Download In vitro studies of histone tail cross

In vitro studies of histone tail cross-talk effects on histone
demethylases. Deciphering epigenetic marks on the molecular level.
Brian Lohse*, Jan Kristensen, Charlotte Helgstrand, Ulrike Leurs, Paul A. C. Cloos, Rasmus P. Clausen, Jesper L. Kristensen.
University of Copenhagen, Universitetsparken 2, 2100 København Ø, Denmark.
Introduction
Highlights
DNA is wound tightly around histone
complexes (see figure 1) and from the
histone complexes long peptides are
protruding perpendicularly, called histone
tails (indicated by arrow on figure 1).
We did the following observations for the
enzymatic activity of the lysine demethylases:
PTM cross-talk effects in vitro for
Full length JMJD2C
JMJD2A, JMJD2C and PHF8
1) H3K9(Me)3 > H3K9(Me)2 > H3K9(Me)1
2) H3K9(Me)3 < H3K9(Me)3K14(Ac)
3) H3K4(Me)3K9(Me)3 > H3K9(Me)3
4) H3K9(Me)3T11(ph) = No activity
Enzyme Kinetics
The correlation between the catalytic activity
of 2C (see fig. 3), 2A & PHF8 (see fig. 4) as
a function of peptide length on di- and
trimethylated substrates on H3K9:
Figure 1: DNA wound around a histone complex. Arrow
indicates a histone tail.
0,9
JMJD2C (Ac)1: (Me)3
0,8
JMJD2C (Ac)1: (Me)2
0,7
JMJD2C (Ac)0: (Me)3
0,6
kcat (min-1)
These histone tails are constantly modified
by a range of enzymes adding and removing
groups, through chemical modifications
primarily: phosphorylations, acetylations
and methylations. In this study we focus on
histone 3 (H3), it’s tail modifications (see
figure 2) and compare three demethylating
enzymes within the Jumonji family.
0,5
0,4
0,3
0,2
Figure 5: kcat / Km for Full Length JMJD2C without and
with K4(Me)3 Showing a change in kcat / Km
0,1
0
0
5
10
15
20
25
30
35
40
Number of amino acids in the peptides
Figure 2: Histone 3 tail and it’s known modification sites.
Figure 3: kcat for JMJD2C vs. length of substrate. (Ac)1
means K14 is acetylated on (εN). (Me)3 means K9 is
trimethylated and (Me)2 dimethylated both on (εN).
Purpose of the investigation
1.
To try and shead light on the impact of the
chemical modifications, through histone tail
substrate modification and mimicking.
2.
To truncate the natural substrate (H3), to the
shortest peptide possible still giving rise to
lysine demethylation.
3.
Is it possible to observe cross-talk effects in
vitro by monitoring the effect on the histone
lysine demethylases, upon presenting more
than one chemical modification on the same
substrate e.g. H3K9(Me)3-T11(phos).
45
Conclusion
We have shown that it is possible to study
cross-talk effects on histone modifying
enzymes by histone tail mimicking. Through
peptide synthesis, specific combinations can
be synthesized and the effects studied on one
enzyme at the time, providing an important
tool to gain information on the impact of
chemical modifications. We have used these
results to find specific substrate based
inhibitors, for candidates as anticancer drugs.
References
Lohse et al. Angew. Chem. Int. Ed. 2011, 50, 9100.
Figure 4: kcat for JMJD2A and PHF8 vs. length of substrate.
(Ac)1 means K14 is acetylated on (εN). (Me)3 means K9 is
trimethylated and (Me)2 dimethylated both on (εN).
Lohse et al. Bioorg. Med. Chem. 2011, 19, 3625.
*Contact: [email protected]