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 modifications to intercellular signaling[17]. With

the goal of better understanding how regulation at the epigenetic level affects 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 effects 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 sufficient

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.

∗ fliang14@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

earth’s rotation reflects the deepness of the connection between circadian cycles and life (on

earth) itself.

A defining feature of biological processes classified 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].

Scientific 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 CRY’s

mechanism for inhibition also involves epigenetic modification 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 effects of epigenetic modifica-

tions from other regulatory mechanisms such as post-translational modifications and cellular

signaling. Another question we would like to answer is whether histone acetylation alone is

sufficient 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 effects

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 sufficient for generating controlled artificial circadian rhythms.

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B. Significance

Many biological processes can be classified 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 definition 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 efforts 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 identification of causal relationships between epigenetic modifications 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 modification of

the epigenome has high impact potential on how researchers study epigenetic modifications

and their consequences. This is particularly interesting as while both genetic and epigenetic

information is heritable[9], the genome is relatively fixed whereas the epigenome is highly

dependent on environment and regularly modified. Tools for perturbing epigenetic state

can enable targeted modification of parental epigenomes followed by characterization of the

resulting offspring. This can shed light on how the environment of the parent can impact

the offspring’s 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 sufficient

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 Højfeldt et al[8], our proposed design (Figure 5) also utilizes a CID to

recruit a protein to a DNA-binding domain (DBD). Unlike Højfeldt 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 effects 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 effects of our recruited HAT from CLOCK’s endogenous HAT, we will

use CLOCK mutants from Doi et al where CLOCK’s HAT activity is knocked out[5]. EMSA

or ChIP seq can be used to confirm that the constructed DBD has a DNA binding profile

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 significant 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, effectively decoupling the negative

feedback loop from our engineered system. We will verify the disruption in CRY-BMAL

binding affinity 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 affinity 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. Verification 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 fibroblast 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 profile to genes affected 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 sufficient for circadian

rhythms, then we expect the addition of rapalog to cause changes in expression similar to

differential expression due to circadian rhythms in WT cells.

DNA microarray, or alternatively RNA seq, can be used to profile gene expression levels

and identify differentially expressed genes. The advantage of RNA seq is that no specific

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 identification

of genes whose differential 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 profile of CLOCK-BMAL.

This will enable perturbation of circadian rhythms at the epigenetic level, allowing identifi-

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 sufficient 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.

[1] Aguilar-Arnal et al. The circadian epigenome: how metabolism talks to chromatin remodeling.

Curr Opin Cell Biol, 25(2):170–6, 2013.

[2] Bayle et al. Rapamycin analogs with differential binding specificity permit orthogonal control

of protein activity. Chem Biol, 13(1):99–107, 2006.

[3] Chen et al. A conserved dna damage response pathway responsible for coupling the cell

division cycle to the circadian and metabolic cycles. Cell Cycle, 6(23):2906–2912, 2007.

[4] Czeisler et al. Sleep and circadian rhythms in humans. Cold Spring Harb Symp Quant Biol,

72:597–97, 2007.

[5] Doi et al. Circadian regulator clock is a histone acetyltranferase. Cell, 125(3):497–508, 2006.

[6] Galdieri et al. Determination of histone acetylation status by chromatin immunoprecipitation.

Methods in Molecular Biology, 809:255–265, 2012.

[7] Ho et al. Dimeric ligands define a role for transcriptional activation domains in reinitiation.

Nature, 382:822–826, 1996.

[8] Højfeldt et al. Transforming ligands into transcriptional regulators: building blocks for bi-

functional molecules. Chem Soc Review, 40:4286–4292, 2011.

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[9] Jeppeson et al. Histone acetylation: a possible mechanism for the inheritance of cell memory

at mitosis. Bioessays, 19(1):67–74, 1997.

[10] Langmesser et al. Interaction of circadian clock proteins per2 and cry with bmal1 and clock.

BMC Molecular Biology, 9:41, 2008.

[11] Legube et al. Regulating histone acetyltransferaces and deacetylases. EMBO Rep, 4(10):944–

947, 2003.

[12] Masri et al. Circadian acetylome reveals regulation of mitochondrial metabolic pathways.

PNAS, 110(9):3339–3344, 2013.

[13] Rodd et al. Current and emerging therapeutics for cutaneous t-cell lymphoma: Histone

deacetylase inhibitors. Lymphoma, 2012(290685), 2012.

[14] Sahar et al. Metabolism and cancer: the circadean clock connection. Nat Rev Cancer,

9(12):886–96, 2009.

[15] Wang et al. Intermolecular recognition revealed by the complex structure of human clock-

bmal1 basic helix-loop-helix domains with e-box dna. Cell Research, 23:213–224, 2012.

[16] Waterhouse et al. The circadian rhythm of core temperature: origin and some implications

for exercise performance. Chronobiol Int, 22(2):207–25, 2005.

[17] William Bechtel et al. Thinking dynamically about biological mechanisms: Networks of cou-

pled oscillators. Foundations of Science, 18(4):707, 2013.

[18] Yoshihisa et al. Circadian and light-induced transcription of clock gene per1 depends on

histone acetylation and deacetylation. Mol Cell Bio, 24(14):6278–6287, 2004.

[19] Jiang. Epigenetics and human disease. Annu Rev Genomics Hum Genet, 5:479–510, 2004.

[20] Tanya Leise. Personal communication, 2014-04-12.

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