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  1. 1. 3/14/13 Characterizing Novel Transcriptional Outputs with Potentially Circadian Gene Expression in Arabidopsis thaliana Kellen Na Undergraduate, Senior Biochemistry/Cell Biology B.S. University of California, San Diego
  2. 2. Introduction to Circadian Rhythms What Exactly Are Circadian Rhythms? “Extensive circadian clock networks regulate almost every biological process in plants.” - Pruneda-Paz, Kay, 2010 Trends in Plant ScienceOrganisms, from cyanobacteria to humans, havebiological clocks that are used to “tell time.” Byallowing the anticipation and rapid response toexternal changes, circadian clocks provide aninvaluable tool for increased fitness in aconstantly rhythmic environment. One example of environmental rhythms that the biological clock uses to regulate output is seen in the daily oscillations associated with the Earth’s rotation and the periodic changes in light, temperature, and humidity.
  3. 3. What Exactly Are Circadian Rhythms?The field of circadian rhythms seeks to understand the complex systems underlying the properphasing of internally-driven biological activity to the environment. Hallmarks of circadian rhythms include: 1. The ability to continue cycling in constant environmental conditions (endogenous) 2. Period-compensation in different temperatures 3. The ability to change endogenous oscillations to match changes in the environment (entrainment)These rhythms in biological process arise from extremely complex (and not entirely known)systems that work together to generate oscillations in gene expression and observable output.
  4. 4. The Circadian Clock in PlantsThe circadian clock is an endogenous oscillator of most, if not all, plant functions. It plays a keyrole in the ability to respond to various environmental inputs. This is especially important becauseplants rely on the sun for energy, and their fitness directly depends on the ability to maximizeenergy input by catering their physiological processes such as growth, water usage, stomatalaperture, photosynthesis, etc. to follow the daily rhythmicity of sunlight.
  5. 5. The Circadian Clock in Plants Circadian rhythms are crucial to plant survival. Plants need to tightly link their biochemical processes to resource availability. They must be able to predict and adapt to changing seasons , day lengths, temperature, etc. One particularly observable circadian rhythm seen in plants is leaf movement and the ability for plants to anticipate what time the sun will come up every day. Video: Note how right before sunrise the plant positions its leaf towards the direction where the sun will come up.Video will be onthe next slide Roger P. Hangarter, Indiana University
  6. 6. The Core Clock Mechanism in At The core components of the clock in Arabidopsis thaliana (At) have been identified as a part of a transcription-translation feedback loop (TTFL).The TTFL includes:1. Positive element TOC1 (TIMING OF CAB EXPRESSION-1)2. Negative elements LHY (LATE ELONGATED HYPOCOTYL) and CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) McClung C R Plant Cell 2006;18:792-803
  7. 7. The Core Clock Mechanism in AtCCA1 and LHY proteins function asnegative-element transcription factors,repressing the transcription of TOC1.CCA1 and LHY are rhythmicallyexpressed, with peaks of expressionaround dawn. They bind to a conservedsequence known as the “eveningelement” (EE) in the TOC1 promoter.TOC1 is also rhythmically expressed,with its peak of expression around dusk.TOC1 functions as a positive-elementtranscription factor, activation theexpression of CCA1 and LHY by bindingto the “morning element” of theirpromoters, thus completing the TTFL. McClung C R Plant Cell 2006;18:792-803
  8. 8. The Core Clock Mechanism in At
  9. 9. The Core Clock and Circadian-Regulated Phenotypes Abiotic Stress This simple clock core-component ResponsesTTFL, involving two negative elements and one positive element, must Biotic Stress somehow play a role incircadian Responses control of physiological processes in plants. Water Usage ? Flowering Stomatal Apeture Growth How does it do that? Photosynthesis
  10. 10. The Clock Mechanism in AtThe core clock TTFL model isactually a very simplified versionof the entire, known molecularmodel of the At circadianoscillator.Here is a more complex version,to show the core circadianproteins in the TTFL (LHY, CCA1,and TOC1) are involved in manyother interactions, receivemessages from the environment,and carry out differentdownstream effects. McClung C R Plant Cell 2006;18:792-803
  11. 11. The Core Clock and Circadian-Regulated PhenotypesYet this model is still incomplete: transcriptional and post-transcriptional outputpathways of the clock are still being discovered and understood.There are two possibilities that can explain how the cell translate signals from thecore transcription-translation feedback loop… …It is possible that the core clock components directly control all transcriptional output of circadian-regulated cellular processes in At
  12. 12. The Core Clock and Circadian-Regulated Phenotypes Abiotic StressHowever, a second possibility is that there is a Responsescascade of clock transcription factors; proteinsthat regulate transcription of other clock Biotic Stressproteins, which go on to regulate other Responsesdownstream proteins, etc. Water Usage Flowering Stomatal Apeture Growth Photosynthesis
  13. 13. The HypothesisThe hypothesis for my project is that the core circadian clockcontrols circadian output via the second possibility, atranscription factor cascade: the downstream targets whoseexpression rhythms are regulated under this clock cascadefurther the influence of circadian rhythms by allowing theextension of clock output to many different physiologicalprocesses in Arabidopsis thaliana.
  14. 14. The QuestionThus, my research question is an attempt to test my hypothesis regarding adownstream clock transcription factor cascade: What transcription factors help extend circadian clock output? In other words, can we identify a potential candidate factor that works downstream of the clock, and identify its function?
  15. 15. Experimental Data and ResultsFirst, I screened for transcription factors that are potentially a part of a transcriptionfactor cascade downstream of the core clock, aka they cycled strongly under constantconditions but with no known circadian regulation. We searched for genes thatshowed no change between photo- and thermo-cycles. The method used to screen forpotential candidates was through a web-based program called Diurnal. Diurnal is atool that provides genome-wide expression data of diurnal and circadian rhythmic lociin Arabidopsis. It also provides data on expression changes of genes under a variety ofdifferent light, temperature, and stress conditions. More information can be found ontheir website advisor, Dr. Doherty, and I found a candidate transcription factor called WLIM1. Itwas picked because it has the same phase and pattern of expression under constanttemperature and light conditions.
  16. 16. Experimental Data and ResultsWLIM1 (AT1G10200) shows strong cycling under LDHC conditions (Light (12h) Dark(12h)/ Hot (12h) Cold (12h)). Note that WLIM1 expression peaks in the evening.
  17. 17. Experimental Data and ResultsWLIM1 also shows the same strong cycling/peak of expression underconstant conditions, making it a great candidate to do further testing on. Red: Entrained on LDHC and measured in constant light and constant hot temperature Blue: Entrained and measured in LDHC Green: Entrained and measured in constant light and constant hot temperature
  18. 18. Experimental Data and ResultsThe data on WLIM1’s strong cycling under constant conditions was very surprising,especially since there have not been any published studies involving WLIM1’spotential role in the circadian clock.What is WLIM1?WLIM1 is a transcription factor and is a part of a family of LIM proteins (containing aLIM-domain), which are involved in actin bundling and higher-order cytoskeletonassembly. WLIM1 in particular is known to directly bind actin filaments and triggerformation of thick actin bundles in a pH and Ca2+ independent way. It is expressedalmost ubiquitously in the Arabidopsis plant tissue.Because of WLIM1’s known interaction with actin proteins, I decided to see therelationship between WLIM1 and an actin protein that also cycles: ACTIN7 (ACT7).
  19. 19. Experimental Data and ResultsRed: WLIM1Blue: ACT7In LDHC conditions WLIM1 and ACT7 have the same peaks of expression when measured in LDHC conditions.
  20. 20. Experimental Data and ResultsI also compared WLIM1 expression to those of the core clock components LHY, CCA1, and TOC1.WLIM1 is anti-phasic to CCA1 and LHY in LDHC conditions:
  21. 21. Experimental Data and ResultsWLIM1 and TOC1 have the same phase in LDHC:
  22. 22. Experimental Data and ResultsBecause WLIM1 is perfectly anti-phasic to CCA1 and LHY, which are the two negative elements inthe core clock mechanism that suppress the morning expression of genes by binding to an“evening element” (EE) sequence, the next step in my experiment was to perform ChIP-sequencing on WLIM1.The results (next slide) revealed a very weak signal for evening element sequences on the WLIM1locus. I was expecting a strong EE signal due to it’s anti-phasic nature to the core clock proteinsCCA1 and LHY, so this result was surprising.However, this turned my experiment towards a new direction. Other literature has shown thatnot all genes containing the EE upstream are regulated by CCA1/LHY. Some evening genes areshown to continue cycling in lhy and cca1 double mutants, and also LHY and CCA1 can positivelyregulate genes that are expressed in the morning. (Mizoguchi et al., 2005)This shows us that there is much more to the negative element component of the clockmechanism than the simple repression of genes with evening elements by core clock proteinsLHY and CCA1. Does that mean that WLIM1 is a part of a circadian mechanism that interacts withthe core clock components, or other downstream transcription factors, in an unknown way(perhaps via a transcription cascade)?
  23. 23. Experimental Data and ResultsChIP-seq results of the WLIM1 locus shows weak evening element signals:
  24. 24. ConclusionsThe expression data of WLIM1 as well as the surprising results from the ChIP-sequencing of the WLIM1 locus raises a lot of questions regarding exactly how itinteracts with the core clock TTFL mechanism.It’s perfectly anti-phasic expression levels with LHY and CCA1 (in addition to it’sperfectly phased expression with TOC1), and yet it’s lack of a strong evening elementpoint to WLIM1 interacting with other proteins besides the core clock components tomediate its circadian output as an evening-expressed transcription factor. Inaddition, it’s identical expression phase with actin protein ACT7 raises the possibilitythat it may also play a role in mediating the expression of this gene.My initial hypothesis was to ask if there existed a transcription factor cascademechanism responsible for the regulation of gene expression output downstream ofthe core clock TTFL. I believe my findings definitely show that this is a plausiblemechanism, but many more experiments need to be done to fully identify themechanism by which WLIM1 expression is being clock-controlled.
  25. 25. ConclusionsMy future goals/experiments:  I am currently in the process of developing knock out and overexpressor lines of WLIM1. Entraining these lines in different temperatures and light/dark cycles will let me see if the expression levels of known clock components (especially LHY and CCA1) as well as actin proteins are affected in any way by the changes in WLIM1 expression under different conditions.  The overall goals of my future research will be to characterize WLIM1 and it’s role in mediating downstream circadian output and learn more about the exact interactions between WLIM1 and the core circadian clock.
  26. 26. AcknowledgementsI would like to thank Professor Estelle and all the members of the Estelle lab forproviding me with the necessary resources/assistance to conduct my research thisyear.Also, this research project was done through the BISP 199 (Individual Research forUndergraduates) course offered by the Biological Sciences department at UC SanDiego.And last but not least, I would like to sincerely thank my research advisor/mentor, Dr.Doherty, for all the countless amounts of advice, coaching, and words ofencouragement that made this project possible.