2017 Biochemistry Department Retreat

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2nd Annual Biochemistry Department Retreat
Sponsored by the Biochemistry Student Association

The goal of this retreat is to learn more about the research that takes place throughout the Biochemistry Department!


January 31 & February 1st
Student Life Center Board of Trustees Room

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Retreat Feedback

We'd love it if you could fill out our survey whether you attended or not. We'd like to get feedback on what you enjoyed, what we could improve, or why you chose not to attend.

 


Schedule of Events

Tuesday, January 31

1:00-2:00        Poster Setup & Registration

2:00-2:20        Yi Ren, Faculty
                       “Structural analysis of helicase-mediated remodeling of nuclear mRNP prior to
                       nuclear export"

2:20-2:40       Elwood Mullins, Post-doc, Brandt Eichman Lab
                      “Time-resolved molecular snapshots reveal an unprecedented mechanism for
                      recognition and removal of damaged bases in DNA"

2:40-3:00        Romell Gletten, Graduate Student, Kevin Schey Lab
                       “The Structure and Function of Aquaporin 5 in Ocular Lens Fiber Cells"

3:00-3:10        Coffee Break

3:10-3:30        Yan Su, Post-doc, Fred Guengerich Lab
                       “Ribonucleotide Incorporation by Human DNA Polymerase eta”

3:30-3:50        Manny Ascano, Faculty
                       “VIR-CLASP reveals unexpected interactions between host-encoded pioneer
                       RNA-binding proteins and the pre-replicated RNA genomes of Chikungunya
                       and Zika viruses"

3:50-6:00        Posters / Wine & Cheese

Wednesday, February 1

8:30-9:00        Breakfast / Registration

9:00-9:20        Keenan Taylor, Post-doc, Chuck Sanders Lab
                       “Precision Medicine and the Potassium Channel KCNQ1”

9:20-9:40        Aaron Fidler, Graduate Student, Billy Hudson Lab
                       “Collagen IV and the evolutionary dawn of metazoan tissues”

9:40-10:00      Kareem Mohni, Post-doc, David Cortez Lab 
                       “HMCES is a DNA repair or tolerance protein required for error free repair”

10:00-10:20    Lauren Jackson, Faculty
                       “High-resolution crystal structures of the tepsin ENTH and VHS domains”

10:20-10:30    Coffee Break

10:30-10:50    Kelly Barnett, Graduate Student, Emily Hodges Lab
                       “Genomic Methods for Studying Epigenetic and Activity Dynamics at
                       Enhancers During Early Cell Fate Specification”

10:50-11:10    Jeff Spraggins, Faculty, Richard Caprioli Lab 
                       “Pulling data out of the noise: Maximizing sensitivity of molecular imaging
                       experiments through gas phase fractionation and enrichment”

11:10-11:30    Boone Prentice, Post-doc, Richard Caprioli Lab 
                       “The Effect of Metal Content on the Extent of Calprotectin Oligomerization as
                       Revealed by Native Mass Spectrometry”

11:30-1:30      Lunch & Poster Session

1:30-1:50        Adrian Olivares, Faculty
                       “Direction matters: Mechanical protein destruction by AAA+ proteases”

1:50-2:10        Johanna Schafer, Graduate Student, Jennifer Pietenpol Lab
                       “Mechanisms of Bromodomain and Extra-Terminal motif inhibitor (BETi)
                       sensitivity in Triple-Negative Breast Cancer (TNBC)”

2:10-2:30        Carl Sedgeman, Graduate Student, Fred Guengerich Lab
                       “Formation of N6-Deoxyadenosinyl-glutathione and Replication Past the
                       Adduct by Translesion DNA Polymerases"

2:30-2:40        Coffee Break

2:40-3:10        Cunningham Award Talk

3:10-3:40        Busenlehner Award Talk

3:40-3:50        Coffee Break

3:50-4:10        Shilpa Sampathi, Post-doc, Scott Hiebert Lab
                       “High resolution mapping of RNA polymerase dynamics upon inhibition of
                       CDKs in t(8;21) AML"

4:10-4:30        James Dewar, Faculty
                       “The termination of DNA replication.”

4:30-4:50        Rose Follis, Graduate Student, Bruce Carter Lab
                       "A role for NF-kB in demyelinating neuropathies"

4:50-5:10        Kristy Stengel, Post-doc, Scott Hiebert Lab
                       “HDAC3 is required for FOXO1 activity in germinal centers and B cell
                       lymphoma”

5:10-5:30        Break / Walk to Banquet & Awards at University Club

5:30-7:30        Dinner at University Club

6:20                Announce Talk & Poster Winners

Award Winners

Leon Cunningham Award: Thomas Bass, graduate student in the Dave Cortez lab.
Laura Busenlehner Award: Rachel Ashley, graduate student in the Neil Osheroff lab.
Best graduate student talk: Kelly Barnett
Best post-doc talk: Keenan Taylor
Best graduate student poster: Kami Bhat
Best post-doc poster: Clayton Marshall
Honorable mention graduate student talk: Johanna Schafer
Honorable mention post-doc talk: Kristy Stengel
Honorable mention graduate student poster: Meredith Frazier
Honorable mention post-doc poster: Orrette Wauchope

 

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Yi Ren, faculty. “Structural analysis of helicase-mediated remodeling of nuclear mRNP prior to nuclear export.”

mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2’s intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1.

 

Elwood Mullins, post-doc, lab of Brandt Eichman. “Time-resolved molecular snapshots reveal an unprecedented mechanism for recognition and removal of damaged bases in DNA.”

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. Previous studies have shown that a hallmark of DNA glycosylases and other DNA repair enzymes is their use of base flipping to pull modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. We recently described the first DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the bacterial DNA glycosylase AlkD, we reconstructed steps along the reaction coordinate through structures representing substrate, intermediate, and product complexes, as well as crystallographically monitored excision of an alkylpurine substrate as a function of time. These structures showed that instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic CH–π interactions that preferentially stabilize the transition state. We now show through a combination of crystallographic, biochemical, and cellular techniques how this unique mechanism enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin and CC-1065 family of antimicrobial and antitumor natural products. Bulky adducts of this, or any type, are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new paradigm for DNA repair and have potentially far-reaching implications for DNA damage recognition.

 

Romell Gletten, graduate student, lab of Kevin Schey. “The Structure and Function of Aquaporin 5 in Ocular Lens Fiber Cells.”

The lens contains several types of transmembrane water channels or aquaporins (AQPs) which mediate lens transparency. Among these, AQP5 exhibits cytoplasmic subcellular localization in newly differentiating lens fiber and increasingly translocates to the plasma membrane during lens fiber cell maturation. This spatiotemporal trend of AQP5 membrane insertion occurs in concert with AQP0 C-terminal truncation, which is implicated in water pore restriction and thereby reduced AQP0 water permeability. AQP5 deletion studies suggest that lenticular AQP5 compensates for perturbations to lens osmotic balance. Together, these data suggest that AQP5 functions as a compensatory water channel to mitigate reductions in C-terminally truncated AQP0 water permeability in the lens. However, the mechanism(s) that regulates AQP5 plasma membrane insertion in the lens is unclear. AQP5 phosphorylation and palmitoylation are examined here as both have been implicated to regulate AQP plasma membrane insertion. In this study, peptides from human or bovine lens membrane fractions were generated following enzymatic digestion and analyzed via LC-MS/MS. Cysteine palmitoylation was prescreened via acyl-biotin exchange. AQP5 spatial expression during lens fiber cell maturation was analyzed via immunohistochemistry. Water permeability in lens fiber cell plasma membrane vesicles was quantified via vesicle swelling assays. Our data demonstrate that bovine lenticular AQP5 spatiotemporal protein expression is analogous to that of human and rodent lenses with cytoplasmic expression in newly differentiating fiber cells and increasing plasma membrane insertion with fiber cell maturation. Additionally, AQP5 is palmitolyated at Cys6 and phosphorylated on Thr259. Furthermore, AQP5 is inserted into the plasma membrane of newly differentiating lens fiber cells following ex vivo culture. Plasma membrane vesicles derived from ex vivo cultured lens fiber cells demonstrate a mercury-sensitive increase in water permeability. These data demonstrate that lenticular AQP5 is subject to phosphorylation and palmitoylation and suggest that AQP5 can be induced to traffick to the plasma membrane in vivo.

 

Yan Su, post-doc, lab of Fred Guengerich. “Ribonucleotide Incorporation by Human DNA Polymerase eta.”

Ribonucleotides and 2´-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2´-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2´-OH group. Y-family human DNA polymerase eta (hpol eta) is of interest owing to its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol eta maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2´-deoxyguanosine (8-oxodG) and cyclobutane pyrimidine dimer (CPD) but with rates that are 1000-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that hpol eta scaffolds the incoming rNTP to pair with the template base (dG) or 8-oxodG with a significant propeller twist. As a result, the 2´-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.

 

Manny Ascano, faculty. “VIR-CLASP reveals unexpected interactions between host-encoded pioneer RNA-binding proteins and the pre-replicated RNA genomes of Chikungunya and Zika viruses.”

By the time viruses have co-opted host machinery to replicate its genome, host transcription and translation has largely been relegated to viral production, greatly limiting any ability to mount a cellular immune response. Thus a critical window of time exists, prior to viral replication, in which interactions between the initial viral genome and host proteins can lead to either a facultative infection or an effective anti-viral response. However, our molecular understanding of the early events that lead to the deployment of the virus inside the cell remains incomplete, particularly with respect to the interactions between the infecting viral genome and the host factors that act to limit infection or are co-opted for pro-viral purposes. In an effort to characterize pre-replication viral infection dynamics, we developed VIR-CLASP (Virus-induced ribonucleoside analog-enhanced crosslinking and solid phase purification), which can comprehensively identify direct interactions between host-encoded proteins and first-generation viral RNA genomes. We will discuss our current progress with Chikungunya and Zika viruses, the associations that we have identified, and our unexpected discovery of host proteins with novel RNA-binding capacity and their potential roles during infection.

 

Keenan Taylor, post-doc, lab of Chuck Sanders. “Precision Medicine and the Potassium Channel KCNQ1.”

The human potassium channel KCNQ1 is a polytopic α-helical membrane protein. KCNQ1 is expressed in both epithelial and heart tissue. In the heart, KCNQ1, in association with KCNE1, mediates the Iks current responsible for the repolarization of the cardiac action potential. Mutations that cause a loss-of-function of KCNQ1 result in a congenital condition known as long-QT syndrome (LQTS). LQTS predisposes an individual to cardiac arrhythmia and can result in sudden death. Genome sequencing provides a powerful diagnostic tool for congenital disease such as LQTS. However, when a genetic variation of unknown significance is uncovered accurate functional and diagnostic interpretation of the data is often difficult. At the molecular level, genetic variants can alter the function, structure, or dynamics of the encoded protein molecules. In some cases, genetic variation gives rise to increased likelihood of disease, while in other cases variation is completely benign. Such complexities highlight the need for a substantial biochemical understanding in order to accurately predict the likelihood of disease outcomes based on genetic information. We employ a multi-faceted approach where the trafficking, structural, and electrophysiological consequences of KCNQ1 mutations are measured. Ultimately, these data will be used in predictive models to evaluate the likelihood of disease outcomes for genetic variations of unknown significance. This is a multi-group effort involving the Meiler, Sanders, and George labs. We present trafficking, electrophysiology, and NMR characterization of 51 KCNQ1 mutations.
 

Aaron Fidler, graduate student, lab of Billy Hudson. “Collagen IV and the evolutionary dawn of metazoan tissues.”

The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as the earliest-branching extant animal phylum, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of Ctenophora and other basal metazoan phyla. We identified basement membrane (BM) and collagen IV in Ctenophora, and show that the structural and genomic features of collagen IV are homologous to those of basal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Collectively, we conclude that collagen IV and its variant are primordial components of the extracellular microenvironment, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis.

 

Kareem Mohni, post-doc, lab of David Cortez. “HMCES is a DNA repair or tolerance protein required for error free repair.”

The DNA damage response kinase ATR (ATM and Rad3 related) and its effector kinase CHEK1 (Checkpoint kinase 1, CHK1) are required for cancer cells to survive oncogene induced replication stress. ATR inhibitors exhibit synthetic lethal interactions with ERCC1, ATM, and XRCC1 deficiency as well as with Cyclin E over-expression. Here we report a systematic whole genome screen to identify synthetic lethal interactions with ATR-pathway targeted drugs, which might be useful for guiding their use in the cancer clinic. Reduced function of the ATR pathway itself, as well as reduced levels of DNA replication proteins provided the strongest synthetic lethal interactions. We hypothesize that genes exhibiting synthetic lethality with ATR-pathway targeted drugs may function in DNA replication or repair. Indeed 71 genes exhibiting synthetic lethal interactions were localized to replication forks via isolation of proteins on nascent DNA (iPOND) in a concurrent screen. Of these, 29 have no known functions in DNA replication or repair. HMCES exhibited synthetic lethality with 4/4 siRNAs and localized to replication forks in iPOND. HMCES is conserved through bacteria and predicted to be a SOS-response protein, although it has not been studied in any organism. Preliminary data show that HMCES binds structured DNA, interacts with PCNA, and loss of function sensitizes cells to ATR inhibition, MMS, and UV.

 

Lauren Jackson, faculty. “High-resolution crystal structures of the tepsin ENTH and VHS domains.”

Tepsin is the only known accessory protein in adaptor-related protein 4 (AP4) coated vesicles that assemble at the trans-Golgi network (TGN). Tepsin depends upon AP4 for its membrane recruitment, but the biological role of tepsin remains unknown. We have determined high resolution X-ray crystal structures of both folded domains found in tepsin, the tepsin Epsin N-Terminal Homology (tENTH) and tepsin Vps27/Hrs/Stam (tVHS)-like domains. Both domains are found in other trafficking proteins, but our data reveal unexpected structural features in each domain. ENTH domains usually contain nine -helices, including an amphipathic helix0 that forms a lipid binding pocket and allows the epsin to bind membranes directly. Our tENTH structure (1.4 Å) indicates tepsin lacks helix0 and thus the lipid binding pocket. These data explain why tepsin is the only epsin that requires an adaptor for membrane recruitment. tENTH further lacks helix8, making it the smallest known ENTH domain. VHS domains contain eight -helices and have two primary roles in other trafficking proteins: they bind short linear acidic dileucine-based motifs (DxxLL/I) in transmembrane proteins cargoes or bind ubiquitin to send proteins to the proteasome. Our tVHS-like structure (1.7 Å) instead contains six -helices. Loss of helix8 explains why tepsin cannot bind dileucine-based motifs, which we have confirmed in vitro using calorimetry. Key differences in residues located in helix2 prevent tVHS from interacting with monomeric ubiquitin, which we confirmed using NMR spectroscopy. One striking observation is that these domains are more structurally similar to each other than to other members of the families to which they have been assigned. We are currently pursuing candidate protein binding partners for both domains in order to understand the role of tepsin in cells.

 

Kelly Barnett, graduate student, lab of Emily Hodges. “Genomic Methods for Studying Epigenetic and Activity Dynamics at Enhancers During Early Cell Fate Specification.”

Gene regulatory elements such as enhancers form networks that are potent guides for cell specification and organismal development. Epigenetic changes such as DNA methylation and chromatin remodeling have been shown to reflect the modulation of these gene regulatory elements, yet little is known about the precise timing of these changes at the earliest stages of cell fate specification. While this is believed to be an ordered process, very few studies have captured the relationship between chromatin remodeling, transcription and DNA demethylation at enhancers. We have coupled sequencing methodologies such as ATAC-seq, STARR-seq and Bisulfite sequencing to systematically map these network-level events during early cell fate specification.

 

Jeff Spraggins, faculty, lab of Richard Caprioli. “Pulling data out of the noise: Maximizing sensitivity of molecular imaging experiments through gas phase fractionation and enrichment.”

In the analysis of biological tissue, sensitivity and dynamic range are of paramount importance in obtaining experimental results that provide insight into underlying biological processes. Many important biomolecules are present in the tissue environment in low concentrations and in complex mixtures with other species of widely ranging abundances, pushing the limits of analytical technologies. A common approach to overcoming challenges associated with mass spectrometric analysis of complex biological samples is to use LC-based fractionation to reduce the overall complexity of the mixture. Analogous gas phase fractionation techniques can be used in imaging mass spectrometry (IMS) experiments to improve performance for target analytes detected from tissue samples. Here we demonstrate the capabilities of gas phase fractionation and enrichment to maximize the effective sensitivity and dynamic range (>100-fold) for low intensity ions for molecular imaging experiments.

All imaging experiments were performed using a 15T MALDI Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a Smartbeam-II Nd:YAG laser. Gas phase fractionation and enrichment was done using continuous accumulation of selected ions (CASI) allowing for detection and enhancement of low abundant analytes. As a case study for this enrichment technology, we applied CASI FTICR IMS for the analysis of human squamous cell carcinoma (SCC) biopsies. We explored the phospholipidomes of 30 human SCCs using both traditional LC-based lipidomics and MALDI imaging mass spectrometry. LC-MS experiments found that, almost invariably (in 96.7% of cases), SCCs contain phospholipids with longer acyl chains compared to matched normal tissues. Molecular imaging experiments were used to confirm that observed long chain phospholipids were spatially localized specifically to tumor cell nests. Although traditional “full-scan” MALDI IMS experiments were able to detect many of the target lipids, gas phase fractionation and enrichment was required to elucidate the spatial distributions of lowly abundant long chain phospholipids.

 

Boone Prentice, post-doc, lab of Richard Caprioli. “The Effect of Metal Content on the Extent of Calprotectin Oligomerization as Revealed by Native Mass Spectrometry.”

The structural characterization of protein complexes is crucial to understanding the biochemical function of these assemblies. Classical biophysical technologies such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are excellent at providing high resolution structural information. However, the throughput and sensitivity of these methods are limited, especially when studying large heterogeneous complexes. Increasingly, mass spectrometry (MS) analyses of intact protein complexes (i.e., “native MS” workflows) are used to determine the stoichiometry, binding arrangement, ligand interactions, and dynamics of these macromolecular assemblies. One protein complex in particular, calprotectin (CP), has been identified as a critical factor in the innate immune response. CP, a known calcium-binding protein, is a ~24 kDa heterodimer complex consisting of S100A8 and S100A9 subunits. In addition to calcium, it has also been shown that this protein has affinities for other nutrient metals such as zinc, manganese, and copper. Despite the extensive work being done on CP, surprisingly little is known about its ability to former higher-order oligomer states. Herein, we demonstrate the ability of native MS to accurately characterize the stoichiometry of a number of protein complexes, including, the heat shock protein Mj 16.5, the membrane protein aquaporin-0 (AQP0), and the innate immune protein CP. Specifically, we show that CP readily forms heterodimer, heterotetramer, and octamer states. The ability of CP to form higher-order oligomer states is shown to be dependent on protein concentration and calcium concentration. Interestingly, we note here that the addition of sub-stoichiometric amounts of zinc almost completely disrupts the heterodimer state, while higher-order oligomer states appear unaffected. Coupled with dynamic light scattering (DLS) measurements, these results provide new insights on the stoichiometry of CP complexation, which is likely related to its functionality and role in cell signaling. In general, native MS represents a unique and complementary approach to existing structural biology workflows.

 

Adrian Olivares, faculty. “Direction matters: Mechanical protein destruction by AAA+ proteases.”

AAA+ proteases maintain protein homeostasis and regulate protein-protein and protein-nucleic acid interactions, largely in response to changes in cellular environment such as stress. These machines couple the hydrolysis of ATP to mechanical unfolding and translocation of substrate proteins following initial recognition of degradation tags called degrons. Recent single-molecule studies have uncovered differences in the mechanochemical behaviors of two members of this destructive family from E. coli, ClpXP and ClpAP, using model substrates containing C-terminal degrons. However, both enzymes recognize and process cellular substrates bearing N-terminal degrons in addition to the well-characterized C-terminally tagged substrates. How mechanical protein degradation occurs from the N-terminus of a protein is largely unknown. We therefore examined how ClpXP and ClpAP mechanically unfold and translocate protein substrates from the N-terminus using single-molecule optical trapping. We discover that both enzymes unfold a multidomain model substrate more rapidly from the N-terminus while translocation speed is slightly reduced compared to similar substrates containing a C-terminal degron. These measurements directly establish the role of directionality on mechanical protein degradation, demonstrate that the local stability near the site of enzyme-mediated pulling determines the rate of mechanical substrate unfolding, and provide insight into the placement of degrons on proteins.

 

Johanna Schafer, graduate student, lab of Jennifer Pietenpol. “Mechanisms of Bromodomain and Extra-Terminal motif inhibitor (BETi) sensitivity in Triple-Negative Breast Cancer (TNBC).”

With currently no FDA-approved targeted therapies for triple-negative breast cancers (TNBCs), patients with TNBC have a higher risk of local and distant recurrence, and an overall increased rate of mortality.  Poor prognosis is due, at least in part, to clonal evolution during tumorigenesis and the resulting high degree of heterogeneity.  At time of diagnosis, most TNBCs consist of subpopulations of cells that will either respond or be inherently resistant to therapy. Cells that respond often undergo apoptosis, senesce, or acquire resistance through either genetic or epigenetic modifications.  Here we demonstrate TNBC cell lines harboring a PIK3CA mutation consist of a subpopulation of cells with high N-Myc expression that acquire resistance to phosphoinositol 3-kinase inhibition (PI3Ki).  To determine targeted therapy for N-Myc+ PI3Ki-resistant disease, we developed clonal populations from the parental and PI3Ki-resistant cell lines and subjected them to the NCI library of FDA-approved oncology drugs as well as several inhibitors under clinical development to target N-Myc. One class of inhibitors targeting the bromodomain and extraterminal (BET) family of proteins ranked high among drugs with efficacy in the PI3Ki-resistant clones.  Furthermore, precision run-on and sequencing (PRO-seq) demonstrated a significant decrease in N-Myc enhancer and gene expression after 15 min of BET-inhibitor (BETi) treatment.  Additional TNBC cell lines with N-Myc+ subpopulations also decreased N-Myc expression in the presence of BETi. Targeting these cells early is critical due to the prevalence of MYCN amplifications in the metastatic setting and the overall poor prognosis of patients with high N-Myc-expressing breast cancer.  Our data suggest a potential biomarker of BETi sensitivity in naïve and PI3Ki-resistant disease that may be of value in further pre-clinical studies.

 

Carl Sedgeman, graduate student, lab of Fred Guengerich. “Formation of N6-Deoxyadenosinyl-glutathione and Replication Past the Adduct by Translesion DNA Polymerases.”

1,2-Dibromoethane (DBE) is a potent carcinogen due at least in part to its DNA crosslinking effects. DBE crosslinks glutathione (GSH) to DNA, notably to sites on adenosine and guanosine [Cmarik, J.L. et al. (1991) J. Biol. Chem. 267, 6672-6679]. However, adduction to the N6-position of adenosine had not been detected, but this is an adduction site for the linkage of O6-alkylguanine DNA alkyltransferase [Chowdhury, G., et al. (2013) Angew. Chem. Int. Ed. 52, 12879-12882]. We identified and quantified a new adduct, S-[2-(N6-deoxyadenosinyl)ethyl]GSH, in calf thymus DNA by LC-MS/MS. Replication studies were also performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) η, ι, and κ, as well as with Sulfolobus solfataricus Dpo4. hPols η and ι and Dpo4 were able to bypass the adduct with only slight impedance; hPol κ was completely blocked by the adduct. hPol η and ι showed increased misincorporation opposite the adduct compared to unmodified dA. LC-MS/MS analysis of full-length primer extension products by hPol η confirmed the incorporation of dC opposite S-[2-(N6-deoxyadenosinyl)ethyl]GSH and also showed the production of an n-1 frameshift. These results reveal the significance of N6-adenosine GSH-DBE adducts in blocking replication, as well as producing misinsertions, by human translesion DNA polymerases.

 

Shilpa Sampathi, post-doc, lab of Scott Hiebert. “High resolution mapping of RNA polymerase dynamics upon inhibition of CDKs in t(8;21) AML.”

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). The ETO/MTG8 component of the encoded fusion protein is recruited to genes by DNA binding transcription factors and bridges these factors to co-repressors containing histone deacetylases, but also CDK9, which stimulates transcription elongation. The expression of the encoded RUNX1-ETO fusion protein is controlled by RUNX1 regulatory elements and the inhibition of CDK7 suppressed RUNX1 expression, displayed potent anti-tumor activity in T cell acute lymphocytic leukemia. In our study we found that t(8;21) AML cells are exquisitely sensitive to CDK9 and CDK7 inhibition and after only 4 hrs of treatment the cells displayed hallmarks of apoptosis. To get mechanistic insights into transcriptional control by CDKs, we performed precision global run-on transcription sequencing (PRO-Seq). This technique creates high resolution maps of the active RNA polymerases within t(8;21) containing cells to define the global effects on RNA polymerase dynamics in response to inhibitors of the “transcriptional” CDKs. Broad spectrum CDK inhibitors (Flavopridol and Dinaciclib), as well as more selective inhibitors of CDK9 (PHA767491) caused RNA polymerase pausing at nearly 80% of the expressed genes. Conversely, inhibition of CDK7 by using THZ1 caused a wide-spread loss of paused RNA polymerase at promoters on about half of the expressed genes. Within this larger cohort of affected genes, inhibition of CDK7 increased the rate of transcription of a subset and decreased the transcription of other genes, while having modest effects on genes regulated by “super enhancers”. Strikingly, on the genes displaying increased rates of transcription after a 1 hr treatment with THZ1, RNA polymerases termination was skewed towards the earliest (most 5’) termination and poly(A) addition sites.

 

James Dewar, faculty. “The termination of DNA replication.”

Faithful duplication of the genome is crucial for viability and, in multicellular organisms, to safeguard against disease. DNA replication is intensively studied, but the final stages, ‘termination’, are relatively uncharacterized. This knowledge-gap exists because the timing and location of termination is essentially random in cells, which makes it challenging to study. In the past couple of years, great strides have been made in understanding eukaryotic termination, including elucidation of a mechanistic model for termination (Dewar et al, 2015, Nature), and identification of a dedicated pathway for removal of replication proteins from DNA (Maric et al, 2014, Science, Moreno et al, 2014, Science). Despite this progress, many questions are unanswered and few termination proteins are known.

I will review the insights gleamed from a biochemical system termination (Dewar et al, 2015, Nature), and describe subsequent work that identified termination proteins and investigated their roles.
 

Rose Follis, graduate student, lab of Bruce Carter. "A role for NF-kB in demyelinating neuropathies."

Charcot Marie Tooth disease (CMT) is the most common group of hereditary peripheral neuropathies, affecting 1 in 2500 people. Trisomy of peripheral myelin protein 22 (PMP22) gene is the most prevalent form, giving rise to CMT type 1A. Both duplication and point mutations in PMP22 lead to dys-/demyelination and secondary axon degeneration. It is still not clear how duplication or point mutations lead to the pathology. PMP22 is a tetraspan membrane protein found primarily in the ER with a small fraction expressed at the plasma membrane. The role of PMP22 in Schwann cells is poorly understood; however, our data suggest a novel function of PMP22 in the ER, regulating calcium flux through Store Operated Calcium (SOC) channels. Several PMP22 point mutations, including the L16P, Trembler J (TrJ) mutation, increase protein misfolding and excess ER accumulation. We found that Schwann cells from TrJ mice have increased Ca+2 influx through SOC channels. Similarly, increased Ca+2 influx has been detected in Schwann cells from PMP22 over expressing rats. We also detected activation of the ER stress response protein IRE1 in the TrJ nerves. Both calcium influx through SOC channels and stimulation of IRE1 lead to activation of the transcription factor NFkB. In normal development, NFkB is activated in immature Schwann cells as they differentiate into a myelinating phenotype, but is inactive in mature, myelinating cells. However, we find abnormal activation of NFkB in the nerves of adult TrJ mice and C3-PMP mice, which overexpress PMP22 and have a phenotype similar to CMT1A. Moreover, forced activation of NFkB in myelinating cells in culture and in vivo resulted in demyelination. These findings lead us to hypothesize that excess or mutant PMP22 protein leads to increased calcium influx through SOC channels and IRE1 activation, which causes Schwann cell de-differentiation and demyelination through activation of NFkB.

 

Kristy Stengel, post-doc, lab of Scott Hiebert. “HDAC3 is required for FOXO1 activity in germinal centers and B cell lymphoma.”

B cell activation upon antigen encounter triggers the initiation of a germinal center (GC) reaction, in which iterative rounds of immunoglobulin mutation and selection generate high affinity antibodies. In addition to a critical role in adaptive immunity, GC B cells are the cell of origin for the majority of diffuse large B cell lymphomas (DLBCLs). The transcription factor, BCL6, is required for GC formation, where it represses transcription of genes associated with the DNA damage response, cell cycle checkpoints and terminal B cell differentiation by recruiting co-repressor complexes including SMRT:HDAC3. Importantly, aberrant BCL6 function following chromosomal translocation is frequently observed in human DLBCL. Here, we utilize mouse models of Hdac3 deletion to determine the requirement of this co-repressor for germinal center formation and Bcl6-mediated transcriptional function. We find that unlike Bcl6, Hdac3 is not required for germinal center formation, suggesting that Hdac3 is not necessary for the full complement of Bcl6 activities. Rather, Hdac3 deletion resulted in the formation of abnormal germinal centers characterized by a loss of dark zone GC B cells (also known as centroblasts). This phenotype was strikingly similar to that reported upon Foxo1 deletion from GC B cells. Consistently, Hdac3 deletion resulted in the up-regulation of a subset of Bcl6 target genes that are co-bound by Foxo1 and generally associated with light zone centrocytes and plasma cell differentiation. We further demonstrate an interaction between HDAC3 and FOXO1, suggesting that HDAC3 may regulate FOXO1 function. Consistently, treatment of DLBCL cell lines with an HDAC3 selective inhibitor revealed highly varied responses; however, those cell lines with reported FOXO1 mutations proved particularly sensitive, and HDAC3 knockdown in FOXO1 mutant cells impaired tumor growth. Thus, Hdac3 is required for Foxo1 transcriptional activities within the germinal center and represents a promising therapeutic target for B cell lymphomas with altered FOXO1 activity.

Bill Martin, graduate student, lab of Nick Reiter. “Structure-Specific Recognition of a G-Quadruplex RNA by the Histone Demethylase LSD1.”

 

Thomas Bass, graduate student, lab of David Cortez. “ETAA1 maintains genome integrity through activation of ATR.”

 

Justin Tyler Marinko, graduate student, lab of Chuck Sanders. “Elucidation of Limiting Interactions within the Cellular Quality Control Pathway of Peripheral Myelin Protein 22.”

 

Lisa Poole, graduate student, lab of David Cortez. “SMARCAL1 maintains telomere integrity during DNA replication.”

 

Tyson Rietz, graduate student, lab of Steve Fesik. “Therapeutic targeting of TIGIT with novel small-molecule inhibitors.”

 

Meredith Frazier, graduate student, lab of Lauren Jackson. “Structure and function studies of the VHS-like domain from the AP4 accessory protein tepsin.”

 

Tara Archuleta, post-doc, lab of Lauren Jackson. “Characterization of the structure and function of the tENTH domain from the AP4 accessory protein tepsin.”

 

Sarah Arcos, graduate student, lab of Manny Ascano. “N6-methyladenosine-dependent regulation of messenger RNA during innate immune activation.”

 

Elizabeth Gibson, graduate student, lab of Neil Osheroff. ““Mycobacterium Gyrase Inhibitors” (MGIs): A Novel Class of Gyrase Poisons.”

 

Alex Trevisan, graduate student, lab of Bruce Carter. “The role of Jedi in the development and function of the dorsal root ganglia.”

 

Katie Rothamel, graduate student, lab of Manny Ascano. “The role of RNA-binding proteins in Immunity.”

 

Clayton Marshall, post-doc, lab of Jennifer Pietenpol. “p73 is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network.”

 

Petria Thompson, graduate student, lab of David Cortez. “Determining DNA Substrate Specificity of HMCES.”

 

Hui Huang, post-doc, lab of Chuck Sanders. “Comprehensive Assessment of Disease Mutant Forms of the Human KCNQ1 Potassium Channel.”

 

Orrette Wauchope, post-doc, lab of Larry Marnett. “Oxidative stress increases M1dG, a major peroxidation DNA adduct, in mitochondrial DNA (mtDNA).”

 

Vaughn Thada, graduate student, lab of David Cortez. “Mechanism of action of ETAA1, a novel ATR activator.”

 

Monica Bomber, graduate student, lab of Scott Hiebert. “Characterizing C-terminal domain binding partners in AML1-ETO induced leukemia.”

 

Gabriela Santos Guasch, graduate student, lab of Jennifer Pietenpol. “The Role of p73 in Hormonally-Regulated Tissues”

 

Rongxin Shi, graduate student, lab of Brandt Eichman. “Base excision repair of cationic nucleobases by the bacterial DNA glocosylase AlkC.”

 

Sarah Wessel, post-doc, lab of David Cortez. “ATR and Wee1 inhibition in OCI-LY-19 and DB DLBCL cell lines.”

 

Kami Bhat, graduate student, lab of David Cortez. “XSB antagonizes RAD51 to maintain replication fork stability and modulate chemosensitivity.”

 

Tim Shaver, graduate student, lab of Jennifer Pietenpol. “Targeting the p53 mutant-adapted state in triple-negative breast cancer.”

 

Matt Albertolle, graduate student, lab of Fred Guengerich. “The ω-Hydroxylation Activity of Cytochrome P450 4A11 is Attenuated in the Presence of Hydrogen Peroxide.”

 

Adrian Olivares, faculty. “Mechanical protein remodeling at the single-molecule level: From proteolysis to nuclear function.”

 

James Dewar, faculty. “The Dewar Lab.”

 

Bob Chen, staff, lab of Emily Hodges. “Breadth and Depth in the Exploration of Putative Enhancers.”

 

Bradley Clarke, graduate student, John York Lab. "Structural and Functional Characterization of the Clu1-IP6 Complex."