||Shelby Biomedical Rsch Building
19th Street South
Birmingham, AL 35294-0024
Current Research Projects
The overall theme of the Giles lab is to understand transcriptional control of the rRNA and tRNA genes. We are focused on two distinct questions: What are the molecular mechanisms that allow differential expression of tRNA and rRNA? and How do changes in tRNA and rRNA levels impact cell fate? It is our long-term goal to answer these questions and use that knowledge to improve the application of stem cells as a therapeutic approach to a wide-range of human diseases.
A) The regulation of tRNA gene transcription by CTCF-mediated chromatin structure.
Human tRNA genes are transcribed by RNA Polymerase III (Pol III) (1). The regulation of Pol III activity has been linked to cancer progression (2-6), neuronal disease (7, 8), and overall energy metabolism (9-11). The precise mechanisms for this health-relevance is unknown, however recent discoveries have indicated many novel roles for tRNA in regulating cellular physiology (12-16). Active tRNA genes can function as a chromatin insulator, which interferes with enhancer-promoter interactions (14, 17-19). Similarly, active tRNA genes can also suppress nearby Pol II-transcribed genes in cis, in a mechanism that is distinct from insulator function (see below for our contribution to understanding this phenomena) (20-23). tRNA can also be processed into a myriad small RNA (< 35 nts) that impact many distinct cellular processes, such as apoptosis and translation efficiency (16, 24). These functions indicate the importance of understanding the molecular basis for the cell-type specific regulation of tRNA genes.
Despite the wealth of information on the basal Pol III transcriptional apparatus, there is virtually nothing known about how the cell can distinguish and differentially regulate individual tRNA genes. It is our primary hypothesis that this process is regulated by CTCF-Cohesin chromatin loops. We are also interested in how altered tRNA levels impact cellular physiology. Recent studies have shown that experimentally altering tRNA levels in S. cerevisiae can impact translation efficiency of specific mRNA (25). The general model for this observation is that the longer it takes a tRNA to recognize an actively translating ribosome, the slower the overall translation rate will be. If a given open reading frame is enriched for codons that are recognized by a rare tRNA isoacceptor, then that gene could be strongly influenced by changes in tRNA abundance. We have discovered that significant changes to tRNA levels can occur during rapid transitions in cell fate.
B) How does rRNA synthesis rate control pluripotency?
The promise of stem cells as a therapeutic agent critically depends on the ability to control the growth and differentiation of ESCs. This control requires an increased understanding of the pathways that regulate pluripotency. The rRNA synthesis rate in mammalian ESCs is a critical regulator of pluripotency, and any molecular or chemical perturbation of the normal rRNA synthesis rate can induce the loss of pluripotency and induce differentiation (26, 27). Our group has shown that the reduction in rRNA synthesis occurs very early during ESC differentiation, within 2 hours of ACTIVIN A treatment (28). This change correlates with the reduced occupancy of the Pol I transcription factor UBF but precedes any significant changes in gene expression or increases to heterochromatin-associated histone modifications (H3K9me3, H3K27me3, and H4K20me3). Furthermore, the direct inhibition of rRNA synthesis with a Pol I inhibitor is sufficient to induce cellular differentiation. We hypothesize that the transcription factor UBF is subject to phosphorylation upon ACTIVIN A signaling. This reduces the affinity of UBF for the rRNA gene and thus causes a reduction in Pol I recruitment. In addition, UBF has been shown to regulate the promoter escape and overall elongation rate of Pol I. We have developed an in vitro transcription system to test the ability of UBF to regulate each step in the Pol I transcription cycle. The goal of this work is to not only increase our knowledge regarding the regulation of rRNA synthesis during a critical developmental transition, but also make significant advancements to the general understanding of transcription-control mechanisms.
C) How do miRNA regulate Pol I transcription?
We have recently shown that AGO2-miRNA complexes can base-pair to nascent Pol I transcripts (29). This interaction reduces the overall levels of pre-rRNA. Our current hypothesis is that interactions between the AGO2-miRNA complexes recruit heterochromatin modifying complexes that reduce the elongation rate of Pol I. We are currently testing this hypothesis as well as investigating the extent to which miRNA-mediated control of the rRNA synthesis rate participates in cancer formation.
Assistant Professor University of Alabama at Birmingham 2011
Post-Doctoral NIDDK/NIH 2011
PhD The Johns Hopkins University 2004
BS State University of New York at Buffalo 1999
- Giege R. 2008. Toward a more complete view of tRNA biology. Nature structural & molecular biology 15:1007-1014.
- White RJ. 2004. RNA polymerase III transcription and cancer. Oncogene 23:3208-3216.
- Hunemeier T, Salzano FM, Bortolini MC. 2009. TCOF1 T/Ser variant and brachycephaly in dogs. Animal genetics 40:357-358.
- Masotti C, Ornelas CC, Splendore-Gordonos A, Moura R, Felix TM, Alonso N, Camargo AA, Passos-Bueno MR. 2009. Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients. BMC medical genetics 10:136.
- Richter CA, Amin S, Linden J, Dixon J, Dixon MJ, Tucker AS. 2010. Defects in middle ear cavitation cause conductive hearing loss in the Tcof1 mutant mouse. Human molecular genetics 19:1551-1560.
- Pavon-Eternod M, Gomes S, Geslain R, Dai Q, Rosner MR, Pan T. 2009. tRNA over-expression in breast cancer and functional consequences. Nucleic acids research 37:7268-7280.
- Schaffer AE, Eggens VR, Caglayan AO, Reuter MS, Scott E, Coufal NG, Silhavy JL, Xue Y, Kayserili H, Yasuno K, Rosti RO, Abdellateef M, Caglar C, Kasher PR, Cazemier JL, Weterman MA, Cantagrel V, Cai N, Zweier C, Altunoglu U, Satkin NB, Aktar F, Tuysuz B, Yalcinkaya C, Caksen H, Bilguvar K, Fu XD, Trotta CR, Gabriel S, Reis A, Gunel M, Baas F, Gleeson JG. 2014. CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration. Cell 157:651-663.
- Karaca E, Weitzer S, Pehlivan D, Shiraishi H, Gogakos T, Hanada T, Jhangiani SN, Wiszniewski W, Withers M, Campbell IM, Erdin S, Isikay S, Franco LM, Gonzaga-Jauregui C, Gambin T, Gelowani V, Hunter JV, Yesil G, Koparir E, Yilmaz S, Brown M, Briskin D, Hafner M, Morozov P, Farazi TA, Bernreuther C, Glatzel M, Trattnig S, Friske J, Kronnerwetter C, Bainbridge MN, Gezdirici A, Seven M, Muzny DM, Boerwinkle E, Ozen M, Baylor Hopkins Center for Mendelian G, Clausen T, Tuschl T, Yuksel A, Hess A, Gibbs RA, Martinez J, Penninger JM, Lupski JR. 2014. Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function. Cell 157:636-650.
- Li H, Zhang X, Li Z, Chen J, Lu Y, Jia J, Yuan H, Han D. 2012. [Clinical and genetic analysis of a patient with Treacher Collins syndrome in TCOF1 gene]. Lin chuang er bi yan hou tou jing wai ke za zhi = Journal of clinical otorhinolaryngology, head, and neck surgery 26:459-462.
- Ciccia A, Huang JW, Izhar L, Sowa ME, Harper JW, Elledge SJ. 2014. Treacher Collins syndrome TCOF1 protein cooperates with NBS1 in the DNA damage response. Proceedings of the National Academy of Sciences of the United States of America 111:18631-18636.
- Sethy I, Moir RD, Librizzi M, Willis IM. 1995. In vitro evidence for growth regulation of tRNA gene transcription in yeast. A role for transcription factor (TF) IIIB70 and TFIIIC. The Journal of biological chemistry 270:28463-28470.
- NE II, Heward JA, Roux B, Tsitsiou E, Fenwick PS, Lenzi L, Goodhead I, Hertz-Fowler C, Heger A, Hall N, Donnelly LE, Sims D, Lindsay MA. 2014. Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes. Nature communications 5:3979.
- Kirchner S, Ignatova Z. 2015. Emerging roles of tRNA in adaptive translation, signalling dynamics and disease. Nature reviews. Genetics 16:98-112.
- Raab JR, Chiu J, Zhu J, Katzman S, Kurukuti S, Wade PA, Haussler D, Kamakaka RT. 2012. Human tRNA genes function as chromatin insulators. The EMBO journal 31:330-350.
- Li Z, Ender C, Meister G, Moore PS, Chang Y, John B. 2012. Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs. Nucleic acids research 40:6787-6799.
- Gebetsberger J, Polacek N. 2013. Slicing tRNAs to boost functional ncRNA diversity. RNA biology 10:1798-1806.
- van den Boogaard M, Barnett P, Christoffels VM. 2014. From GWAS to function: genetic variation in sodium channel gene enhancer influences electrical patterning. Trends in cardiovascular medicine 24:99-104.
- Jambunathan N, Martinez AW, Robert EC, Agochukwu NB, Ibos ME, Dugas SL, Donze D. 2005. Multiple bromodomain genes are involved in restricting the spread of heterochromatic silencing at the Saccharomyces cerevisiae HMR-tRNA boundary. Genetics 171:913-922.
- Van Bortle K, Corces VG. 2012. tDNA insulators and the emerging role of TFIIIC in genome organization. Transcription 3:277-284.
- Woolnough JL, Atwood BL, Giles KE. 2015. Argonaute 2 binds directly to tRNA genes and promotes gene repression in cis. Molecular and cellular biology.
- Wang L, Haeusler RA, Good PD, Thompson M, Nagar S, Engelke DR. 2005. Silencing near tRNA genes requires nucleolar localization. The Journal of biological chemistry 280:8637-8639.
- Pratt-Hyatt M, Pai DA, Haeusler RA, Wozniak GG, Good PD, Miller EL, McLeod IX, Yates JR, 3rd, Hopper AK, Engelke DR. 2013. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing. Proceedings of the National Academy of Sciences of the United States of America 110:E3081-3089.
- Good PD, Kendall A, Ignatz-Hoover J, Miller EL, Pai DA, Rivera SR, Carrick B, Engelke DR. 2013. Silencing near tRNA genes is nucleosome-mediated and distinct from boundary element function. Gene 526:7-15.
- Haussecker D, Huang Y, Lau A, Parameswaran P, Fire AZ, Kay MA. 2010. Human tRNA-derived small RNAs in the global regulation of RNA silencing. Rna 16:673-695.
- Weinberg DE, Shah P, Eichhorn SW, Hussmann JA, Plotkin JB, Bartel DP. 2016. Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation. Cell reports 14:1787-1799.
- Watanabe-Susaki K, Takada H, Enomoto K, Miwata K, Ishimine H, Intoh A, Ohtaka M, Nakanishi M, Sugino H, Asashima M, Kurisaki A. 2014. Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells. Stem cells 32:3099-3111.
- Savic N, Bar D, Leone S, Frommel SC, Weber FA, Vollenweider E, Ferrari E, Ziegler U, Kaech A, Shakhova O, Cinelli P, Santoro R. 2014. lncRNA maturation to initiate heterochromatin formation in the nucleolus is required for exit from pluripotency in ESCs. Cell stem cell 15:720-734.
- Woolnough JL, Atwood BL, Liu Z, Zhao R, Giles KE. 2016. The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells. PloS one 11:e0157276.