To determine if the BAIT transcript has a function in regulating PUMA expression, the BAIT transcript was knocked down using an antisense oligonucleotide (ASO) manufactured by ISIS Pharmaceuticals®. Five ASOs targeting sequences at various distances relative to the transcription start site (TSS) of BAIT were tested (+18, +22, +46, +182, +610) (Figure 14).
Figure 14: Location of ASOs relative to the transcription start site. The ASOs were generated by ISIS Pharmaceuticals®. This strand is the sense orientation of the PUMA locus around Exon 3. The top purple strand is the ASO sequence that binds to the RNA sequence produced from antisense transcription of the bottom purple strand.
The ASOs have a DNA sequence that is identical to the sense strand of the PUMA sequence, thus complementarily binding the BAIT antisense RNA transcript. The ASOs have a phosphorothioate backbone that protects from nuclease degradation. When the ASO-DNA duplex is formed with the BAIT RNA, the duplex is degraded by the nuclear RNase-H enzyme.
A successful transfection with minimal cell death of the non-targeting ASO tagged with Texas Red Malemide (NT-TR) into cells was observed with microscopy using 15 uL of Lipofectamine LTX + reagent (Supplementary Figure 2). Lipofectamine LTX + treatment alone did not significantly change the level of BAIT or PUMA expression (Supplemental Figure 3). The 5 ASOs were then transfected into cells using Lipofectamine LTX + (vehicle), then treated with either DMSO or Nutlin at 12 hours transfection and harvested at 24 hours post-transfection (Figure 15A). BAIT expression, PUMA expression at the 2:3 and 3:4 exon junctions, and p21 expression was measured using qRT-PCR analysis (Figure 15B). As anticipated, BAIT expression decreased in Nutlin compared to DMSO in conditions with a NT ASO. +18 and +46 ASOs significantly knocked down BAIT expression in cells exposed to Nutlin. This decrease in BAIT expression caused at least 40% reduction of PUMA exon 2:3 and 3:4 junctions in Nutlin.
Although the knockdown of BAIT in DMSO, by +18, +22, +46, and +182, was reduced by about 50%, it was not statistically significant. However, the DMSO BAIT knockdowns caused about a 70% reduction in PUMA exon 2:3 and exon 3:4 junction by 70%. The knockdown of BAIT showed a decreased expression in PUMA, suggesting that BAIT contributes to a positive regulation of PUMA expression.
Figure 15: BAIT expression knockout decreases expression of PUMA E2:E3 and E3:E4 junctions. (A) Timeline of ASO experiment. The HCT116 cells were transfected with 200uM of the ASO. At 24 hours they were treated with DMSO or Nutlin.
Cells were harvested 12 hours post-treatment. (B) Significant reduction in expression: BAIT in Nutlin – +18 (57%) and +46 (63%).
PUMA E2:E3 junction in DMSO – +18 (73%), +22 (68%), +46 (75%), +182 (67%); in Nutlin – +82 (50%), +46 (47%), +182 (39%).
PUMA E3:E4 junction in DMSO – +18 (75%), +46 (70%); in Nutlin – +18 (37%), +22 (29%). * Signifies a difference relative to NT – specific to DMSO (blue) or Nutlin (red) by T-Test with p < .05.
DISCUSSION
The transcription factor p53 is a key tumor suppressor protein that regulates a transcriptional program mediating diverse cellular responses to stress including cell cycle arrest
and programmed cell death. p21 and PUMA are two main targets of p53 that are induced in the presence of cellular stress signals, such as radiation, genotoxic agents, hypoxia, or mutations resulting in oncogenic signaling. Both p21 and PUMA harbor the potential to tip the cell towards arrest (p21) or apoptosis (PUMA). Since the p53 network is inactivated in over 90% of cancers (either by p53 mutation or alterations upstream of p53), these pathways downstream of p53 are important to understand for targeted cancer therapies. Investigating p53s gene networks could provide insight for new therapeutic strategies that bypass p53 to artificially activate desired outcomes such as cell death of cancer cells. This idea has been demonstrated by a class of cancer drugs named BH3 mimetics, which act like PUMA and are able to inhibit the pro-survival mitochondrial proteins of the intrinsic apoptotic pathway (Chonghaile and Letai 2009). Attention has also turned to PUMA in hopes of understanding p53-autonomous regulatory mechanisms for possible exploitation of new cancer therapies. Using GRO-seq data to measure active and strand-specific RNA Pol II movement along the genomic locus of PUMA, we found evidence of antisense transcription initiating 6 Kb into the locus (Allen et al. 2014). In general, natural antisense transcripts (NATs), such as this one are characterized as being non-coding RNAs (ncRNAs), which in turn constitute 90% of the transcribed material within the genome (Mattick and Makunin 2006; Prasanth and Spector 2007). Currently, much effort is devoted to understanding the potential functions these antisense transcripts may have in regulating their corresponding sense transcript. Some of the investigated members of this class of ncRNAs function by regulating the sense transcript through RNA masking, RNA interference, transcriptional interference, and epigenetic modifications (Beiter 2008). Here, I investigated a newly identified NAT, dubbed BAIT, as a possible gene-specific regulator of the PUMA locus.
The identification of two putative TATA boxes in close proximity to the transcription start site for BAIT antisense transcription identified by GRO-seq prompted an investigation to understand if this region was a functional promoter region for BAIT. The proximal and distal TATA boxes were engineered to contain single point mutations that modify the third and fourth base in the motif, which was previously shown to diminish their activity within the promoter (T159A, A160G, T175A, and A176G) (Wobbe and Struhl 1990). The activity of these mutated TATA boxes were compared to the WT region using a reporter assay. We observed that the activity of the promoter region decreases by about half when either the proximal or distal TATA boxes were mutated at the fourth position and when the proximal TATA box contained a T switched to an A at the third position. This result suggests that the proximal TATA box within the BAIT promoter region is necessary to drive expression in this system, but is inconclusive regarding the distal TATA box.
Previously it has been reported that the level of antisense transcription fluctuates in different cell types and in response to different stimuli (Beiter 2008). Similarly we demonstrated this variability in the expression of BAIT. We detected BAIT in all eight cell lines tested; however, the level of BAIT expression varied widely among these eight lines. The expression ranged over a four-fold difference from A549s, which express BAIT at the lowest levels, to HCT116s, which demonstrate the highest levels of BAIT expression. Not only were BAIT levels different between cell lines, but its expression level changed in response to different stimuli in a single cell line, HCT116. We demonstrated that BAIT expression decreases in response to gamma-IR and is unaltered after UVC exposure. These conditions do not increase PUMA expression at the RNA level, but they do increase PUMA induction at the protein level.
However, this experiment is confounded by the low level of p53 induction in the presence of gamma-IR and UVC.
Although we observed that BAIT consistently decreases in response to Nutlin, we found it increases in response to 5FU. This surprising result increases the complexity of the PUMA/BAIT relationship since there are similar increases in p53, PUMA, and p21 at the RNA and protein level of cells treated with both Nutlin and 5FU. We expected that activating the PUMA locus by p53 would have a similar effect on BAIT regardless of the activating agent. We thus wanted to address the expression of BAIT in the absence of p53. We took advantage of RNA extracts previously harvested from HCT116 p53 +/+ and -/- cells treated with DMSO, 5FU, or Dox, generated by Veronica Dengler of the Espinosa lab. BAIT expression dramatically increased as expected in the p53 +/+ cells, but unexpectedly, BAIT expression after 5FU exposure in the p53 -/- cells increased immensely. This result demonstrates that BAIT expression is not activated by p53 and suggests that additional unknown regulators contribute to its expression. The toxic nature of 5FU, a genotoxic agent employed in chemotherapy, could potentially induce an additional transcription factor, beyond p53-induced by Nutlin, that is promoting or stabilizing the expression of BAIT.
To explore the function of BAIT we used antisense oligonucleotides to knockdown BAIT in HCT116 p53 +/+ cells exposed to DMSO or Nutlin. Knockdown of BAIT expression by about 50% in both DMSO and Nutlin, in turn decreased mature PUMA RNA expression as measured by both exon 2:3 and 3:4 splice junctions. These data suggest that BAIT contributes to the up-regulation of PUMA at some point upstream of the 2:3 junction splicing, perhaps at the PUMA promoter region. BAIT could potentially do this by recruiting or guiding epigenetic modifiers,
such as histone modifications or chromatin remodeling complexes, to regions which promotes PUMA expression. To test this hypothesis, chromatin immunoprecipitation (ChIP) analysis of histone modificatiers and chromatin complexes at the PUMA locus in cells with the BAIT ASO compared to NT, could be employed. This hypothesis would have to be validated in multiple cell lines and in alternative stress conditions. It would also be important to observe BAIT knockdown in 5FU, since its expression is upregulated in that condition. Unfortunately, I have been unable to achieve a consistent stable knockdown in 5FU. Another angle to manipulate BAIT expression would be to create a knockout of BAIT, using CRISPRs designed to delete the proximal functional TATA box within the BAIT promoter region.
Investigating BAIT as a regulatory NAT for PUMA has provided more questions than answers. Regrettably, I am unable to provide a specific mechanism in which knockdown of BAIT causes a reduction of PUMA expression, but this study has provided initial evidence for a functional regulatory SAS pair within the PUMA locus. Adding natural antisense transcription as an additional level to gene regulation provides an alternative way to approach cancer therapies.
Thus, if further investigation revealed a mechanism by which BAIT is able to regulate PUMA expression, it could add an alternative angle to activate PUMA, and thus cell death, in cancer cells.
SUPPLEMENTARY FIGURES
Supplementary Figure 1: BBC3 (PUMA) Locus. The PUMA locus showing a compilation of the important features and regions referred to in this thesis.
Supplementary Figure 2: 15ul Lipofectamine is optimal for an effective transfection and minimal cell death. The NT ASO with a Texas Red tag was created for a control to observe which amount (15μl of 25 μl) of Lipofectamine LTX + was required for uptake of the ASO into the cell with the least toxicity.
Supplementary Figure 3: Lipofectamine LTX + reagent does not affect BAIT, PUMA, or p21 expression. qRT-PCR analysis showed no significant change in BAIT, PUMA exon 2:3 and 3:4, and p21 levels in Lipo / LTX + compared to untreated. This is the same blot as Figure 11 but including the Lipo/LTX well. * Signifies a difference from the untreated by T-Test with p < .05.
ACKNOWLEDGMENTS
Thank you to Dr. Joaquín Espinosa for the incredible opportunity to learn and work in his laboratory as well as his support throughout my educational experience and project. His passion for science and research is motivational.
Thank you to Dr. Hestia Mellert for being an invaluable, inspirational, and supportive mentor. This project would not have been possible without her guidance, teachings, and assistance with experimental design. Thank you for allowing me to make mistakes and through each one I have learned to be better scientist and ask more questions.
Thank you to Amber Johnson, Liz Terhune and Veronica Dengler for helping finalize my thesis and their enthusiastic continual support. Thank you to the entire Espinosa lab for sharing your passion for science, expertise, and knowledge of cancer biology. Each one of them has helped motivate my interest in research and taught me the devotion required to be a research scientist.
Thank you to my family (Jim, Katy, and Jake Michael) and David Franklin for their endless moral support.
BIBLIOGRAPHY
Allen, Mary Ann et al. “Global Analysis of p53-Regulated Transcription Identifies Its Direct Targets and Unexpected Regulatory Mechanisms.” eLife 3 (2014): n. pag. PubMed Central. Web.
American Cancer Society. Cancer Facts & Figures 2014. Atlanta: American Cancer Society, 2014.
Print.
“Apoptosis Signaling Pathways.” R&D Systems. N.p., 2012. Web.
Attardi, Laura D. et al. “PERP, an Apoptosis-Associated Target of p53, Is a Novel Member of the PMP-22/gas3 Family.” Genes & Development 14.6 (2000): 704–718. genesdev.cshlp.org.
Web.
Ball, Kathryn L. et al. “Cell-Cycle Arrest and Inhibition of Cdk4 Activity by Small Peptides Based on the Carboxy-Terminal Domain of p21WAF1.” Current Biology 7.1 (1997): 71–80.
www.cell.com. Web.
Barski, Artem et al. “High-Resolution Profiling of Histone Methylations in the Human Genome.”
Cell 129.4 (2007): 823–837. ScienceDirect. Web.
Beiter, T. et al. “Antisense Transcription: A Critical Look in Both Directions.” Cellular and Molecular Life Sciences 66.1 (2008): 94–112. link.springer.com. Web.
Beltran, Manuel et al. “A Natural Antisense Transcript Regulates Zeb2/Sip1 Gene Expression during Snail1-Induced Epithelial–mesenchymal Transition.” Genes & Development 22.6 (2008): 756–769. PubMed Central. Web.
Borner, Christoph. “The Bcl-2 Protein Family: Sensors and Checkpoints for Life-or-Death Decisions.” Molecular Immunology 39.11 (2003): 615–647. ScienceDirect. Web.
Borsani, Omar et al. “Endogenous siRNAs Derived from a Pair of Natural Cis-Antisense Transcripts Regulate Salt Tolerance in Arabidopsis.” Cell 123.7 (2005): 1279–1291.
PubMed Central. Web.
Bouvard, V. et al. “Tissue and Cell-Specific Expression of the p53-Target Genes: Bax, Fas, mdm2 and waf1/p21, before and Following Ionising Irradiation in Mice.” Oncogene 19.5 (2000):
649–660. NCBI PubMed. Web.
Brown, J. P., W. Wei, and J. M. Sedivy. “Bypass of Senescence after Disruption of p21CIP1/WAF1 Gene in Normal Diploid Human Fibroblasts.” Science (New York, N.Y.) 277.5327 (1997):
831–834. Print.
Chen, Jianjun et al. “Genome-Wide Analysis of Coordinate Expression and Evolution of Human Cis-Encoded Sense-Antisense Transcripts.” Trends in Genetics 21.6 (2005): 326–329.
ScienceDirect. Web.
Chonghaile, T. Ni, and A. Letai. “Mimicking the BH3 Domain to Kill Cancer Cells.” Oncogene 27.S1 (2009): S149–S157. www.nature.com. Web.
Consortium, International Human Genome Sequencing. “Finishing the Euchromatic Sequence of the Human Genome.” Nature 431.7011 (2004): 931–945. www.nature.com. Web.
Gomes, Nathan P., and Joaquín M. Espinosa. “Gene-Specific Repression of the p53 Target Gene PUMA via Intragenic CTCF-Cohesin Binding.” Genes & Development 24.10 (2010): 1022–
1034. NCBI PubMed. Web.
Han, J. et al. “Expression of bbc3, a pro-Apoptotic BH3-Only Gene, Is Regulated by Diverse Cell Death and Survival Signals.” Proceedings of the National Academy of Sciences of the United States of America 98.20 (2001): 11318–11323. NCBI PubMed. Web.
Hastings, Michelle L. et al. “Post-Transcriptional Regulation of Thyroid Hormone Receptor Expression by Cis-Acting Sequences and a Naturally Occurring Antisense RNA.” Journal of Biological Chemistry 275.15 (2000): 11507–11513. www.jbc.org. Web.
Haupt, Susan et al. “Apoptosis - the p53 Network.” Journal of Cell Science 116.Pt 20 (2003):
4077–4085. NCBI PubMed. Web.
Heckman, Karin L., and Larry R. Pease. “Gene Splicing and Mutagenesis by PCR-Driven Overlap Extension.” Nature Protocols 2.4 (2007): 924–932. www.nature.com. Web.
Heinz, S. et al. “Impact of Natural Genetic Variation on Enhancer Selection and Function.”
Nature 503.7477 (2013): 487–492. PubMed Central. Web.
Hikisz, Paweł, and Zofia M. Kiliańska. “PUMA, a Critical Mediator of Cell Death--One Decade on from Its Discovery.” Cellular & Molecular Biology Letters 17.4 (2012): 646–669. NCBI PubMed. Web.
Hobson, David J. et al. “RNA Polymerase II Collision Interrupts Convergent Transcription.”
Molecular Cell 48.3 (2012): 365–374. PubMed Central. Web.
Kapranov, Philipp, Aarron T. Willingham, and Thomas R. Gingeras. “Genome-Wide Transcription and the Implications for Genomic Organization.” Nature Reviews Genetics 8.6 (2007):
413–423. www.nature.com. Web.
Karp, Gerald. Cell and Molecular Biology: Concepts and Experiments. 6th edition. Hoboken, NJ:
Wiley, 2009. Print.
Kouzarides, Tony. “Chromatin Modifications and Their Function.” Cell 128.4 (2007): 693–705.
ScienceDirect. Web.
Lander, Eric S. et al. “Initial Sequencing and Analysis of the Human Genome.” Nature 409.6822 (2001): 860–921. www.nature.com. Web.
Lapidot, Michal, and Yitzhak Pilpel. “Genome-Wide Natural Antisense Transcription: Coupling Its Regulation to Its Different Regulatory Mechanisms.” EMBO Reports 7.12 (2006):
1216–1222. PubMed Central. Web.
Li, Yuan-Yuan et al. “In Silico Discovery of Human Natural Antisense Transcripts.” BMC Bioinformatics 7 (2006): 18. PubMed Central. Web.
Ma, Wenzhe et al. “A Pivotal Role for p53: Balancing Aerobic Respiration and Glycolysis.”
Journal of Bioenergetics and Biomembranes 39.3 (2007): 243–246. NCBI PubMed. Web.
MacLachlan, Timothy K., and Wafik S. El-Deiry. “Apoptotic Threshold Is Lowered by p53 Transactivation of Caspase-6.” Proceedings of the National Academy of Sciences of the United States of America 99.14 (2002): 9492–9497. PubMed Central. Web.
Mattick, John S., and Igor V. Makunin. “Non-Coding RNA.” Human Molecular Genetics 15.suppl 1 (2006): R17–R29. hmg.oxfordjournals.org. Web.
Ming, Lihua et al. “Sp1 and p73 Activate PUMA Following Serum Starvation.” Carcinogenesis 29.10 (2008): 1878–1884. PubMed Central. Web.
Moll, Ute M., and Oleksi Petrenko. “The MDM2-p53 Interaction.” Molecular Cancer Research 1.14 (2003): 1001–1008. Print.
Munroe, S. H., and J. Zhu. “Overlapping Transcripts, Double-Stranded RNA and Antisense Regulation: A Genomic Perspective.” Cellular and Molecular Life Sciences CMLS 63.18 (2006): 2102–2118. link.springer.com. Web.
Muzio, M. “Signalling by Proteolysis: Death Receptors Induce Apoptosis.” International Journal of Clinical & Laboratory Research 28.3 (1998): 141–147. Print.
Nakano, K., and K. H. Vousden. “PUMA, a Novel Proapoptotic Gene, Is Induced by p53.”
Molecular Cell 7.3 (2001): 683–694. Print.
Neeman, Yossef et al. “Is There Any Sense in Antisense Editing?” Trends in Genetics 21.10 (2005): 544–547. ScienceDirect. Web.
Oeder, Sebastian et al. “Uncovering Information on Expression of Natural Antisense Transcripts in Affymetrix MOE430 Datasets.” BMC Genomics 8 (2007): 200. PubMed Central. Web.
Olivier, Magali, Monica Hollstein, and Pierre Hainaut. “TP53 Mutations in Human Cancers:
Origins, Consequences, and Clinical Use.” Cold Spring Harbor Perspectives in Biology 2.1 (2010): n. pag. PubMed Central. Web.
Prasanth, Kannanganattu V., and David L. Spector. “Eukaryotic Regulatory RNAs: An Answer to the ‘genome Complexity’ Conundrum.” Genes & Development 21.1 (2007): 11–42.
genesdev.cshlp.org. Web.
“Research Apoptosis.” Genetech BioOncology. N.p., n.d. Web.
Sax, Joanna K. et al. “BID Regulation by p53 Contributes to Chemosensitivity.” Nature Cell Biology 4.11 (2002): 842–849. www.nature.com. Web.
Shearwin, Keith E., Benjamin P. Callen, and J. Barry Egan. “Transcriptional Interference – a Crash Course.” Trends in genetics : TIG 21.6 (2005): 339–345. PubMed Central. Web.
Tommasino, Massimo et al. “The Role of TP53 in Cervical Carcinogenesis.” Human Mutation 21.3 (2003): 307–312. Wiley Online Library. Web.
Trojer, Patrick, and Danny Reinberg. “Facultative Heterochromatin: Is There a Distinctive Molecular Signature?” Molecular Cell 28.1 (2007): 1–13. ScienceDirect. Web.
Vassilev, Lyubomir T. et al. “In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2.” Science (New York, N.Y.) 303.5659 (2004): 844–848. NCBI PubMed. Web.
Vousden, Karen H., and Carol Prives. “Blinded by the Light: The Growing Complexity of p53.”
Cell 137.3 (2009): 413–431. NCBI PubMed. Web.
Wang, Peng, Jian Yu, and Lin Zhang. “The Nuclear Function of p53 Is Required for
PUMA-Mediated Apoptosis Induced by DNA Damage.” Proceedings of the National Academy of Sciences of the United States of America 104.10 (2007): 4054–4059. NCBI PubMed. Web.
Watanabe, Toshiaki et al. “Endogenous siRNAs from Naturally Formed dsRNAs Regulate Transcripts in Mouse Oocytes.” Nature 453.7194 (2008): 539–543. www.nature.com.
Web.
Weber, Michael et al. “Distribution, Silencing Potential and Evolutionary Impact of Promoter DNA Methylation in the Human Genome.” Nature Genetics 39.4 (2007): 457–466.
www.nature.com. Web.
Weinberg, Robert A. The Biology of Cancer. 1st ed. New York: Garland Science, 2006. Print.
Wight, Megan, and Andreas Werner. “The Functions of Natural Antisense Transcripts.” Essays in biochemistry 54 (2013): 91–101. PubMed Central. Web.
Wobbe, C R, and K Struhl. “Yeast and Human TATA-Binding Proteins Have Nearly Identical DNA Sequence Requirements for Transcription in Vitro.” Molecular and Cellular Biology 10.8 (1990): 3859–3867. Print.
World Health Organization. Noncommunicable Diseases (NCD) Country Profiles. N.p., 2014.
Web.
Yelin, Rodrigo et al. “Widespread Occurrence of Antisense Transcription in the Human Genome.” Nature Biotechnology 21.4 (2003): 379–386. www.nature.com. Web.
You, Han et al. “FOXO3a-Dependent Regulation of Puma in Response to Cytokine/growth Factor Withdrawal.” The Journal of Experimental Medicine 203.7 (2006): 1657–1663. PubMed Central. Web.
Yu, J. et al. “PUMA Induces the Rapid Apoptosis of Colorectal Cancer Cells.” Molecular Cell 7.3 (2001): 673–682. Print.
Yu, J., and L. Zhang. “PUMA, a Potent Killer with or without p53.” Oncogene 27 Suppl 1 (2008):
S71–83. NCBI PubMed. Web.
Yu, Wenqiang et al. “Epigenetic Silencing of Tumour Suppressor Gene p15 by Its Antisense RNA.” Nature 451.7175 (2008): 202–206. PubMed Central. Web.