1. Engineered histone acetylation using DNA-binding
domains (DBD), chemical inducers of dimerization
(CID), and histone acetyltransferases (HAT)
BCBP Research Proposal
Feynman Liang
Amherst College
fliang14@amherst.edu
5/2/2014
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 1 / 14

2. Dynamical systems in biology
Figure 1: Negative feedback in PER
regulation [1]
Figure 2: Resultant limit cycle [1]
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 2 / 14

3. The mammalian circadean clock regulatory network
Figure 3: Focus on E-Box,
CLK:BMAL1, PER:CRY [1]
Circadian acetylome shows
CLOCK-dependent
oscillatory patterns[2]
CLOCK is a HAT,
inhibition of CLOCK HAT
activity disrupts circadian
rhythmicity [3]
PER:CRY binds
CLK:BMAL1 and recruits
SIN3:HDAC [4]
HDAC inhibitor relieves
repression of CRY and
PER [5]
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 3 / 14

4. So it seems that transcriptional regulation at the epigenetic level is a
pretty big deal. But what exactly is histone acetylation?
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 4 / 14

5. Epigenetic regulation: histone acetylation
Figure 4: Acetylation in histone tails upregulates gene expression [6]
Figure 5: Mechanism of lysine acetylation [8]
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 5 / 14

6. Goals
Disrupt the circadian rhythm at the epigenetic level
Construct a DNA-binding domain (DBD) mimicking DNA binding
profile of CLOCK:BMAL1
Use CID/CID inhibitor to toggle recruitment of HAT
Prototype a modular method for specifically modifying the histone
code
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 6 / 14

7. Technicalities
Model organism: mouse cell cultures
Prerequisite recombineering
KO the WT CLOCK:BMAL1 proteins
Insert recombinant CLOCK-FKBP fusion protein, BMAL1, and
HAT-FRB fusion protein (to be described)
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 7 / 14

8. Designing the DNA-Binding Domain (DBD)
Figure 6: CLOCK and BMAL1
recognize canonical E-box (CACGTG)
[7]
Reuse CLOCK mutant without
HAT activity [3], engineer
BMAL1 mutant resistant to
CRY inhibition
Effectively decouple negative
feedback
Good: DNA-sequence
affinity matches desired
target
Bad: Don’t have PER:CRY
crystal structure
Verify DBD competitively
inhibits WT with EMSA
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9. Recruiting HAT using CID
Heterodimerization: FKBP-Rapamycin-FRB
Competitively inhibited by FK1012 [11] (likely AP1903 [10] as well)
Figure 7: Cartoon of proposed DBD, CID, HAT system
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 9 / 14

10. The Histone Acetyltransferase (HAT)
DBD-mutated CLOCK?
Type A: Gcn5, p300/CBP, TAFII250
Located in nucleus
Contain bromodomain to recognize acetylated lysines
Type B
Acetylation of newly synthesized histones
Found in cytoplasm
Feynman Liang (AC) DBDs, CIDs, and HATs 5/2/2014 10 / 14

11. Future steps (failure plans)
Optimize dimerization ligand’s lipophilicity and bioorthogonality
(e.g.g bump-hole)
Use another TF that binds the canonical E-box (e.g. USF [9]) as DBD
Try other HATs
Bromodomain ligand for CID conjugate partner
Recruit endogeneous HAT
“. . . not inhibit any enzymatic function in the recruited [HAT] and
would even be predicted to recruit complexes in a natural orientation”
[12]
Bromodomain inhibitor already discovered [13]
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12. References I
William Bechtel et al.
Thinking Dynamically about Biological Mechanisms: Networks of Coupled Oscillators.
Foundations of Science 18(4), 707 (2013)
Masri et al.
Circadian acetylome reveals regulation of mitochondrial metabolic pathways.
PNAS 110(9), 3339–3344 (2013)
Doi et al.
Circadian regulator CLOCK is a histone acetyltranferase.
Cell 125(3), 497–508 (2006)
Duong et al.
A molecular mechanism for circadian clock negative feedback
Science 332(6036) (2011)
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)
Rodd et al.
Current and Emerging Therapeutics for Cutaneous T-Cell Lymphoma: Histone Deacetylase Inhibitors
Lymphoma 2012(290685), (2012)
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)
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13. References II
Trcum
Lysine acetylation, “https://en.wikipedia.org/wiki/File:Lysine acetylation.png”
Wikipedia, The Free Encyclopedia, (2014), Accessed 4-29-2014
Ferre´e-D’Amar´e AR et al.
Structure and function of the b/HLH/Z domain of USF.
EMBO J, 13(1), 180-9 (1994)
Clackson et al.
Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.
PNAS, 95:10437–10442 (1998)
Ho et al.
Dimeric ligands define a role for transcriptional activation domains in reinitiation
Nature, 382:822-826 (1996)
Højfeldt et al.
Transforming ligands into transcriptional regulators: building blocks for bifunctional molecules
Chem Soc Review, 40:4286–4292 (2011)
Filippakopoulos et al.
Selective inhibition of BET bromodomains
Nature, 468:1067–1073 (2010)
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14. The End
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