For
many years we thought that DNA was the last word and that the whole information
for gene expression and its regulation during development and cellular
differentiation was coded in the DNA itself. However, an unexpected role in
these processes was discovered for chromatin.
Chromatin is a complex assembly formed by the
DNA sequence and histones, proteins that tightly pack DNA into the chromosomes
to fit into the nucleus. This
assembly can be modified by the addition of chemical groups such as methyl or
acetyl groups, enzymes and some forms of RNA such as microRNAs and siRNAs, that
can alter chromatin structure to influence gene expression, holding an extra
layer of instructions.
The
word “epigenetic” literally means “above” or “on top of” genetics and it
includes any changes in chromatin that alters gene activity without changing
the DNA sequence. So epigenetic modifications affect how cells “read” the
information coded in genes.
The
scientific interest in epigenetics has been growing at an exponential rate for
the past few years and its growing awareness has gained medical interest. The
reason is obvious: while genes can't readily be changed, proteins involved in
chromatin modifications can. Moreover they are excellent potential drug targets
since their action is reversible. As of 2013, there were four epigenetic
therapies approved for patients in the United States, all to treat cancers, and
there are currently more than a dozen in U.S. clinical trials1.
MicroScale
Thermophoresis has become an invaluable tool for the analysis of a series of
epigenetic proteins, revealing crucial information regarding to their
properties and function.
Interaction between
histones and proteins
The
primary protein components of chromatin are histones that associate with DNA,
forming an octamer of the histones H2A, H2B, H3 and H4. The resulting
structure, the nucleosome is the basic structural component of chromatin.
Although the nucleosome is a very stable protein-DNA complex, it is not static
and has been shown to undergo a number of different structural re-arrangements
(Längst and Becker, 2001)2. In addition, the nucleosome represents a
binding partner for various proteins and serves as scaffold to establish multi-protein
complexes at chromatin.
In
a recent study, the Drosophila Decondensation factor 31 (Df31) was shown to be
involved in regulating chromatin compaction and to be tethered to chromatin
(Schubert et al., 2012)3. Using MicroScale Thermophoresis, it was
demonstrated that Df31 does not only interact with histone molecules but also
with soRNA (small nucleolar RNAs) and revealed that this complex regulates
accessibility of chromatin at specific genomic regions.
Analysis of epigenetic
protein specificity and function
Histone
post-translational modifications, which hold one type of epigenetic
information, are catalyzed by enzymes generally known as “writers” while
proteins that recognize and bind to those modifications and interpret the
information coded in them are known as “readers”.
Histone
methylation is so far the most complex modification, since it can have
different functional outcomes depending on the precise methylation site and the
degree of modification.
MicroScale
Thermophoresis has recently been used for
examining issues of specificity with respect to methyl recognition. A study by
Greer et al.4 examined H3K9 methylation, a modification
involved in euchromatic gene silencing as well as in heterochromatin formation.
Using this technology, Greer showed
that EAP-1 (epigenetic memory
antagonism protein 1) bound most tightly to H3K9me3 (Kd =
157 nM), followed by H3K9me2 (Kd =
2.05 μM) and H3K9me1 (Kd =
6.14 μM) and revealed its function as a H3K9 reader.
Analysis of protein-small
molecule interactions
Potent,
selective and cell-permeable inhibitors that target key regulators of cellular
signalling ("chemical probes") are essential tools for target
validation and provide starting points for translational research projects.
Among them, probes against epigenetic proteins are thought to be
the next frontier in medicinal chemistry.
The
histone methyltransferase (HMT) G9a plays a crucial role in epigenetic
regulation and has been implicated in cancer. Thus, G9a inhibitors are expected
to exert synergistic effects in epigenetic cancer therapy.
MicroScale
Thermophoresis was used to assess the direct interaction between G9a and its
inhibitor BIX-01294 and to gain insight into its mode of action (Seidel et al.,
2013)5. With this
technology it was possible to detect direct binding interactions between G9a
and BIX-01294. Moreover it
was shown that the BIX-01294 competes with G9a for binding to the histone
binding site but does not compete with G9a cofactor SAM.
MicroScale
Thermophoresis appears as an excellent tool for the analysis of biomolecular
interaction within epigenetic molecules.
1.
http://www.cihrirsc.gc.ca/e/documents/show_me_evidence_v2_I4_en.pdf
2.
Längst, G., and Becker, P. B. (2001). Mol Cell. 8: 1085-1092.
3.
Schubert, T. et al (2012). Mol Cell. 48: 434-444.
4.
Greer, E. et al. (2014). Cell
Rep. 7(1): 113–126.
5.
Seidel, S. et al. (2013). Methods. 59(3): 301–315.
