Tools for Screening Chromatin

Modifying Enzymes

Meera Kumar Thomas Hengstl

Pre-calibrated enzyme

≤ 20% substrate

depletion

Substrates @ Km

Robust Signal (Z’ > 0.7)

ID and source

Reagents/

Technology

Validate activity with different

isoform and Lots Assay Development

What is Epigenetics?

A. Modification at the DNA level

1. Cytosine Methylation

B. Histone modification - the Histone Code

1. Histone Acetylation

2. Histone Methylation

3. Histone Phosphorylation

4. Histone Ubiquitination/Sumoylation

Sukesh R Bhaumik, Edwin Smith & Ali Shilatifard Nature Structural & Molecular Biology 14, 1008 - 1016 (2007)

Transcreener® & Epigenetic Modifications

5

Transcreener relies on direct

detection of ADP. Binding of tracer

to antibody causes a change in

fluorescence. There are just two

components, and no intermediate

steps.

Simplified protocol

Transcreener ADP2 Assay:

Direct detection of ADP means less chance for interference

0.01 0.1 1 100

50

100

150

200

250

0 hr

1hr

4hr

8hr

24hr

ADP (µM)

m

P

0.01 0.1 1 100

50

100

150

200

250

RT

37°C

Control

-80°C

-20°C

4°C

ADP (µM)

m

P

21 Day Reagent Stability

Signal Stability % ATP Conversion 0.1 µM 1 µM 10 µM 100 µM 1000 µM100 0.96 0.95 0.95 0.94 0.93

60 0.94 0.94 0.95 0.94 0.93

30 0.89 0.94 0.94 0.92 0.9

20 0.87 0.94 0.93 0.92 0.9

15 0.87 0.93 0.92 0.91 0.89

10 0.78 0.91 0.93 0.88 0.89

7.5 0.74 0.87 0.92 0.89 0.85

5 0.65 0.86 0.88 0.87 0.83

3 0.53 0.76 0.85 0.82 0.8

1.5 -4.22 0.36 0.6 0.63 0.62

0.75 -2.24 0.25 0.48 0.54 0.6

0.0001 0.001 0.01 0.1 1 10 100 10000

50

100

150

200

250

1000 M

100 M

10 M

1 M

0.1M

ADP M

m

P

Transcreener ADP2 Assay:

Stable, Robust and Reproducible

http://bricker.tcnj.edu/Amb/le10/cellproph.jpg

Simple Detection of Histone Phosphorylation

0 50 100 150 200 2500

50

100

150

200

Kemptide-50 M

Histone H1-3 M

Histone H3-10M

[PKA] (ng/mL)

m

P

Histone phosphorylation can be interrogated

using the Transcreener ADP2 assay.

Exploring the sumoylation/ubiquitination cascade using the

Transcreener® AMP2/GMP2 Assay

8

• Transcriptional regulation

• Genome organization and repair

• Nucleocytoplasmic translocalization- Ex:Sumoylation of RanGAP1 is

required for proper targeting to the NPC.

• Protein-Protein interactions

• Protein DNA binding

• Protein stability

Monitoring AMP Flux with the Transcreener Assay

From Tang Z., et al, 2008. FEBS Journal 275: 3003-3015.

Sumoylation, Neddylation, and Ubiquitination Pathways:

Transcreener Assay:

In this study: SUMO= SUMO-1

E1= SAE1/SAE2

E2 = Ubc9

E3= RanBP2

AMP Formation is SUMO-dependent and stoichiometric with E1

Eq.1 E1 + ATP + Sumo E1.AMP~Sumo + PPi

Eq.2 E1.AMP~Sumo E1-S-CO-Sumo + AMP

Eq.3 E1-S-CO-Sumo + ATP + Sumo E1-S-CO-Sumo + PPi.AMP-Sumo

0 50 100 150 200 250 3000

50

100

150

200

250

0

100

200

300

400

+ Sumo

- Sumo

E1, nM

m

P

AM

P, n

M

Reactions were allowed to go to completion, thus AMP should be produced

in stoichiometric amounts with E1, as was observed.

E1, nM

ExperimentalAMP, nM

TheoreticalAMP, nM

150 134 (±8.6) 150

75 69 (±4.2) 75

37.5 41 (±7.4) 37.5

18.7 19 (±4.1) 18.7

9.3 8 (±3.1) 9.3

AMP Production by E1 is enhanced by E2 and E3

E1-S-CO-Sumo.AMP-Sumo .AMP-Sumo

Eq.5 + RanBP2-NH2 E2-SH + RanBP2-NH-CO-Sumo

A. B.

Reactions were again run to completion. Addition of E2 resulted in a stoichiometric increase in

AMP, as expected (Eq. 4). Addition of E3 to the reaction resulted in >5-fold increase in AMP

production, suggesting that multiple SUMOs are being transferred to E3.

E2, nM

Experimental+ 50 nM E1AMP, nM

Theoretical*+ 50 nM E1AMP, nM

Experimental+ 50 nM E1 + 75 nM E3

AMP, nM

Theoretical*+50 nM E1+ 75 nM E3

AMP, nM

50 119 (±7.2) 100 758 (±4.8) 175

25 86 (±4.3) 75 600 (±6.2) 150

12.5 61 (±5.4) 62.5 368 (±7.1) 137.5

0 47 (±3.4) 50 53 (±1.4) 50

AMP formation by SUMO ligase enzymes is readily detected using the

Transcreener AMP Assay.

In reactions with E1 and E1 + E2 that are allowed to run to completion, the

expected stoichiometric amounts of AMP are produced.

Addition of E3 results in an increase in AMP formation several fold higher

than stoichiometric, suggesting that it is sumoylated at more than one

lysine.

AMP Flux can be interrogated with Transcreener AMP/GMP

Assay

A B

Transcreener EPIGEN Methyltransferase Assay

0.01 1 1000

50

100

150

200G9a/H3(1-21)

SET8/H4 (15-24)

Set7/9/H3(1-21)

NSD2/Nucleosomes

MLL4 Complex/H3(1-21)

EZH2 Complex/H3(21-44)

SUV39H1/H3(1-21)

Dot1L/nucleosomes

GLP/H3(1-21)

G9a/GLP/H3(1-21)

[E], ng/L

m

P

0.01 0.1 1 100

50

100

150

200

PRMT1/H4(1-20)

PRMT3/H4(1-20)

PRMT8/GST-GAR

PRMT4/H3-3

[E] ng/L

m

P

0.1 1 10 1000

50

100

150

DNMT1

DNMT3b

ng/L

m

P

Methylation of native proteins & peptides

with Methyltransferases

Enzyme, ng/L

SA

H,

M

0.0 0.5 1.0 1.5 2.0 2.50.0

0.1

0.2

0.3

0.4

0.5

G9a

SET8

SET7/9

PRMT3

Tranzyme Methyltransferase Assay Kit

• A kit that includes everything-Enzyme, Substrate, Buffer, Detection Mix

•Quick guide tells you how much enzyme you need to use, how much

substrate and detection Mix you need for the most optimal signal.

•The assay is a simple mix-and-read format, and the signal is stable for

many hours.

We now offer 13 Transzyme Methyltransferase kits.

Advantages of EPIGEN Methyltransferase Assay

• Homogenous and Generic Assay.

• Developed for measuring Initial velocity conditions.

• Stable and reliable signal for up to 48 hours.

• Detection mixture has an excellent deck stability.

• Robust Z` and good assay window at initial velocity conditions.

• Low interference (0.7%) was observed due to use of far red tracer and the two coupling enzymes were several fold excess of what is required for a good signal.

• Uses very specific antibody that has been extensively validated in HTS labs.

0 50 100 150 200 250 300 350

0

25

50

75

100

125

without acceptor substrate

1 M Histone H3 (full length)

10 M Histone H3 Peptide 1-25

100 M Histone H3 Peptide 1-25

10 M Histone H3 Peptide 15-39

[G9a] ng/mL

m

P

Acetyltransferases

Active Inactive

17

Acetylated histones open up the chromatin

and enable transcription.

Histones are acetylated by HAT (Histone

acetylases) which are parts of many

chromatin remodeling and transcription

complexes.

0 2 4 6 8 100

50

100

150

200

0.0

0.2

0.4

0.6

0.8

1.0

CoA, M

m

P

Z' V

alu

e

0 20 40 60 80 1000

50

100

150

200

0.0

0.2

0.4

0.6

0.8

1.0

CoA, M

mP

Z' V

alu

e

Excellent Z’ Values (< 10% Acetyl CoA Conversion)

10 µM Acetyl CoA

Standard Curve

100 µM Acetyl CoA

Standard Curve

18

Signal Stability for Acetyl CoA/CoA Standard Curve

19

0 20 40 60 80 1000

50

100

150

200

0.5 Hr

1 Hr

2 Hr

3 Hr

24 Hr

CoA M

m

P

0 10 20 30 40 50 600

25

50

75

100

2

1

0.5

0.25

0.13

0.060

[pCAF] ng/L

Time, min

m

P

Enzyme & Time Dependent Production of CoA

10 µM peptide + 100 µM Acetyl CoA

0 10 20 30 40 50 600

2

4

6

8

10

12

0.5

0.25

0.125

0.0625

0

1

[pCAF] ng/L

Time, min

Co

A,

M

20

21

Universal: Any Enzyme, Any substrate, Any ATP/SAM concentration

Sensitive: low substrate consumption, use less enzyme

Direct detection, far red fluors: less interference

Single addition, mix and read format: easy automation

Three fluorescent readouts, instrument-validated: flexibility, confidence

> 12hr reagent and signal stability: easy automation

Transcreener technology offers simple solutions for

interrogating epigenetic mechanisms

Transcreener® Platform: 4 Assays … Thousands of Targets

Methyltransferases

Acetyltransferases

G protein/RGS

22

Transzyme Methyltransferase Assay Kits (200-1000 Assays; 384 well format)

Transzyme Assay Kit Catalog #DNMT1 9002

HMT-DOT1L 9024

HMT-G9a 9008

HMT-MLL4 9010

HMT-PRMT1 9014

HMT-PRMT3 9016

HMT-SET7/9 9018

HMT-SET8 9020

HMT-SUVH391 9022

HMT-GLP 9054

HMT-G9a-GLP 9056

HMT-PRMT4 (CARM1) 9058

HMT PRMT8 9060

For more information on Transzyme Methyltransferase Assay Kits, email us

at meera.kumar@bellbrooklabs.com or call toll free 866-313-7881.

Visit our website at www.bellbrooklabs.com to learn more about the technology.

http://www.bellbrooklabs.com/products-services/enzymes-and-assay-kits/transzyme-methyltransferase-assay-kitsmailto:info@bellbrooklabs.comhttp://www.bellbrooklabs.com/