Epigenetic perturbation of circadian rhythms using

chemically-induced histone acetylation

Feynman Liang

Amherst College
(Dated: May 14, 2014)
Abstract
Circadian rhythms serve as endogenous biological clocks. They are responsible for a diverse
set of biological processes, including body temperature regulation[16], the sleep/wake cycle[4],
and metabolism[12]. The characteristic 24-hour periodic oscillations are due to tightly coupled
regulatory interactions spanning from epigenetic modications to intercellular signaling[17]. With
the goal of better understanding how regulation at the epigenetic level aects circadian rhythms,
we propose the design of a modular system for ligand-induced reversible acetylation of histones.
Such a tool provides ligand-gated control over the circadian cycle and can distinguish the eects of
histone acetylation from other mechanisms of regulation. This permits isolation of the physiological
changes caused by histone acetylation and will determine if histone acetylation alone is sucient
for generating circadian oscillations. Our proposed system is modular, and as a result can be easily
generalized to study epigenetic regulation in other biological processes.

iang14@amherst.edu
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(a) A negative feedback regulatory motif. Transcription
increases mRNA levels, increasing protein abundance.
The protein then feeds back and downregulates
transcription[17]
(b) Phase-plane plot of the transient
response and limit cycle (solid line)
for an oscillatory system. One turn of
the limit cycle takes 24 hours[17].
FIG. 1: Regulation of PER1, a protein involved in circadian rhythms, is an example of a
dynamical system exhibiting oscillations
I. BACKGROUND
Circadian rhythms are biological processes which are entrainable (may be reset with
external stimuli) and exhibit oscillations on the 24-hour timescale[1]. As a result, organisms
are able to anticipate periodic changes such as transitions between day and night, which
enables regulation and coordination of internal biological processes. Believed to originate
from early cells protecting DNA from increased UV exposure during the daytime[3], the
connection between the 24-hour periodicity of circadian rhythms and the period of the
earths rotation reects the deepness of the connection between circadian cycles and life (on
earth) itself.
A dening feature of biological processes classied as circadian rhythms is the presence of
oscillations. One particular condition for creating oscillations is through negative feedback
(Figure 1a). In this regulatory motif, species A (PER mRNA in Figure 1a) increases the
abundance of species B (PER protein in Figure 1a). Species B in turn inhibits species A
activity (by inhibiting transcription of PER mRNA in Figure 1a), which closes the negative
feedback loop. This results in decreased species B abundance, relieving the inhibition of
species A and marks one full oscillation. Over time, the system converges to a steady state
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FIG. 2: Model of the regulatory network for mammalian circadian rhythms[17]
limit cycle where the period and amplitude of oscillations remain constant (Figure 1b).
At the heart of the current model for mammalian circadian cycles is negative feedback
at the transcriptional level (Figure 2). CLOCK (CLK in Figure 2) and BMAL (BMAL1
in Figure 2) are two regulatory proteins which form a DNA-interacting heterodimer that
recognizes the canonical E-box sequence (CACGTG) and upregulates transcription of CRY
protein[15]. CRY subsequently binds BMAL and inhibits the transcriptional upregulation
caused by the CLOCK-BMAL complex[10].
Scientic evidence suggests that histone acetylation is involved in both CLOCK-BMAL
and CRY regulation of transcription. PER1 is a protein whose expression is regulated by
CLOCK-BMAL[17] which Masri et al have shown (amongst many other proteins) to be
correlated with oscillatory histone acetylation[12]. Acetylation of histone tails results in re-
laxation of chromatin, which increases the accessibility of DNA to transcriptional complexes
and upregulates transcription (Figure 3). As CLOCK possesses intrinsic histone acetyltrans-
ferase (HAT) activity, which has been shown to be required for circadian oscillations[5], it is
highly probable that histone acetylation is involved in the upregulation of CLOCK-BMAL
controlled genes. Also, Yoshihisa et al report that transcriptional repression by CRY is re-
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FIG. 3: Left: The positively-charged lysine residues in non-acetylated histone tails binds
the negatively-charged DNA phosphate backbone tightly, resulting in tightly packed
closed chromatin. Right: Acetylation of lysines neutralizes charge, relaxing the
chromatin and enabling transcription complexes to easily access the DNA[13]
lieved by histone deacetylase (HDAC) inhibitors[18]. This result demonstrates that CRYs
mechanism for inhibition also involves epigenetic modication through HDACs. After bind-
ing to the CLOCK-BMAL complex, CRY localizes SIN3-HDAC complexes to the same
regions of DNA, which results in deacetylation and therefore downregulation of the same
target genes[18].
A. Problem
It is clear that amongst other things, histone acetylation is an important regulatory com-
ponent. Indeed, it is a necessary condition for circadian oscillations to exist[5]. What is
less clear are the tools available for studying the dynamic physiological response caused by
histone acetylation. In particular, we would like to isolate the eects of epigenetic modica-
tions from other regulatory mechanisms such as post-translational modications and cellular
signaling. Another question we would like to answer is whether histone acetylation alone is
sucient for circadian oscillations to occur. These factors motivate the design of a tool for
ligand-induced reversible histone acetylation. Such a tool will enable isolation of the eects
caused by histone acetylation from other regulatory interactions. Furthermore, by alternat-
ing between induction and reversal of histone acetylation, we can answer whether oscillatory
acetylation alone is sucient for generating controlled articial circadian rhythms.
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B. Signicance
Many biological processes can be classied as circadian rhythms. An immediately rele-
vant example is how you slept last night. The human sleep/wake cycle is 24-hour periodic
and is also entrainable (can be resynchronized) after exposure to light[4], satisfying the two
requirements in the denition of a circadian rhythm. Improving our understanding of circa-
dian rhythm regulation has implications in the modeling and treatment of sleep disorders.
In particular, as histone acetylation is reversible and therefore malleable, it may provide a
mechanism for entrainability of sleep cycles.
More recent research eorts have uncovered a link between metabolic pathways and cir-
cadian rhythms through epigenetic regulation[1]. Studies suggest that abnormal metabolism
in certain cancers may be due to disrupted circadian rhythm regulation[14]. This suggests
that identication of causal relationships between epigenetic modications and circadian
rhythms will yield exciting opportunities for using pharmacological modulation of circadian
rhythms in treating metabolic disorders and human cancers.
Outside of circadian rhythms, tools which enable ligand-gated reversible modication of
the epigenome has high impact potential on how researchers study epigenetic modications
and their consequences. This is particularly interesting as while both genetic and epigenetic
information is heritable[9], the genome is relatively xed whereas the epigenome is highly
dependent on environment and regularly modied. Tools for perturbing epigenetic state
can enable targeted modication of parental epigenomes followed by characterization of the
resulting ospring. This can shed light on how the environment of the parent can impact
the osprings epigenetic state, potentially elucidating the mechanism for heritable disease
states not explained by changes in DNA sequence[19].
II. RESEARCH DESIGN AND METHODS
Chemical inducers of dimerization (CID) are a reliable method for colocalization of two
proteins upon addition of a small molecule inducer. Using a CID system comprised of
FK1012 (dimer of FK506, a dimerizing ligand) and FK binding protein (FKBP), Ho et al[7]
have shown that localization of transcription initiation factors to target genes is sucient
for transcriptional upregulation (Figure 4). We will assume that this is true for HATs as
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FIG. 4: CID system used by Ho et al[7] for activating transcription. Top: No dimerization
in absence of ligand. Middle: Addition of ligand induces dimerization and promotes
formation of preinitiation complex, resulting in upregulated transcription. Bottom:
Addition of competitive binder inhibits dimerization and downregulates transcription.
well: localization of HAT to chromatin should result in histone acetylation. Under this
assumption, the problem reduces to designing a tool for localizing HAT to target sequences.
Whereas Ho et al used FKBP102 to homodimerize two proteins, we need a CID system
to form heterodimers. Borrowing from a ligand-induced transcriptional activation system
demonstrated by Hjfeldt et al[8], our proposed design (Figure 5) also utilizes a CID to
recruit a protein to a DNA-binding domain (DBD). Unlike Hjfeldt et al, rather than re-
cruiting a transcription activation domain we will recruit a HAT. Upon addition of the
ligand, dimerization between the DBD and HAT will localize the HAT to target regions of
chromatin, which leads to targeted acetylation of histone tails. Dimerization is reversed by
addition of a competitive binder for the CID, resulting in a decrease in localized HATs and
consequentially decreased acetylation.
Most HATs do not possess a DNA-interacting domain and are recruited by transcription
factors which recognize consensus sequences in the DNA[11]. Aiming to isolate the eects of
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FIG. 5: Proposed design. CLOCK protein (dimerized to BMAL1) serves as the DBD.
Gcn5 serves as the HAT. FKBP fused to CLOCK and mFRB fused to Gcn5 are
chemically-induced to heterodimerize by the addition of a rapamycin analogue (rapalog).
histone acetylation in circadian rhythms, the DBD must recruit HATs to the same regions
of DNA as that of WT CLOCK-BMAL. Accordingly, we will use CLOCK-BMAL as the
DBD. To isolate the eects of our recruited HAT from CLOCKs endogenous HAT, we will
use CLOCK mutants from Doi et al where CLOCKs HAT activity is knocked out[5]. EMSA
or ChIP seq can be used to conrm that the constructed DBD has a DNA binding prole
similar to WT CLOCK-BMAL.
To ensure the induced acetylation is robust from endogenous inhibition, the interaction
between CLOCK-BMAL and its negative regulators must be disrupted. As the CRY-BMAL
interaction is the most signicant and the interaction domain has already been mapped to
the BMAL protein sequence[10], we will develop a mutant BMAL resistant to CRY inhibition
by engineering point mutations to disrupt the interaction domain. This should inhibit the
binding of CRY and subsequent recruitment of HDACs, eectively decoupling the negative
feedback loop from our engineered system. We will verify the disruption in CRY-BMAL
binding anity using isothermal titration calorimetry (ITC).
CID protein domains will then be fused to CLOCK and to the HAT protein Gcn5, re-
sulting in the chimeric proteins CLOCK-FKBP12 and mFRB-Gcn5. Gcn5 is a ubiquitous
HAT found in many cell types and conserved across species. Therefore, we expect its HAT
activity to be independent of particular cellular conditions or other proteins. In any case,
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the modularity of our design enables us to easily swap the HAT out for another. mFRB
is a mutated FRB (FKBP-rapamycin binding domain) developed by the Crabtree lab to
heterodimerize with FKBP12 upon the addition of a bio-orthogonal bumped rapamycin
analogue (rapalog)[2]. Unlike natural rapamycin, which is a potent immunosupressor, rapa-
log has been optimized for strong binding anity with mFRB but minimal interaction with
endogenous proteins[2]. The dimerization event can be competitively reversed by addition
of FKBP506[2], a ligand which competitively binds FKBP12 and inhibits rapalog induced
dimerization. Verication of successful and reversible dimerization can be accomplished with
gel electrophoresis and Western blot.
After construction and validation of each component, the integrated system can be val-
idated in vitro. Conversations with researchers in circadian cycles[20] gives evidence for
circadian oscillations lasting up to 6 weeks for some mouse broblast cultures. Cell cultures
of CLOCK and BMAL knockout mouse cells will be used. Mouse cells are used because
simpler model organisms (e.g. cyanobacteria or yeast) do not possess endogenous CLOCK
or BMAL proteins. Furthermore, the mouse genome is completely sequenced, so mapping
from acetylation prole to genes aected is possible. Knockout of endogenous proteins is
necessary to remove possible dimerization between endogenous and recombinant proteins
as well as endogenous circadian oscillations. If histone acetylation is sucient for circadian
rhythms, then we expect the addition of rapalog to cause changes in expression similar to
dierential expression due to circadian rhythms in WT cells.
DNA microarray, or alternatively RNA seq, can be used to prole gene expression levels
and identify dierentially expressed genes. The advantage of RNA seq is that no specic
cDNA array is required, reducing experimental bias and possibly identifying novel regulatory
interactions. To ensure the change in transcription is due to acetylation, ChIP seq can
be used determine histone acetylation status[6]. As we have isolated the induced histone
acetylation from the rest of the circadian rhythm regulatory network, the combined RNA seq
and ChIP seq data can be compared to WT expression levels and should enable identication
of genes whose dierential expression is caused by, rather than merely correlated to, histone
acetylation.
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III. CONCLUSION
Histone acetylation is a key regulatory mechanism in circadian oscillations. To enable
ligand-gated induction of histone acetylation as well as to isolate its consequences from other
regulatory mechanisms, we propose a design for ligand-induced reversible histone acetylation
utilizing a CID system which exploits the native DNA-binding prole of CLOCK-BMAL.
This will enable perturbation of circadian rhythms at the epigenetic level, allowing identi-
cation of causal relationships between histone acetylation and physiological response. Alter-
nating induction and reversal of acetylation can be performed to test if histone acetylation
alone is sucient for driving circadian rhythms. As each component functions indepen-
dently, the design of this system is modular. Therefore, the DBD and HAT may be easily
generalized to perturb and better understand other biological processes at the epigenetic
level.
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