I have generated several reagents to be used for overexpression and inhibition experiments of Gtl2 lncRNA in cardiomyocytes and in the murine heart.
Furthermore, I have established the efficacy of these expression vectors. However, the effectiveness of the additional Gtl2 constructs remain to be
determined. Future directions include pursuing the use of shMEG3 for knocking down Gtl2 lncRNA expression levels in NRVMs. Since shMEG3 was only used once to knockdown Gtl2 expression levels basally (Figure 3.1.2), this experiment would need to be replicated several times to confirm that the knockdown is reproducible. Once robust knockdown of Gtl2 basal expression levels is confirmed, shMEG3 can be used in place of the GapmeRs for the in vitro hypertrophy experiments. Expression analysis of hypertrophic markers Nppa (ANF) and Nppb (BNP) will be performed to determine if the reduction of Gtl2 lncRNA expression levels causes attenuation of hypertrophy. To validate the expression analysis, immunohistochemistry against α-actin and DAPI will be performed to measure cardiomyocyte surface area. Additionally, Gtl2
overexpression experiments will be performed in NRVMs utilizing the adenovirus encoding for MEG3. Similar, to the knockdown experiments, expression analysis will be done to confirm the overexpression of MEG3 and other marker genes, such as those for proliferation and cell death. Immunohistochemistry will be used to validate the expression analysis.
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To compliment the in vitro experiments, the rAAV9/MEG3 virus will be administered with intraperitoneal injection. Both mice pups and adult mice will be injected with the virus. The hearts will be dissected and observed for any
phenotypic changes. If phenotypic changes are apparent, immunohistochemistry can be performed using cross sections of the heart tissue. Additionally, to confirm MEG3 overexpression as well as the dysregulation of other marker genes for proliferation, hypertrophy, and cell death, qRT-PCR will be performed. Similar knockdown in vitro experiments with rAAV9 encoding a shGtl2 will be done and the results will be compared. Taken together, the results from these experiments will aide us in developing a deeper understanding of Gtl2’s role in the heart.
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LIST OF JOURNAL ABBREVIATIONS Cell Mol Life Sci
Circ Res Dev Cell Dev Dyn Genes Dev Hum Mol Genet J Biol Chem J Mol Endocrinol Mol Cell
Mol Cell Biol Nat Med
Nat Rev Cardiol Nat Rev Genet
Nat Rev Mol Cell Biol
Cellular and Molecular Life Sciences Circulation Research
Developmental Cell
Developmental Dynamics Genes and Development Human Molecular Genetics Journal of Biological Chemistry Journal of Molecular Endocrinology Molecular Cell
Molecular and Cellular Biology Nature Medicine
Nature Reviews. Cardiology Nature Reviews. Genetics
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REFERENCES
Benetatos, L., Hatzimichael, E., Londin, E., Vartholomatos, G., Loher, P., Rigoutsos, I., and Briasoulis, E. (2013) The microRNAs within the DLK1-DIO3 genomic region: Involvement in disease pathogenesis. Cell. Mol. Life Sci. 70, 795–814
Clark, A. L., and Naya, F. J. (2015) MicroRNAs in the myocyte enhancer factor 2
(MEF2)-regulated Gtl2-Dio3 noncoding RNA locus promote cardiomyocyte proliferation by targeting the transcriptional coactivator Cited2. J. Biol. Chem. 290, 23162–23172 Clark, Amanda. (2016) MEF2-Regulated Gtl2-Dio3 Noncoding RNAs in Cardiac Muscle and Disease. (unpublished doctoral dissertation). Boston University, Boston, MA
Devaux, Y., Zangrando, J., Schroen, B., Creemers, E. E., Pedrazzini, T., Chang, C.-P., Dorn, G. W., Thum, T., and Heymans, S. (2015) Long noncoding RNAs in cardiac development and ageing. Nat. Rev. Cardiol. 12, 415–25
Geisler, S., and Coller, J. (2013) RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat. Rev. Mol. Cell Biol. 14, 699–712
Gossett, L. A., Kelvin, D. J., Sternberg, E. A., and Olson, E. N. (1989) A new myocyte- specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol. Cell. Biol. 9, 5022–5033
Greco, C. M., and Condorelli, G. (2015) Epigenetic modifications and noncoding RNAs in cardiac hypertrophy and failure. Nat Rev Cardiol. 12, 488–497
Grote, P., Wittler, L., Hendrix, D., Koch, F., Währisch, S., Beisaw, A., Macura, K., Bläss, G., Kellis, M., Werber, M., and Herrmann, B. G. (2013) The Tissue-Specific lncRNA Fendrr Is an Essential Regulator of Heart and Body Wall Development in the Mouse. Dev. Cell. 24, 206–214
Han, P., Li, W., Lin, C.-H., Yang, J., Shang, C., Nurnberg, S. T., Jin, K. K., Xu, W., Lin, C.-Y., Lin, C., Xiong, Y., Chien, H.-C., Zhou, B., Ashley, E., Bernstein, D., Chen, P.-S., Chen, H.-S. V., Quertermous, T., and Chang, C. (2014) A long noncoding RNA protects the heart from pathological hypertrophy. Nature. 514, 102–106
Kallen, A. N., Zhou, X. B., Xu, J., Qiao, C., Ma, J., Yan, L., Lu, L., Liu, C., Yi, J. S., Zhang, H., Min, W., Bennett, A. M., Gregory, R. I., Ding, Y., and Huang, Y. (2013) The Imprinted H19 LncRNA Antagonizes Let-7 MicroRNAs. Mol. Cell. 52, 101–112
63
Kaneko, S., Bonasio, R., Saldaña-Meyer, R., Yoshida, T., Son, J., Nishino, K., Umezawa, A., and Reinberg, D. (2014) Interactions between JARID2 and Noncoding RNAs
Regulate PRC2 Recruitment to Chromatin. Mol. Cell. 53, 290–300
Klattenhoff, C. A., Scheuermann, J. C., Surface, L. E., Bradley, R. K., Fields, P. A., Steinhauser, M. L., Ding, H., Butty, V. L., Torrey, L., Haas, S., Abo, R., Tabebordbar, M., Lee, R. T., Burge, C. B., and Boyer, L. A. (2013) Braveheart, a long noncoding RNA required for cardiovascular lineage commitment. Cell. 152, 570–583
Kole, R., Krainer, A. R., and Altman, S. (2012) RNA therapeutics: Beyond RNA
interference and antisense oligonucleotides. Nat. Reviews. Drug Discovery. 11, 125–140 Lin, S.-P., Coan, P., da Rocha, S. T., Seitz, H., Cavaille, J., Teng, P.-W., Takada, S., and Ferguson-Smith, A. C. (2007) Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region. Development. 134, 417–26 Matsumoto, E., Sasaki, S., Kinoshita, H., Kito, T., Ohta, H., Konishi, M., Kuwahara, K., Nakao, K., and Itoh, N. (2013) Angiotensin II-induced cardiac hypertrophy and fibrosis are promoted in mice lacking Fgf16. Genes to Cells. 18, 544–553
Mercer, T. R., Dinger, M. E., and Mattick, J. S. (2009) Long non-coding RNAs: insights into functions. Nat. Rev. Genet. 10, 155–159
Mohammad, F., Mondal, T., and Kanduri, C. (2009) Epigenetics of imprinted long noncoding RNAs. Epigenetics. 4, 277–286
Olson, E. N. (2006) Gene Regulatory Networks in the Evolution and Development of the Heart. Science. 313, 1922–1927
Pacak, C. A., Mah, C. S., Thattaliyath, B. D., Conlon, T. J., Lewis, M. A., Cloutier, D. E., Zolotukhin, I., Tarantal, A. F., and Byrne, B. J. (2006) Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo. Circ. Res.
10.1161/01.RES.0000237661.18885.f6
Pon, J. R., and Marra, M. A. (2015) MEF2 transcription factors: developmental regulators and emerging cancer genes. Oncotarget. 10.18632/oncotarget.6223 Potthoff, M. J., and Olson, E. N. (2007) MEF2: a central regulator of diverse developmental programs. Development. 134, 4131–4140
Schmidt, J. V, Matteson, P. G., Jones, B. K., Guan, X.-J., and Tilghman, S. M. (2000) The Dlk1 and Gtl2 genes are linked and reciprocally imprinted. Genes Dev. 14, 1997– 2002
Schuster-Gossler, K., Bilinski, P., Sado, T., Ferguson-Smtth, A., and Gossler, A. (1998) The mouse Gtl2 gene is differentially expressed during embryonic development, encodes
64
multiple alternatively spliced transcripts, and may act as an RNA. Dev. Dyn. 212, 214– 228
Snyder, C. M., Rice, A. L., Estrella, N. L., Held, A., Kandarian, S. C., and Naya, F. J. (2013) MEF2A regulates the Gtl2-Dio3 microRNA mega-cluster to modulate WNT signaling in skeletal muscle regeneration. Development. 140, 31–42
Takahashi, N., Okamoto, A., Kobayashi, R., Shirai, M., Obata, Y., Ogawa, H., Sotomaru, Y., and Kono, T. (2009) Deletion of Gtl2, imprinted non-coding RNA, with its
differentially methylated region induces lethal parent-origin-dependent defects in mice. Hum. Mol. Genet. 18, 1879–1888
Wang, K. C., and Chang, H. Y. (2011) Molecular Mechanisms of Long Noncoding RNAs. Mol. Cell. 43, 904–914
Wang, K., Liu, F., Zhou, L. Y., Long, B., Yuan, S. M., Wang, Y., Liu, C. Y., Sun, T., Zhang, X. J., and Li, P. F. (2014) The long noncoding RNA CHRF regulates cardiac hypertrophy by targeting miR-489. Circ. Res. 114, 1377–1388
Wang, Z., Zhang, X.-J., Ji, Y.-X., Zhang, P., Deng, K.-Q., Gong, J., Ren, S., Wang, X., Chen, I., Wang, H., Gao, C., Yokota, T., Ang, Y. S., Li, S., Cass, A., Vondriska, T. M., Li, G., Deb, A., Srivastava, D., Yang, H.-T., Xiao, X., Li, H., and Wang, Y. (2016) The long noncoding RNA Chaer defines an epigenetic checkpoint in cardiac hypertrophy. Nat. Med. 10.1038/nm.4179
Zhang, X., Rice, K., Wang, Y., Chen, W., Zhong, Y., Nakayama, Y., Zhou, Y., and Klibanski, A. (2010) Maternally expressed gene 3 (MEG3) noncoding ribonucleic acid: Isoform structure, expression, and functions. Endocrinology. 151, 939–947
Zhou, Y., Zhang, X., and Klibanski, A. (2012) MEG3 noncoding RNA: A tumor suppressor. J. Mol. Endocrinol. 48, 45–53
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