WLIM1 is a candidate transcription factor downstream of the core circadian clock that may help extend circadian output in Arabidopsis thaliana. WLIM1 shows strong cycling under constant conditions and is anti-phasic to core clock proteins LHY and CCA1. However, ChIP-seq revealed only weak evening element binding, suggesting WLIM1 interacts with other proteins. Future experiments will characterize WLIM1's role and interactions with the core clock.

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
http://www.ccmb.res.in/staff/imran/Arabidopsis.jpg

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 Science
Organisms, from cyanobacteria to humans, have
biological clocks that are used to “tell time.” By
allowing the anticipation and rapid response to
external changes, circadian clocks provide an
invaluable tool for increased fitness in a
constantly 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.
http://4.bp.blogspot.com/-ZANPW1vK_C4/UJCOrpizJwI/AAAAAAAAUuc/X9vY0H8qslQ/s1600/AxialTiltObliquity.png

3. What Exactly Are Circadian Rhythms?
The field of circadian rhythms seeks to understand the complex systems underlying the proper
phasing 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. The Circadian Clock in Plants
The circadian clock is an endogenous oscillator of most, if not all, plant functions. It plays a key
role in the ability to respond to various environmental inputs. This is especially important because
plants rely on the sun for energy, and their fitness directly depends on the ability to maximize
energy input by catering their physiological processes such as growth, water usage, stomatal
aperture, photosynthesis, etc. to follow the daily rhythmicity of sunlight.

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 on
the next slide
Roger P. Hangarter, Indiana University
http://plantsinmotion.bio.indiana.edu/plantmotion/starthere.html

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. The Core Clock Mechanism in At
CCA1 and LHY proteins function as
negative-element transcription factors,
repressing the transcription of TOC1.
CCA1 and LHY are rhythmically
expressed, with peaks of expression
around dawn. They bind to a conserved
sequence known as the “evening
element” (EE) in the TOC1 promoter.
TOC1 is also rhythmically expressed,
with its peak of expression around dusk.
TOC1 functions as a positive-element
transcription factor, activation the
expression of CCA1 and LHY by binding
to the “morning element” of their
promoters, thus completing the TTFL.
McClung C R Plant Cell 2006;18:792-803

8. The Core Clock Mechanism in At

9. The Core Clock and Circadian-Regulated
Phenotypes
Abiotic Stress
This simple clock core-component
Responses
TTFL, 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. The Clock Mechanism in At
The core clock TTFL model is
actually a very simplified version
of the entire, known molecular
model of the At circadian
oscillator.
Here is a more complex version,
to show the core circadian
proteins in the TTFL (LHY, CCA1,
and TOC1) are involved in many
other interactions, receive
messages from the environment,
and carry out different
downstream effects.
McClung C R Plant Cell 2006;18:792-803

11. The Core Clock and Circadian-Regulated
Phenotypes
Yet this model is still incomplete: transcriptional and post-transcriptional output
pathways of the clock are still being discovered and understood.
There are two possibilities that can explain how the cell translate signals from the
core 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. The Core Clock and Circadian-Regulated
Phenotypes
Abiotic Stress
However, a second possibility is that there is a Responses
cascade of clock transcription factors; proteins
that regulate transcription of other clock Biotic Stress
proteins, which go on to regulate other Responses
downstream proteins, etc.
Water Usage
Flowering
Stomatal Apeture
Growth
Photosynthesis

13. The Hypothesis
The hypothesis for my project is that the core circadian clock
controls circadian output via the second possibility, a
transcription factor cascade: the downstream targets whose
expression rhythms are regulated under this clock cascade
further the influence of circadian rhythms by allowing the
extension of clock output to many different physiological
processes in Arabidopsis thaliana.

14. The Question
Thus, my research question is an attempt to test my hypothesis regarding a
downstream 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. Experimental Data and Results
First, I screened for transcription factors that are potentially a part of a transcription
factor cascade downstream of the core clock, aka they cycled strongly under constant
conditions but with no known circadian regulation. We searched for genes that
showed no change between photo- and thermo-cycles. The method used to screen for
potential candidates was through a web-based program called Diurnal. Diurnal is a
tool that provides genome-wide expression data of diurnal and circadian rhythmic loci
in Arabidopsis. It also provides data on expression changes of genes under a variety of
different light, temperature, and stress conditions. More information can be found on
their website dirunal.mocklerlab.org.
My advisor, Dr. Doherty, and I found a candidate transcription factor called WLIM1. It
was picked because it has the same phase and pattern of expression under constant
temperature and light conditions.

16. Experimental Data and Results
WLIM1 (AT1G10200) shows strong cycling under LDHC conditions (Light (12h) Dark
(12h)/ Hot (12h) Cold (12h)).
 Note that WLIM1 expression peaks in the evening.

17. Experimental Data and Results
WLIM1 also shows the same strong cycling/peak of expression under
constant 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. Experimental Data and Results
The data on WLIM1’s strong cycling under constant conditions was very surprising,
especially since there have not been any published studies involving WLIM1’s
potential role in the circadian clock.
What is WLIM1?
WLIM1 is a transcription factor and is a part of a family of LIM proteins (containing a
LIM-domain), which are involved in actin bundling and higher-order cytoskeleton
assembly. WLIM1 in particular is known to directly bind actin filaments and trigger
formation of thick actin bundles in a pH and Ca2+ independent way. It is expressed
almost ubiquitously in the Arabidopsis plant tissue.
Because of WLIM1’s known interaction with actin proteins, I decided to see the
relationship between WLIM1 and an actin protein that also cycles: ACTIN7 (ACT7).

19. Experimental Data and Results
Red: WLIM1
Blue: ACT7
In LDHC conditions
WLIM1 and ACT7 have the same peaks of expression when measured in LDHC conditions.

20. Experimental Data and Results
I 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. Experimental Data and Results
WLIM1 and TOC1 have the same phase in LDHC:

22. Experimental Data and Results
Because WLIM1 is perfectly anti-phasic to CCA1 and LHY, which are the two negative elements in
the 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 WLIM1
locus. I was expecting a strong EE signal due to it’s anti-phasic nature to the core clock proteins
CCA1 and LHY, so this result was surprising.
However, this turned my experiment towards a new direction. Other literature has shown that
not all genes containing the EE upstream are regulated by CCA1/LHY. Some evening genes are
shown to continue cycling in lhy and cca1 double mutants, and also LHY and CCA1 can positively
regulate 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 clock
mechanism than the simple repression of genes with evening elements by core clock proteins
LHY and CCA1. Does that mean that WLIM1 is a part of a circadian mechanism that interacts with
the core clock components, or other downstream transcription factors, in an unknown way
(perhaps via a transcription cascade)?

23. Experimental Data and Results
ChIP-seq results of the WLIM1 locus shows weak evening element signals:

24. Conclusions
The 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 it
interacts with the core clock TTFL mechanism.
It’s perfectly anti-phasic expression levels with LHY and CCA1 (in addition to it’s
perfectly phased expression with TOC1), and yet it’s lack of a strong evening element
point to WLIM1 interacting with other proteins besides the core clock components to
mediate its circadian output as an evening-expressed transcription factor. In
addition, it’s identical expression phase with actin protein ACT7 raises the possibility
that 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 cascade
mechanism responsible for the regulation of gene expression output downstream of
the core clock TTFL. I believe my findings definitely show that this is a plausible
mechanism, but many more experiments need to be done to fully identify the
mechanism by which WLIM1 expression is being clock-controlled.

25. Conclusions
My 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. Acknowledgements
I would like to thank Professor Estelle and all the members of the Estelle lab for
providing me with the necessary resources/assistance to conduct my research this
year.
Also, this research project was done through the BISP 199 (Individual Research for
Undergraduates) course offered by the Biological Sciences department at UC San
Diego.
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 of
encouragement that made this project possible.