keith gilesAssistant Professor

Research Areas
Transcriptional control of rRNA and tDNA


Research Interests

The overall theme of the Giles lab is to understand transcriptional control of the rRNA and tDNA.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 answerthese 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 tDNA transcription.

Human tDNAare 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 tDNA can function as a chromatin insulator, which interferes with enhancer-promoter interactions (14, 17-19).Similarly, active tDNAcan 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 tDNA.

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 tDNA. We are currently investigating the role of chromatin structure in this process. Our preliminary studies have shown that CpG methylation is enriched at silent tDNA and that activated tDNA are enriched within chromatin loops anchored by CTCF and COHESIN.These data suggest a strong relationship between chromatin structure and the regulation of overall cellular physiology via controlling tRNA levels.

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 bestrongly 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 ribosome biogenesis 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. Our current goal is to understand the signaling pathway(s) that allow reductions in ribosome biogenesis to control pluripotency.

C) Understanding how a miRNA-network regulatesribosome biogenesis.

We have recently discovered that miR92a, in complex with human AGO2, reduces overall rRNA synthesis rate through base-pairing with nascent rDNA transcripts (29). Furthermore, the localization of AGO2 to the rDNA promotes high levels H3K9me2/3. MiR92a is one of seven miRNA that we have identified to engage in direct base-pairs with the rRNA. In addition, these miRNA form a network that is highly enriched for targets among ribosomal proteins. Our current hypothesis is that this miRNA network controls ribosome biogenesis through regulating both rDNA transcription and the translation of ribosomal proteins. We are currently testing this hypothesis by combining genome editing with CRISPR-Cas9, metabolic labeling, and mass-spectrometry. Ribosome levels are regulated according to cellular needs during growth and proliferation. Therefore it is critical to understand this newly discovered regulatory mechanism and how it is coordinated with other known pathways to control cell fate.

1. Giege R. 2008. Toward a more complete view of tRNA biology. Nature structural & molecular biology 15:1007-1014.
2. White RJ. 2004. RNA polymerase III transcription and cancer. Oncogene 23:3208-3216.
3. Hunemeier T, Salzano FM, Bortolini MC. 2009. TCOF1 T/Ser variant and brachycephaly in dogs. Animal genetics 40:357-358.
4. 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.
5. 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.
6. 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.
7. 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, AltunogluU, 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.
8. 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.
9. 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.
10. 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.
11. Sethy I,Moir RD, Librizzi M, Willis IM. 1995. In vitro evidence for growth regulation of tDNA transcription in yeast. A role for transcription factor (TF) IIIB70 and TFIIIC. The Journal of biological chemistry 270:28463-28470.
12. NE II, HewardJA, 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.
13. Kirchner S, Ignatova Z. 2015. Emerging roles of tRNA in adaptive translation, signalling dynamics and disease. Nature reviews. Genetics 16:98-112.
14. Raab JR, Chiu J, Zhu J, Katzman S, Kurukuti S, Wade PA, Haussler D, Kamakaka RT. 2012. Human tDNA function as chromatin insulators. The EMBO journal 31:330-350.
15. 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.
16. Gebetsberger J, Polacek N. 2013. Slicing tRNAs to boost functional ncRNA diversity. RNA biology 10:1798-1806.
17. 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.
18. 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.
19. Van Bortle K, Corces VG. 2012. tDNA insulators and the emerging role of TFIIIC in genome organization. Transcription 3:277-284.
20. Woolnough JL, Atwood BL, Giles KE. 2015. Argonaute 2 binds directly to tDNA and promotes gene repression in cis. Molecular and cellular biology.
21.Wang L, Haeusler RA, Good PD, Thompson M, NagarS, Engelke DR. 2005. Silencing near tDNArequires nucleolar localization. The Journal of biological chemistry 280:8637-8639.
22. 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 tDNAcomplexes and affects local transcriptional silencing. Proceedings of the National Academy of Sciences of the United States of America 110:E3081-3089.
23. Good PD, Kendall A, Ignatz-Hoover J, Miller EL, Pai DA, Rivera SR, Carrick B, Engelke DR. 2013. Silencing near tDNAis nucleosome-mediated and distinct from boundary element function. Gene 526:7-15.
24. 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.
25. Weinberg DE, Shah P, Eichhorn SW, Hussmann JA, Plotkin JB, BartelDP. 2016. Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation. Cell reports 14:1787-1799.
26. 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.
27. 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.
28. 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.
29. Atwood BL, Woolnough JL, Lefevre GM, Saint Just Ribeiro M, Felsenfeld G,Giles KE. 2016. Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions. The Journal of biological chemistry 291:17919-17928.

Education

Graduate School
Ph.D., The Johns Hopkins University

Postdoctoral Fellowship
NIH/NIDDK

Contact

Office
Kaul Human Genetics
Building Room 502
720 20th Street S.
Birmingham, AL 35294-0024

Phone
(205) 934-4745

Email
kegiles@uab.edu

Committed to exploring new frontiers in basic and translational research.

The Department of Biochemistry and Molecular Genetics is an integral part of the vibrant biomedical research community at the University of Alabama at Birmingham (UAB). UAB ranks among the top public institutions of higher education in terms of research and training awards. Research conducted by the faculty, staff, and students of the Department of Biochemistry and Molecular Genetics is currently supported by more than $4.3 million per year in extramural, investigator-initiated grants.

Research

The Department of Biochemistry and Molecular Genetics carries out cutting-edge basic and translational research. Research strengths in the department includes cancer biology, chromatin and epigenetic signaling, metabolism and signaling, regulation of gene expression, structural biology, DNA synthesis and repair, and disease mechanisms.

Education

Graduate students and postdoctoral fellows in the Department of Biochemistry and Molecular Genetics are trained to carry out hypothesis-driven research using advanced research techniques. This training will prepare our graduates for a career in not just biomedical research, but also in other diverse fields that require critical thinking. Our faculty also proudly trains professional (MD, DDS, & DO) students, as well as undergraduate students at UAB.

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