Uridine Incorporation Research Articles

Abstract 1702: Loss of KEAP1 promotes R-loop formation independent of NRF2

Abstract This study investigates the mechanism of R-loop formation as a consequence of KEAP1 depletion. R-loops, described as three-stranded nucleic acid structures consisting of a DNA:RNA hybrid and a non-template single-stranded DNA, are assemblies often attributed with pathological consequences like genomic instability and replication stress. However, they have significant physiological roles, inter alia, regulation of transcription initiation, splicing and termination. Loss in R-loop resolution factors (like RNaseH nucleases, senataxin) can result in “unscheduled” or “pathological” R-loops, which can compromise genome stability. R-loops are commonly associated with an active transcription bubble, with nascent RNA trailing the RNA polymerase re-annealing to the template DNA strand. Increased transcriptional stress can lead to accumulation of R-loops. In a 2016 eLife study by Stork et al., estrogen induction resulted in concomitantly increased transcription and R-loop formation in the same subset of genes. Similar to estrogen induction, activation of the NRF2/KEAP1 pathway upon oxidative stress causes NRF2 to be released from KEAP1 and enter the nucleus to turn on transcription of its target genes. Kelch-like ECH associated protein 1, encoded by the KEAP1 gene, is a canonical binding partner of NRF2. Under basal conditions, KEAP1 binds NRF2 and targets it for degradation. Mutations in KEAP1 and epigenetic modifications have been reported in lung and breast cancer phenotypes. In lieu of chemical manipulation, NRF2 can be activated by knocking down KEAP1 via RNAi. We therefore asked whether transcription activation downstream of the NRF2/KEAP1 pathway can lead to accumulation of R-loops.We have observed the consequences of depleting KEAP1 (for NRF2 activation) with RNAi on global transcription using ethynyl uridine (EU) incorporation with click-iT chemistry and flow cytometry, on R-loop levels using S9.6 antibody immunocytochemistry and on transcriptional profile changes with RNA-seq. We observe that loss of KEAP1 leads to an increased accumulation of R-loops. Surprisingly, this is not accompanied by an expected increase in transcription as measured quantitatively with EU flow cytometry. This phenomenon was found to consistently occur in various cell lines across cancer types (breast, lung). Interestingly, depleting NRF2 cannot mitigate the effects of KEAP1 loss, suggesting that KEAP1 loss-associated R-loop formation is independent of NRF2 activation. Analysis of RNA-seq data shows that TOP2 is downregulated in KEAP1 deficient cells. As TOP2 has been previously known to regulate R-loop formation, we predict that KEAP1 loss can sensitize cells to topoisomerase inhibitors.In conclusion, interaction of KEAP1 with other players in its interactome outside of NRF2 may regulate R-loop formation and therefore R-loop-induced genome instability. Citation Format: Pramiti Mukhopadhyay, Nicklas Bassani, Alexander J. Bishop. Loss of KEAP1 promotes R-loop formation independent of NRF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1702.

A refined culture system of oocytes from early antral follicles promotes oocyte maturation and embryo development in cattle.

The proposed culture system improves the current state of in vitro culture of growing oocytes in the bovine species and allows access to the untapped gamete reserve, thus improving reproductive efficiency. The present study aimed to improve the in vitro culture of bovine oocytes collected from early antral follicles (EAFs) to support the progressive acquisition of meiotic and developmental competence. The rationale that drove the development of such a culture system was to maintain as much as possible the physiological conditions that support the oocyte growth and differentiation in vivo. To this extent, oocytes were cultured for 5 days, which parallels the transition from early to medium antral follicles (MAFs) in the bovine, and supports promoting a 3D-like structure were provided. Additionally, the main hormones (follicle-stimulating hormone, estradiol, progesterone, and testosterone) were added in concentrations similar to the ones previously observed in bovine EAFs. The meiotic arrest was imposed using cilostamide. The cultured cumulus-oocyte complexes (COCs) reached a mean diameter of 113.4 ± 0.75 µm and showed a progressive condensation of the chromatin enclosed in the germinal vesicle (GV), together with a gradual decrease in the global transcriptional activity, measured by 5-ethynyl uridine incorporation. The described morpho-functional changes were accompanied by an increased ability to mature and develop to the blastocyst stage in vitro, although not matching the rates obtained by MAF-retrieved oocytes. The described system improves the current state of in vitro culture of growing oocytes in the bovine species, and it can be used to increase the number of gametes usable for in vitro embryo production in animals of high genetic merit or with specific desirable traits.

C5-Propynyl modified 2'-fluoroarabinonucleic acids form stable duplexes with RNA that are RNase H competent.

The clinical success of the antisense approach for the treatment of genetic disorders is indisputably the result of chemical modifications along the oligonucleotide (ON) scaffold, which impart desirable properties including high RNA affinity, nuclease stability and improved drug delivery. While effective, many modifications are not capable of eliciting an RNase H response limiting their application in antisense systems. To contribute to the structural design and inventory of nucleoside analogues with favorable antisense properties, herein we describe the synthesis of C5-propynyl-2'-fluoroarabinonucleic acids (FANAP). Incorporation of individual and multiple uridine (FaraUP) and cytidine (FaraCP) inserts into ONs revealed, both stabilized duplexes formed with RNA. In contrast, these modifications demonstrated a negligible (FaraUP) or reduced (FaraCP) effect on DNA binding. Moreover, modified ONs containing these analogues supported E. coli RNase H cleavage of RNA with an altered cleavage pattern observed relative to controls. Moreover, a 2'-O-methoxyethyl (2'-O-MOE) gapmer with a FANAP core was able to elicit RNA cleavage at an increased rate compared to C5-propynyl-arabinonucleic acids (ANAP). Enzymatic hydrolysis of these gapmers was assessed with nuclease S1 digestion and revealed greater stability of ANAP compared to FANAP. These results suggest C5-propynyl ANA/FANA modifications demonstrate promising potential for the design of therapeutic ONs.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent invitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis

An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λPR promoter complexes. Strong position-dependences of overall rate constants (kcat/Km analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.

Hells Promotes Transcription in Lymphoma By Resolving Topological Conflicts and By Preventing DNA Damage

T cell lymphomas (TCLs) are a rare and heterogeneous group of non Hodgkin Lymphoma with aggressive courses and prognoses. TCLs are characterized by genomic instability and massive transcriptional activity that is required to sustain cancer cell proliferation. Indeed, high rate of transcription increases DNA topological tension resulting in accumulation of DNA-RNA hybrid structures called R-Loops, in which the nascent RNA pairs with its DNA-template, favouring structural alterations and DNA-lesions.DNA helicases are a class of enzyme which major function is to alleviate DNA topological stresses by solving R-Loops and protecting DNA from damage. DNA helicases also facilitate RNA-PolymeraseII (RNAPII) progression along the gene body during transcription thus being the major determinant of gene expression.We recently identified the DNA-helicase HELLS - a member of the SWI/SNF2 family- as a new genetic vulnerability of TLCs. HELLS orchestrates a transcriptional program essential to the survival and proliferation of TCLs and its genetic ablation profoundly impairs mitosis and cell proliferation.To understand how HELLS manages and coordinates transcription and to define the HELLS-associated gene expression program in TCLs, we performed RNA-seq in TLBR-2 after the depletion of HELLS (HELLS KD). Gene expression profiling showed that HELLS KD affects the expression of 728 genes (413 downregulated and 315 upregulated genes). These genes were mainly involved in the regulation of cytoskeleton, cell cycle, cytokinesis, chromatin remodelling and DNA repair as indicated by a gene ontology analysis. Next, to evaluate the distribution of chromatin markers after HELLS KD, we performed Chromatin Immunoprecipitation (ChIP) sequencing against active (H3K4me3) and repressive (H3K9me3) histone markers. Depletion of HELLS results in a modest but significant change in the H3K4me3 level at promoters and distal intergenic regions. Differential analysis identified 1,571 bound sites corresponding to 1,278 genes deregulated after HELLS KD. Notably, 59/1,278 genes resulted concordantly deregulated in RNA-seq analysis. Accordingly with the role of HELLS in T lymphocytes development, gene ontology analysis on identified 59 genes reveals enrichment in T cell activation, T-helper 17 differentiation and lineage commitment processes. Instead, no significant changes in H3K9me3 level at examined regulatory regions were observed after HELLS KD, suggesting that HELLS does not modify the distribution of this marker.To deeply study the transcriptional function of HELLS, we investigated its ability to promote transcription by solving DNA topological tension. By performing S9.6 antibody staining on a panel of TCL cells, we showed that loss of HELLS leads to an accumulation of R-Loops that co-localized with the active form of RNAPII (S2-CTD), suggesting that HELLS alleviates RNAPII stall upon collision with R-loops during elongation. Co-immunoprecipitation (Co-IP) experiments showed that HELLS interacts with RNAPII and Chromatin Immunoprecipitation (ChIP) assays in TLBR-2 cells demonstrating that the loss of HELLS leads to changes in the distribution of active form of RNAPII that accumulates on transcriptional starting sites of selective target genes. By immunofluorescence staining, we detected that the decreased RNAPII activity after HELLS KD was associated with a significant decrease in the incorporation of 5-ethynyl uridine (EU) into nascent RNA confirming that transcription process is attenuated across all transcriptome.As R-loops accumulation and persistence is strictly associated with DNA damage, we assessed by immunofluorescences, the level of yH2AX (marker of DNA-damage) upon depletion of HELLS. We observed a significant increase in the intensity of nuclear yH2AX signal and a formation of yH2AX foci in TCL HELLS KD cell lines. Noticeably, accumulating yH2AX foci were observed in correspondence of R-loops and in co-localization with the active form RNAPII (S2-CTD).Collectively, our results indicate that HELLS plays an essential function in supporting TCLs progression promoting gene expression by resolving DNA topological conflicts, easing RNAPII progression and protecting DNA from damaging events simultaneously. These key functions qualify HELLS as a new dependency of TCLs and therefore as potential vulnerability of these lymphomas. DisclosuresNo relevant conflicts of interest to declare.

Mitochondrial Potentiation Ameliorates Age-Related Heterogeneity in Hematopoietic Stem Cell Function.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs invivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

Histone Lysine Demethylases KDM5B and KDM5C Modulate Genome Activation and Stability in Porcine Embryos.

The lysine demethylases KDM5B and KDM5C are highly, but transiently, expressed in porcine embryos around the genome activation stage. Attenuation of KDM5B and KDM5C mRNA hampered embryo development to the blastocyst stage in fertilized, parthenogenetically activated and nuclear transfer embryos. While KDM5B attenuation increased H3K4me2-3 levels on D3 embryos and H3K4me1-2-3 on D5 embryos, KDM5C attenuation increased H3K9me1 on D3 embryos, and H3K9me1 and H3K4me1 on D5 embryos. The relative mRNA abundance of EIF1AX and EIF2A on D3 embryos, and the proportion of D4 embryos presenting a fluorescent signal for uridine incorporation were severely reduced in both KDM5B- and KDM5C-attenuated compared to control embryos, which indicate a delay in the initiation of the embryo transcriptional activity. Moreover, KDM5B and KDM5C attenuation affected DNA damage response and increased DNA double-strand breaks (DSBs), and decreased development of UV-irradiated embryos. Findings from this study revealed that both KDM5B and KDM5C are important regulators of early development in porcine embryos as their attenuation altered H3K4 and H3K9 methylation patterns, perturbed embryo genome activation, and decreased DNA damage repair capacity.

Enhancing Tumor Targeting Efficiency of Radiolabeled Uridine (via) Incorporation into Nanocubosomal Dispersions.

Background: Several nanosystems are currently being utilized to enhance the targeting efficiency of several cancer chemotherapeutic agents. This study was designed to improve tumor accumulation of iodine-125 (125I)-uridine via incorporation into a nanocubosomal preparation. Materials and Methods: Nanocubosomes were prepared with the aid of Glycerol mono-oleate and Pluronic F127. Each prepared nanocubosomal preparation was adequately characterized by testing their particle size, polydispersity index (PDI), ζ potential (ZP), and transmission electron microscopy. The radiolabeling of uridine with 125I was attempted using several oxidizing agents to achieve a high radiochemical yield, and the factors affecting the reaction yield were studied in detail. A comparative biodistribution study of free 125I-uridine and 125I-uridine loaded nanocubosomes was performed in normal and tumor bearing mice. The biodistribution was evaluated by intravenous injection of the sterile test solution, and animals were anesthetized and dissected at different time intervals postinjection (p.i.). Results: 125I-uridine was obtained in a high radiochemical yield (92.5% ± 0.8%). Afterward, 125I uridine was incorporated in a selected nanocubosome formulation, which showed nanosized cubic particles (178.6 ± 0.90 nm) with PDI (0.301 ± 0.04) and a ZP (34.35 ± 0.4). The biodistribution studies revealed that 125I-uridine nanocubosomes showed higher tumor localization (3.1 ± 0.4%IA/g at 2 h p.i. and a tumor/muscle ratio of 6.2) compared with the free 125I-uridine (2.7% ± 0.4%IA/g at 2 h p.i. and a tumor/muscle ratio of 3.3). Conclusion: The results of this study confirmed that 125I-uridine loaded nanocubosome had better efficiency in targeting the tumor site, which makes it an adequate targeting agent for tumor imaging.

Imaging nascent transcription in wholemount vertebrate embryos to characterize zygotic genome activation.

A major event in early embryo development is the awakening of the embryonic genome, a process of large-scale transcriptional induction termed zygotic genome activation (ZGA). To understand how ZGA is controlled temporally and spatially, tools are required to image and quantify nascent transcription in wholemount embryos. In this chapter, we describe a metabolic labeling approach that leverages 5-ethynyl uridine (5-EU) incorporation into newly transcribed RNAs. Subsequently, click chemistry is used to conjugate these nascent transcripts to fluorophores for wholemount confocal imaging or biotin for RNA sequencing. Such an approach facilitates direct visualization of the global transcriptional state of each cell during early embryogenesis and provides a spatial map of gene expression activity. We describe this procedure for imaging nascent transcription in a vertebrate embryo Xenopus laevis, and use it as our model the onset of large-scale ZGA. Unlike cell culture systems in which 5-EU can be added to the media, metabolic labeling in Xenopus embryos requires microinjection in one-cell or two-cell stage embryos. This method is a powerful tool to quantify the nascent transcriptome at a single-cell level and to dissect mechanisms that control ZGA. We propose that this methodology can be applied broadly in other embryonic systems, and demonstrate the feasibility using zebrafish cleavage stage embryos. Finally, we demonstrate how to sequence the nascent transcriptome via 5-EU incorporation and separation of zygotic vs maternal RNAs. Altogether, our generalizable methodology will facilitate new insights into gene regulation and spatial patterning of ZGA during early embryogenesis.

CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane‐induced alopecia

Taxanes are a leading cause of severe and often permanent chemotherapy‐induced alopecia. As the underlying pathobiology of taxane chemotherapy‐induced alopecia remains poorly understood, we investigated how paclitaxel and docetaxel damage human scalp hair follicles in a clinically relevant ex vivo organ culture model. Paclitaxel and docetaxel induced massive mitotic defects and apoptosis in transit amplifying hair matrix keratinocytes and within epithelial stem/progenitor cell‐rich outer root sheath compartments, including within Keratin 15+ cell populations, thus implicating direct damage to stem/progenitor cells as an explanation for the severity and permanence of taxane chemotherapy‐induced alopecia. Moreover, by administering the CDK4/6 inhibitor palbociclib, we show that transit amplifying and stem/progenitor cells can be protected from paclitaxel cytotoxicity through G1 arrest, without premature catagen induction and additional hair follicle damage. Thus, the current study elucidates the pathobiology of taxane chemotherapy‐induced alopecia, highlights the paramount importance of epithelial stem/progenitor cell‐protective therapy in taxane‐based oncotherapy, and provides preclinical proof‐of‐principle in a healthy human (mini‐) organ that G1 arrest therapy can limit taxane‐induced tissue damage.

A Metabolic Engineering Approach to Incorporate Modified Pyrimidine Nucleosides into Cellular RNA.

The incorporation of modified nucleotides into RNA is a powerful strategy to probe RNA structure and function. While a wide variety of modified nucleotides can be incorporated into RNA in vitro using chemical or enzymatic synthesis, strategies for the metabolic incorporation of artificial nucleotides into cellular RNA are limited, largely due to the incompatibility of modified nucleobases and nucleosides with nucleotide salvage pathways. In this work, we develop a metabolic engineering strategy to facilitate the labeling of cellular RNA with noncanonical pyrimidine nucleosides. First, we use structure-based protein engineering to alter the substrate specificity of uridine-cytidine kinase 2 (UCK2), a key enzyme in the pyrimidine nucleotide salvage pathway. Next, we show that expression of mutant UCK2 in HeLa and U2OS cells is sufficient to enable the incorporation of 5-azidomethyl uridine (5-AmU) into cellular RNA and promotes RNA labeling by other C5-modified pyrimidines. Finally, we apply UCK2-mediated RNA labeling with 5-AmU to study RNA trafficking and turnover during normal and stress conditions and find diminished RNA localization in the cytosol during arsenite stress. Taken together, our study provides a general strategy for the incorporation of modified pyrimidine nucleosides into cellular RNA and expands the chemical toolkit of modified bases for studying dynamic RNA behavior in living cells.

Visualization of global RNA synthesis in a human (mini-) organ in situ by click chemistry.

RNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. EU differentially incorporated across the HF epithelium.Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression. By treating human HFs with the cytotoxic cell cycle inhibitor (R)-CR8, which inhibits transcriptional regulators CDK7 and CDK9, it was further shown that this technique can be used to sensitively detect changes in global RNA synthesis in situ. Together, this work delineates new insights into nascent RNA synthesis within a human (mini)- organ and describes a novel read-out parameter that will enrich future ex vivo human tissue research studies.

Peptidylarginine deiminase 1-catalyzed histone citrullination is essential for early embryo development.

Peptidylarginine deiminase (PADI) enzymes are increasingly being associated with the regulation of chromatin structure and gene activity via histone citrullination. As one of the PADI family members, PADI1 has been mainly reported to be expressed in the epidermis and uterus, where the protein in keratinocytes is thought to promote differentiation by citrullinating filament proteins. However, the roles of PADI1 in preimplantation development have not been addressed. Using a PADI1-specific inhibitor and Padi1-morpholino knockdown, we found that citrullination of histone tails at H4R3 and H3R2/8/17 were markedly reduced in the 2- and 4-cell embryos. Consistent with this observation, early embryo development was also arrested at the 4-cell stage upon depletion of PADI1 or inhibition of PADI1 enzyme activity. Additionally, by employing 5-ethynyl uridine (EU) incorporation analysis, ablation of PADI1 function led to a dramatic decrease in overall transcriptional activity, correlating well with the reduced levels of phosphorylation of RNA Pol II at Ser2 observed at 2- or 4-cell stage of embryos under Padi1 knockdown or inhibiting PADI1. Thus, our data reveal a novel function of PADI1 during early embryo development transitions by catalyzing histone tail citrullination, which facilitates early embryo genome transactivation.

Correlation between astrocyte activity and recovery from blood-brain barrier breakdown caused by brain injury.

Glial activation is associated with cell proliferation and upregulation of astrocyte marker expression following traumatic injury in the brain. However, the biological significance of these processes remains unclear. In the present study, astrocyte activation was investigated in a murine brain injury model. Brain injury induces blood-brain barrier (BBB) breakdown and immunoglobulin G (IgG) leak into the brain parenchyma. The recovery of BBB breakdown was evaluated by analyzing immunofluorescent staining with mouse IgG antibody. IgG leakage was greatest at 1 day after stab wound injury and decreased thereafter, and almost diminished after 7 days. Bromodeoxy uridine incorporation was used, and astrocyte proliferation rates were examined by coimmunostaining with anti-bromodeoxy uridine and anti-glial fibrillary acid protein antibodies. Consistent with IgG leakage assays, astrocyte activation was the highest at day 3 and decreased after 7 days. Moreover, in reverse transcriptase-quantitative-PCR experiments, genes associated with BBB integrity were downregulated immediately after BBB breakdown and recovered to basal expression levels within 7 days. These data indicated that astrocyte activation correlated with BBB recovery from breakdown following brain injury.

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability.

KM mutant highlights enhancers in minor ZGA

Before the first cell division, a fertilized embryo, also known as a zygote, contains 2 distinct nuclei called the paternal and maternal pronucleus (PN). During the zygotic period, the paternal PN undergoes massive epigenetic reprogramming characterized by removing protamines and establishing new epigenetic marks to acquire totipotency, whereas the epigenetic alteration of maternal PN is less dynamic. Reflecting this difference, the timing and levels of global gene transcription in the zygotic stage, called “minor zygotic gene activation (ZGA),” differ between paternal and maternal PNs,1 suggesting that the mechanism of transcriptional regulation is asymmetric. Consistent with this idea, several epigenetic events occur uniquely in each PN. For instance, H3.3, an H3 variant that is incorporated into chromatin independently of DNA replication, is the best characterized in zygotes due to its preferential incorporation into paternal chromatin soon after fertilization. A few studies have demonstrated the critical role of H3.3 methylation in early embryonic development by overexpressing the lysine-arginine (K-R) substitutions: e.g., H3.3-K27R and H3.3-K36R, in which the 27th and 36th lysine residues (respectively) are not methylated.2,3 However, unlike in yeast, genetic disruption of specific lysine residues in mammalian histones is impractical, since they are multiply encoded in the genome.

Abstract 343: Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia

Abstract Recently, deep sequencing technology identified SF3B1, a gene encoding a splicing factor, is recurrently mutated in chronic lymphocytic leukemia (CLL). Moreover, its mutation was associated with rapid disease progression and resistance to treatment. SF3B1 is a critical component of the spliceosome, leading to the potential of splicing regulation in CLL pathogenesis. However, the mechanism by which SF3B1 mutations affect CLL disease progression is not known. CLL cells are characterized by their resistance to apoptosis, driven mostly by the overexpression of the Bcl-2 family anti-apoptotic proteins. Many members of the family have multiple splicing variants, sometimes encoding proteins with totally opposite functions. For example, the alternatively spliced, shorter variant of the anti-apoptotic protein Mcl-1, Mcl-1S, resembles BH3-only proteins and induces apoptosis. Using herboxidiene, an inhibitor of SF3B1, we explored the activity of SF3B1 in splicing control of Mcl-1 in primary CLL cells. Primers and probes were designed to specifically detect the Mcl-1 pre-mRNA, the anti-apoptotic Mcl-1 and pro-apoptotic Mcl-1S by real-time RT-PCR. We found that inhibition of SF3B1 reduced the level of Mcl-1 in a concentration dependent manner, which was accompanied by the simultaneous surge of Mcl-1S mRNA by over 10 fold. Immunoblots confirmed reduction of Mcl-1L and concurrent increase of Mcl-1S protein. These results indicated a major role of SF3B1 in Mcl-1 splicing control toward the anti-apoptotic variant. In contrast, although also depleting the Mcl-1 mRNA, the Cdk9 inhibitor SNS-032 reduced both the long and short forms of Mcl-1, as well as its pre-mRNA, consistent with its inhibition on transcription. Uridine incorporation measurements showed inhibition of total RNA synthesis by herboxidiene at concentrations above 30 nM, but dramatic reversion of Mcl-1 splicing was observed at 10 nM, which did not affect RNA synthesis, suggesting that the major mechanism of herboxidiene in CLL cells was modification of Mcl-1 splicing. As CLL cells are critically dependent on Mcl-1 for survival, reducing its level induced mitochondrial membrane permeabilization and apoptosis, which was further accelerated by the accumulation of Mcl-1S. Apoptosis occurred in a time- and concentration- dependent manner, with IC50 of less than 100 nM after 24 hr of incubation. Herboxidiene toxicity was selective to CLL B cells, while sparing the normal lymphocytes from healthy donors. Herboxidiene was equally toxic to CLL cells harboring SF3B1 mutations compared to cells carrying the wild-type SF3B1 (n=5 each). Real-time RT-PCR showed similar increase in the ratio of short to long forms of Mcl-1 by herboxidiene in these two groups. Together, these studies identified SF3B1 mediated splicing control as a novel target for CLL therapy. Further investigations are needed to understand the mechanism of SF3B1 mutations on the control of mRNA splicing. Citation Format: Rong Chen, Yuling Chen, Emanuela M. Ghia, Laura Rassenti, Thomas Kipps, Michael J. Keating, William G. Wierda, William Plunkett. Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2014-343

Differences in immune cell function between tuberculosis positive and negative Asian elephants

Tuberculosis is an important health concern for Asian elephant (Elephas maximus) populations worldwide, however, mechanisms underlying susceptibility to Mycobacterium tuberculosis are unknown. Proliferative responses assessed via brominated uridine incorporation and cytokine expression measured by real-time RT-PCR were evaluated in peripheral blood mononuclear cell (PBMC) cultures from 8 tuberculosis negative and 8 positive Asian elephants. Cultures were stimulated with Mycobacterium bovis purified protein derivative (PPD-B), M.tuberculosis culture filtrate protein (CFP)-10, and Mycobacterium avium PPD (PPD-A). Following stimulation with PPD-B, proliferation was higher (α=0.005) in positive samples; no significant differences were detected following CFP-10 or PPD-A stimulation. Tumor necrosis factor (TNF)-α, interleukin (IL)-12, and interferon (IFN)-γ expression was greater in samples from positive elephants following stimulation with PPD-B (α=0.025) and CFP-10 (α=0.025 TNF-α and IL-12; α=0.005 IFN-γ). Stimulation with PPD-A also produced enhanced IL-12 expression in positive samples (α=0.025). Findings suggested that differences in immune cell function exist between tuberculosis positive and negative elephants. Proliferative responses and expression of TNF-α, IL-12, and IFN-γ in response to stimulation with PPD-B and CFP-10 differ between tuberculosis positive and negative elephants, suggesting these parameters may be important to tuberculosis immunopathogenesis in this species.

Autoradiographic analysis of the effect of cytokinin on protein and RNA syntheses in the Ceratodon purpureus protonema

In the protonema of <em>Ceratodon purpureus</em> (Hedw.) Brid., apical parts of the protonemal filaments (apical cells, initials of protonemal side branches and of gametophore buds) proved to be preferential sites of [<sup>14</sup>C]-leucine incorporation into proteins. In some filaments, a similar preference for [<sup>3</sup>H]-uridine incorporation into RNA was observed, whereas in others there was a rather uniform distribution of label over all cells. A short (0.5-2 h) treatment with cytokinin (N<sup>6</sup>-2-isopentenyladenine) enhanced [<sup>14</sup>C]-leucine incorporation, without changing the relative distribution of label. No such enhancement, as well as no change in label distribution could be observed in [<sup>3</sup>H]-uridine incorporation. No direct relationship seems to exist between the early promotion of protein synthesis by cytokinin in the protonema and cytokinin induction of gametophore buds.