NUCLEUS : CHROMATIN: grainy/rice, DNA loose = chromatin, tight = chromosome
Chromatin Basics
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Transcript of Chromatin Basics
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This slide series has been used for teaching to students of the Montpellier University in 2004.
It explains basic features of the folding of DN in nucleoso!es and chro!atin fibers" and
discusses the regulatory roles of post#translational histone !odificatons
The !aterial displayed is inspired fro! several sources. The literature used is cited $ithin
the slides. Moreover" several slides %indicated by the &'( logo) have been copied or !odified
fro! &acob (aterborg" University of Missouri" *ansas +ity To see his chro!atin !aterial in
full" see the $ebsite, http,--sbs.u!c.edu-$aterborg-
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3-99&'(
DNA compactioncompaction in a human nucleus
1bp (0.3nm)
10,000 nm
30nm
11 nm
Compaction of !" by hi#tone# Compaction by chromo#ome #caffo$% & n'c$ear matri
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Nuclear # chro!oso!e co!paction
Compaction by chromo#ome #caffo$% & n'c$ear matri
ra%ia$ $oop
chro#omo#ome mo%e$
1µm
1 0 µ m
chromatidchromatid
m i t o t i c c h r o m o
s o m e
+ 2M NaCl
histones
+ 2M NaCl
histones
Mitosis!" $oop#
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'I/TN1/are
highly consered!
small! basic proteins
H1
H*
H"
H3
H+
he$i
ariab$e
con#ere%
"istone acetylation
i# a reer#ib$e mo%ification
of $y#ine# in the !-termini
of the core hi#tone#. #esult/ re%'ce% bin%ing to !"/ %e#tabi$iation of chromatin
in2er hi#tone
Core hi#tone#
!
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+ore 'istones
he ba#ic #tr'ct're of " core hi#tone# i# the #ame/ 1 $ong hy%rophobic a$pha-he$i,
bor%ere% by/ #hort hy%rophobic a$pha he$ice#
that form pair#/ H" - H* an% H3 - H+ 4hich interact.
#e$erences% Moudrianakis et al. PNAS 88, 10138 (1991);
PNAS 90, 10489 (1993); PNAS 92, 11170 (199)
The histone#fold
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'istone octa!er asse!bly
"3-"&
tetramer "2A-"2'
dimer
"istone
octamer
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The Nucleoso!e as the funda!entalchro!atin unit
5ig're, co'rte#y from im
6ichmon%, 7H 8ric:4iter$an%
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/ 1+;-1+9 bp !" in a 1.;< t'rn# of a
f$at, $eft-han%e% #'perhe$i/ one p#e'%o t4ofo$% ai# centere% atthe =%ya%> (reference 0 he$ica$ t'rn#)
/ one ba#e-pair preci#e$y at the %ya%/ #harp ben%# at ? 1.< an% ? +-< t'rn#/ Hi#tone-fo% %omain# organie 11 bp
of !". he !" i# bo'n% at 10 bp
intera$# thro'gh many contact#,
inc$'%ing penetration of arginine# at a$$
1+ minor grooe# facing the proteincore/ he grooe# from neighboring !"
t'rn# $ine 'p@ forming channe$#/ H3 an% H* !-termini eit one of
the#e channe$# eery 0bp./ he H+ tai$ e#tab$i#he# contact# 4ith
the net core partic$e.
Nucleosome features
H* H+H3H"
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'istone octa!er organies 34 bp of DN
/ 7ach core hi#tone %imer
ha# ; !" bin%ing #'rface#
that organie 3 !" t'rn#@/ he hi#tone octamer
organie# 1+< bp of !"
in 1 3&+ he$ica$ t'rn of !"+A nm of !" pac2age% in a %i#c of ; 11nm
B ; nm
B
1 1
n m
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(here are the N#ter!ini of the core histones 5
!u"er, Mader,#i$%&ond, Sar"ent ' #i$%&ond
Nature 3(9, 2120 (199)) 6uestion 3: What is the function of histone N-termini ?
6uestion 2: Are all N-termini functionally equivalent ?
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"1*" g$ob'$ar protein %omain
7iner histone '3
,iner histone "1 organi.es e/iting DNA up to 1( - 200 bp
H1 #tabi$ie# interaction bet4een n'c$eo#ome# in compacte% chromatin.
On the OUTSIDE of the DNA gyres ? Asymmetric between the DNA gyres ?*%ou et al. Nature 39! +0 (199() +ole et al. 4cience 2)&! 14 (199)
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Chromatosome
mM
1 mM
7iner 'istone and histone ter!ini
control liner DN entry-exit of chro!atoso!e in chro!atin fiber.
Core histone octamer + 1 Liner !istone + " f#$$ t#rns of DNA %1&' b()
H3
H1
!
C
H3
*%ou, -er$%&an, #a&akris%nan, ra/ers,
Mulder&ans Nature 39, +0 (199()
An, !eua, /an olde, *latano/a
5NA4 9, 339; (199()
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+hro!atin
fibers
+ charged N termini
(bin% !" on neigboring
n'c$eo#ome#)
highly acetylat
core histon
(e#pecia$$y H3 an% H
30 nm
chromatin $iber
11 nm
bead
/ "67" leel o$ histone "1 / #educed leel o$ histone "1
/ N8 gene transcription / 7ene transcription possible
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'istone !odifications
Turner (22!" Cell 111# 2$%
D
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cetylation of conserved lysines
H+ !-termin'#
H3 !-termin'#
"c-:-E-6-E--E-E--E--E--E-E-"--6-H-6-K-F--6--? ? ? ? ? ?? ? ? ?
"c or MeAcAcAcAc
5 8 12 16 20
"-6---G--"-6--:--E-E--"--6--G--"---"-"-6--:-"--
Me "c or Me
Ac Ac Ac Ac
?? ? ? ? ? ? ??
4 9 14 18 23 27
The N#ter!ini of histones '4 and '8" and their acetylation patterns" are absolutely conserved.
'ysine
ε
# N-Acetyl-'ysine
'T %'istone
cetyl#Transferase)'istone
Deacetylasere/ersile rea$tions
8
N
C
C C
C
C
CN+α β
γ δ
ε5
88
8
5
8
-
DN
bacbone
binding
no NA
)in*in+8
N
C
C C
C
C
C
Nε
8
C
C
Acety$*CoA
CoA5-
--
--
---
--
--
--
--
--
-
-
-
-
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e$ectro%e
e$ectro%e
gel type% 4D4
"&
10 aa
"3
13< aa
"3
"&
+
:
9el 1lectrophoresis of 'istones
A; A;<
4eparation by% si.e si.e +charge
si.e + charge +hydrophobicity
"3
"&
"32
"&
"31
01
3
+
0
3
+
1
"&
10
++ +
13<
++ +
"3
0
3
+
1
+ +
: :
7el scans a$fa$fa hi#tone# (+ateror", 90s)
"&
10
++ +
3
13<
++ +
"31
3
3 3 3
3
13<
++ +
"32
3 3
3
013
+1
3
+
0
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Almost ll cetylation is Dyna!ic
+ateror" = 'iol Chem 2)3, D;0 (199()
7amp$e alga
Ch$amyomonas
reinhartii
>
0
10
1
20
2
0
10
1
20
2
0
1
3
+
<
0
1
3
+
<
H3 H4
proteinprotein
,?@?, o$ Ac,ysturnoer
,?@?, o$ Ac,ysturnoer
S t e a d y - s t a t e [ * A c ] l a b e l i n
g
ee$ of hi#tone acety$ation 32 > o$ "3 1 > o$ "&
"erage $ee$ of acety$ation 2& Ac,ys*"3 1 Ac,ys*"&
"erage "cy# t'rnoer I 19 ±0.+ min (H3) 3 ±1.1 min (H+)
"cy# t'rnoer I (min) .A 1.A 1.A 1.; 1.<
:raction of acetylated histone 100 92 10& 10 (2 39 ( (1 100 ) >
sub;ect to c7ys turnover, 300 ±12 < of '8 ±, < of '4
1 " , - . 1 " , - . /AcLys0histone
%=>
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cetylation of +hro!atin Do!ains
ees, laton, %orne, rane#oinson ?M'8 = 13, 1A3 (199&)
1xa!ple, the chicen *g$obin gene omain
l "igh leels o$ chromatin acetylation, acro## comp$ete chromatin %omain#
(!" $oop#), in%'ce# chromatin change# %etecte% a# =general DNase 6 sensitiity>l Within the#e chromatin %omain#, at f'nctiona$ gene# or tran#cription factor#,
the chromatin #tr'ct're i# interr'pte% by #ma$$ =DNase 6 hypersensitie sites>
%omain
bo'n%ary
%omain
bo'n%ary
βρ βH β " βε
0 10 20 30 b
!a#e
hyper-#en#itie #ite
β-g$obin gene#
Ac-,ys antibody
nucleosome ppt
5NA loo6 do&ain
7eneral DNase 6
sensitiity
Contro$
inactie gene
chicken
ovalbumin
0 1 2DNase 6
(U&m$)
D N A r e m a i n i n g
DNase 6
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cetylation at ?ro!oters
Transcritional
./0./NThyroid Hormone Receptor ea&6le o 5NAindin" rans$ri6tion a$tor
co-repressor
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3-99&'(
Deacetylation of +hro!atinTranscri(tiona$ Si$encing 2*chromosome Inacti3ation
Nan et al. MolCell'iol 1, +1+ (199)@ Cell ((, 1 (199))@ ones et al. Nat7enet 19, 1AD (199()
l meC CpE !" mo%ification i# ob#ere% in
repre##e% gene# an% inactiate% L chromo#ome#
l meC CpE-methy$ation i# maintaine% after !"
rep$ication by Maintenance Methy$a#e action on
hemi-methy$ate% !"
l meC bin%# tran#criptiona$ repre##or MeC52
(Methy$C-bin%ing 5rotein-2)
l MeC52 bin%# :in3 4ith #5D3 hi#tone %eacety$a#e
+ +"ypo-acetylated repre##e% chromatin fiber
transcriptional repressor
Histone eacetylase
4in3
#5D3
MeC52
..pCpEp..3 me
≡ ≡ 3 ..pEpCp.. me
co-repressor
Cp7 methylation
'i t M th l ti
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C C
'istone Methylation
'ysine
ε
# N-monomethyl-'ysine
'MT %'istone
Methyl#Transferase)'istone
de!ethylase5
8
N
C
C C
C
C
CN+
α β
γ
δ
ε
8
N
C
C C
C
C
C
ε
C
'MT %'istone
Methyl#Transferase)
/#adenosyl!ethyionine
8
N
C
C C
C
C
C
ε
C
ε
C
'MT %'istoneMethyl#Transferase)
8
N
C C
C
C
ε
C
ε
C
ε# N-*imethyl-'ysine
Cε
# N-trimethyl-'ysine
DN bacbone
binding !ay not bestrongly affected" but
specific proteins !ay
recognie these
!odifications
N+
N+
N+
/#adenosyl!ethyionine
/#adenosyl!ethyionine
"istones can be methylated at lysines or arginines ?/ample% "3 9 methylation
'istone !ethylation , 'istone !ethyltransferases
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'istone !ethylation , 'istone !ethyltransferases
7nhancer of 8e#te KD:i$encing of e'chromatic gene#
Mo%ifie% from
achner an% Jen'
(00). C'rrent N
Ce$$ *io$. 1+, A;
Multiple roles of '8 *@ !ethylation
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Multiple roles of '8 *@ !ethylation
In contrast to histone acetylation" histone !ethylation is /TA71. This !aes of this
!ar a candidate for inheritance of chro!atin states.
N. $rassa 7nhancer of
e#te
:. pombe to man
ri-@ %i-@ mono-HMa#e#O
(ri-met KD)
an% tri- met K9
Mo%ifie% fromachner an% Jen'4e
(00). C'rrent Npin. C*io$. 1+, A;-9A
'eterochro!atin
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'eterochro!atin
irst discoered in Drosophila in genetic studies o$ chromosome rearrangements
"eterochromatin induces gene silencing
:ingh, . *. (199+). Mo$ec'$ar mechani#m# of ce$$'$ar %etermination their re$ation to chromatin #tr'ct're an% parenta$ imprinting. J Ce$$ :ci 107 , ;
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9enes involved in heterochro!atin
:ingh, . *. (199+). Mo$ec'$ar mechani#m# of ce$$'$ar %etermination their re$ation to chromatin #tr'ct're an% parenta$ imprinting. J Ce$$ :ci 107 , ;
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H3
6& 61,
Ac Ac
H36& 61,
HAC
H36& 61,
7ethylation of 6& (Clr,8 uvar3&!
7e
H36& 61,
7e9i .ecruitment of 9i or H0-1
'eterochro!atin for!ation involves histone '8 lysine @ !ethylation by
/u%var)8#@ and recruit!ent of '?#3
.ecruitment of more Clr, (uvar3&! molecul )y 9i (H0-1!
pen chro!atin
+ondensed chro!atin
1pigenetic regulation of centro!ere function and BN
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1pigenetic regulation of centro!ere function and BN
Central *omain uter reeatsuter reeats
" 0om)e Centromere
9i
6& 7e
Nucleosomes
9i
6& 7e
Nucleosomes*s.NAs
This structure requires roteins that are resonsi)le for the henomenon ofBN interference
-; .NA interference is +ene silencin+ me*iate* )y short .NA molecules ro*uce* from
*ou)le stran*e* .NA (*s.NA!"
-; hort .NAs are ro*uce* )y cleava+e of *s.NA )y the nuclease calle* icer" icer
ro*uces short *s.NA calle* si.NA *ule
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pposing functions of '8 *@ versus *4 !ethylation
achner an% Jen'4ein. (00) C'rrent Npin. Ce$$ *io$. 1+, A
functional lin bet$een histone '8 !ethylation and
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functional lin bet$een histone '8 !ethylation and
DN cytosine !ethylation in Arabio(sis tha$iana
'?35
Mo%ifie% from achner an% Jen'4ein. (00) C'rrent Npin. Ce$$ *io$. 1+,
Multiple biological pheno!ena involving histone !ethylation
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/(=!
Multiple biological pheno!ena involving histone !ethylation
H3-KD methy$ation an%
Mo%ifie% from achner an% Jen'4ein. (00) C'rrent Npin. Ce$$ *io$. 1+,
an% H3-KD methy$ation
4 d ti
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4ummary and perspecties
Chromatin i# pac2age% in a hierarchy of #tr'ct're#. 7ach of the#e
$ee$# of pac2aging ha# reg'$atory ro$e# in the genome
he $ee$ of pac2aging 4e 2no4 be#t, a$#o than2# to formi%ab$e
too$# techno$ogy %ee$opment in the recent year#, i# the
n'c$eo#ome.
n partic'$ar, po#t-tran#$ationa$ hi#tone mo%ification# p$ay 2ey ro$e#
in reg'$ation of genome f'nction, an% the combinatoria$ po4er of
the#e mo%ification# i# on$y beginning to be 'nrae$e%.
5't're cha$$enge# 4i$$ be to %ecrypt the %etai$e% meaning of the#e
mo%ification#, an% to 'n%er#tan% the ro$e# of the higher or%er $ee$#
of chromatin an% chromo#ome fo$%ing