MeCP2, an abundant chromosomal protein of the brain

with implications for neurological disorders

Juan Ausió
Department of
Biochemistry and
Microbiology
University of Victoria

MMDR-5, Karachi 2015

 MeCP2 consists of two isoforms (E1 and E2) and is an
intrinsically disordered protein

B
NTD MBD ID TRD CTDα CTDβ

mecp2 C
D

MeCP2
E1
4h

E2
100 h

Random coil α-Helix Extended strand

MeCP2 binds to chromatin and displaces histone H1 in a
DNA methylation-dependent way

Linker Nucleosome
Linker
Linker Nucleosome
Nucleosome
DNA core particle
DNA
DNA core
coreparticle
particle

Me CpG
Me
Me CpG
CpG
Rett syndrome is caused by mutations in MeCP2

Andreas Rett
 MeCP2 chromatin binding domains and PTMs
Mutations throughout are important

a N
C

b
24 25 92 94 131 142 147 161 241 245 261 268 283 286 317-9 333 413 435

Random coil α-Helix Extended strand

K-Acetylation S-Phosphorylation K-Ubiquitination

73 AGKAETSESSGSAPAVPEASASPK 96 269 RKAEADPQAIPKKRGRK 285 390 TKAPMPLLPSPPPPEPESSEDPISPPEPQDLSSSICK 426

c NTD MBD ID TRD CTDα CTDβ

1 PEST1 92 176 215 NLS 326 371 PEST2 498
196 TGRGRGRPKGS 205 276 IPKKRGRKPG 285

d 78 92 176 215
AT hook
276 285 308 384 385 467
MBD DNA binding Binary chromatin
domain binding sites

e
158 304 325 498
Dimerizing domain WDR
MeCP2 PTMs
MeCP2 mutation dependence severity of the Rett
syndrome phenotype

Kerr, Percy and Pineda scores for all mutations—C-terminal deletions are circled (adjusted for age
and data source).

.
Updating the profile of C-terminal MECP2 deletions in Rett syndrome.
Bebbington A, Percy A, Christodoulou J, Ravine D, Ho G, Jacoby P, Anderson A, Pineda M,
Ben Zeev B, Bahi-Buisson N, Smeets E, Leonard H.
J Med Genet. 2010 47:242-248.
 MeCP2 distribution in HeLa cell chromatin

SI SE P SI SE P SI SE P

S1 SE P MeCP2

H3K9me2
H4
H3K27me3
1 2 3
H3K4me3
1 0.25 M sucrose
H3K36me3 10 mM Tris-HCl (pH 8.0)
0.5 mM CaCl2 3 50 mM KCl
pan-Ac H4
2 0.1 M KCl 10 mM Pipes (pH 6.8)
5.0 mM MgCl2
H4 10 mM Tris-HCl (pH 7.5)
1.0 mM CaCl2
1.0 mM CaCl2
Distribution of MeCP2in HeLa cells in response to treatment
with DNA methylating and de-methylating agents
 MeCP2 is present in a 30-60 fold excess in brain over other
tissues and it is present in one of every three nucleosomes

A Liver Brain Testis

CM SI SE P N CM SI SE P N CM SI SE P N CM

MeCP2

Histone H3

B C
MeCP2 chromatin fractionation depends on the extent of nuclease digestion
Most of MeCP2 from brain chromatin is bound to non-
nucleosomal regions in brain
The high nuclease accessibility of the MeCP2-containing
protein question the early models and supports the
notion that MeCP2 is a REGULATOR of gene transcription

MeCP2, a key contributor to neurological disease, activates and represses transcription.

Chahrour M, Jung SY, Shaw C, Zhou X, Wong ST, Qin J, Zoghbi HY.
Science. 2008 May 30;320(5880):1224-1229.
 MeCP2 is distributed throughout the nucleus and is
depleted in the peripheral heterochromatin in brain cells
 MeCP2 is distributed throughout the nucleus and is
depleted in the peripheral heterochromatin in brain cells
Nucleosomal MeCP2 is associated with histone H2A.X
and histone PTMs
A model for MeCP2 chromatin binding in the brain
 CONCLUSIONS

In vivo, MeCP2 is associated with a chromatin fraction readily accessible to

nuclease digestion

Brain chromatin has a 30-60 fold excess of MeCP2 compared to other tissues
and it contains approximately one molecule of MeCP2 for every three
nucleosomes

In brain, MeCP2 is located at the periphery of heterochromatin domains where

it is mainly bound to non-nucleosome regions

The nucleosome-bound MeCP2 fraction is associated with H2A.X and

repressive histone PTMs
MeCP2, Rett syndrome and beyond

Rett
syndrome
Downsyndrome
ADHD/ADD

Cocaine
Alzheimer
Huntington
Depression
Parkinson

Epilepsy

MeCP2
MeCP2, beyond Rett syndrome
 The remaining questions

Where is MeCP2 precisely bound in brain (neurons), and how does all this change during
development? Why is there so much?

Why do different MeCP2 mutation have different severity?

What are the functional differences between the MeCP2 E1 and E2 isoforms?

What is the significance of the different amounts of the two MeCP2 isoforms in different
tissues?

Do the E1 and E2 isoforms have the same average half-life times

What are the roles of PTMs already described, such as ubiquitination and acetylation in
particular, and the still to be uncovered MeCP2 PTMs?

Do the PEST sequences play a role in MeCP2 metabolism?

What are the precise roles of MeCP2 in other neurological disorders?

And many more….

 The people and the $$

Anita
Marlee Ng Thambirajah
Department of Oncology
Michael Hendzel
University of Alberta
Department of Biological Sciences
Philippe Georgel
Marshall University
Genome BC Proteomics Centre
Christoph Borchers
University of Victoria
 The remaining questions

Where is MeCP2 precisely bound in brain (neurons), and how does all this change during
development? Why is there so much?

Why do different MeCP2 mutation have different severity?

What are the functional differences between the MeCP2 E1 and E2 isoforms?

What is the significance of the different amounts of the two MeCP2 isoforms in different
tissues?

Do the E1 and E2 isoforms have the same average half-life times

What are the roles of PTMs already described, such as ubiquitination and acetylation in
particular, and the still to be uncovered MeCP2 PTMs?

Do the PEST sequences play a role in MeCP2 metabolism?

What are the precise roles of MeCP2 in other neurological disorders?

And many more….

 MeCP2 and histone H1 have a short residence time on
chromatin but MeCP2 binds more weakly

1.0

0.8
Recovery

0.6

0.4

0.2

0.0

MeCP2
Histone H1

0 20 40 60 80 100 120
Time (s)

MeCP2 and Histone H1 FRAP MeCP2 and Histone chromatin

dissociation
 MeCP2 and chromatin (in the brain)

(a)
mC

hm/mCpG.[A/T]≥4 H1

MeCP2
200 bp
MeCP2 MBD
AAATT (1)
(b)

Histone H1 WHD
N

(c)
165 bp

Repeat length bp
200
L

H1
A
(2)
A

MeCP2
180
165 bp N
160

(3) 0 7 14
days
Most of MeCP2 from liver chromatin is bound to
nucleosomal regions

0.6

O.D. 260 0.4

0.2

0.0
0 5 10 15 20 25
Fraction number
H3

SI H
H2A
H2B
H4

W MeCP2

SE H1

H
H3
H2A
H2B
H4

W MeCP2