance for stability of highly repetitive genomes. We propose that lack of a timely converging fork or Mus81 may propel genome instability observed in cancer.

Ubp8 and SAGA regulate Snf1 AMP kinase activity.

Posttranslational modifications of histone proteins play important roles in the modulation of gene expression. The Saccharomyces cerevisiae (yeast) 2-MDa SAGA (Spt-Ada-Gcn5) complex, a well-studied multisubunit histone modifier, regulates gene expression through Gcn5-mediated histone acetylation and Ubp8-mediated histone deubiquitination. Using a proteomics approach, we determined that the SAGA complex also deubiquitinates nonhistone proteins, including Snf1, an AMP-activated kinase. Ubp8-mediated deubiquitination of Snf1 affects the stability and phosphorylation state of Snf1, thereby affecting Snf1 kinase activity. Others have reported that Gal83 is phosphorylated by Snf1, and we found that deletion of UBP8 causes decreased phosphorylation of Gal83, which is consistent with the effects of Ubp8 loss on Snf1 kinase functions. Overall, our data indicate that SAGA modulates the posttranslational modifications of Snf1 in order to fine-tune gene expression levels.

Genetic interactions between [PSI+] and nonstop mRNA decay affect phenotypic variation.

Yeast strains can reversibly interconvert between [PSI+] and [psi-] states. The [PSI+] state is caused by a prion form of the translation termination factor eRF3. The [PSI+] state causes read-through at stop codons and can lead to phenotypic variation, although the molecular mechanisms causing those phenotypic changes remain unknown. We identify an interaction between [PSI+]-induced phenotypic variation and defects in nonstop mRNA decay. Nonstop mRNA decay is triggered when a ribosome reaches the 3' end of the transcript. In contrast, we observed little interaction between [PSI+]-induced phenotypic variation and defects in nonsense-mediated decay, which lead to suppression of premature stop codons. These results suggest that at least some of the phenotypic effects of [PSI+] may be due to read-through of "normal" stop codons, thereby producing extended proteins. Moreover, these observations suggest that nonstop mRNA decay may limit [PSI+]-induced phenotypic variation. Such a process would allow periodic sampling of the 3' UTR, which can diverge rapidly, for novel and beneficial protein extensions.