Engineered histone acetylation using DNA-bindingdomains (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

Dynamical systems in biology

Figure 1: Negative feedback in PERregulation [1]

Figure 2: Resultant limit cycle [1]

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The mammalian circadean clock regulatory network

Figure 3: Focus on E-Box,CLK:BMAL1, PER:CRY [1]

Circadian acetylome showsCLOCK-dependentoscillatory patterns[2]

CLOCK is a HAT,inhibition of CLOCK HATactivity disrupts circadianrhythmicity [3]

PER:CRY bindsCLK:BMAL1 and recruitsSIN3:HDAC [4]

HDAC inhibitor relievesrepression of CRY andPER [5]

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So it seems that transcriptional regulation at the epigenetic level is apretty big deal. But what exactly is histone acetylation?

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Epigenetic regulation: histone acetylation

Figure 4: Acetylation in histone tails upregulates gene expression [6]

Figure 5: Mechanism of lysine acetylation [8]

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Goals

Disrupt the circadian rhythm at the epigenetic level

Construct a DNA-binding domain (DBD) mimicking DNA bindingprofile of CLOCK:BMAL1Use CID/CID inhibitor to toggle recruitment of HAT

Prototype a modular method for specifically modifying the histonecode

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Technicalities

Model organism: mouse cell cultures

Prerequisite recombineering

KO the WT CLOCK:BMAL1 proteinsInsert recombinant CLOCK-FKBP fusion protein, BMAL1, andHAT-FRB fusion protein (to be described)

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Designing the DNA-Binding Domain (DBD)

Figure 6: CLOCK and BMAL1recognize canonical E-box (CACGTG)[7]

Reuse CLOCK mutant withoutHAT activity [3], engineerBMAL1 mutant resistant toCRY inhibition

Effectively decouple negativefeedbackGood: DNA-sequenceaffinity matches desiredtargetBad: Don’t have PER:CRYcrystal structure

Verify DBD competitivelyinhibits WT with EMSA

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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

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The Histone Acetyltransferase (HAT)

DBD-mutated CLOCK?

Type A: Gcn5, p300/CBP, TAFII250

Located in nucleusContain bromodomain to recognize acetylated lysines

Type B

Acetylation of newly synthesized histonesFound in cytoplasm

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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] andwould even be predicted to recruit complexes in a natural orientation”[12]Bromodomain inhibitor already discovered [13]

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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 feedbackScience 332(6036) (2011)

Yoshihisa et al.

Circadian and Light-Induced Transcription of Clock Gene Per1 Depends on Histone Acetylation and DeacetylationMol Cell Bio, 24(14), 6278–6287, (2004)

Rodd et al.

Current and Emerging Therapeutics for Cutaneous T-Cell Lymphoma: Histone Deacetylase InhibitorsLymphoma 2012(290685), (2012)

Wang et al.

Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domainswith E-box DNACell Research, 23:213–224, (2012)

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References II

Trcum

Lysine acetylation, “https://en.wikipedia.org/wiki/File:Lysine acetylation.png”Wikipedia, The Free Encyclopedia, (2014), Accessed 4-29-2014

Ferree-D’Amare 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 reinitiationNature, 382:822-826 (1996)

Højfeldt et al.

Transforming ligands into transcriptional regulators: building blocks for bifunctional moleculesChem Soc Review, 40:4286–4292 (2011)

Filippakopoulos et al.

Selective inhibition of BET bromodomainsNature, 468:1067–1073 (2010)

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The End

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