Diferenciace buněk a struktura chromatinu
Buněčná diferenciace je proces při kterém buňky získávají nový fenotyp, který je spojen se specifickou buněčnou funkcí. Pro daný buněčný typ Je charakteristická aktivace (inaktivace) skupiny genů, které jsou zodpovědné za navození terminálni diferenciaci.
Epithelial cell           Nerve cell
G£ phase
Cell Differentiation Cell Growth
Anti-phospho H3
^             ^^^^
Prophase-^
W ■
Cell Cycle
Role of E2Fs
Growth Factors
Al
Al
a
3
S
2 a
V*
es
./^~*^  CONTROL		■	NaBt 72 h
		í	^*fcta.
	sĚWSU		
CONTROL
Gl = 57.2% S = 17.8% G2-M = 25.0%
B2
NaBt 72 hours Gl = 75.1% S = 18.2% G2-M = 6.7%
30           60            90           120          150
Channels (FL2-A)
90           120          150
Channels (FL2-A)
6h
24 h
48 h
72 h
n
*** *. * "
p27KTP1 (27 kDa)
Cycli Dl (34 kDa)
Total protein level
C       NaBt       C       NaBt       C      NaBt       C     NaBt
	-
	|i
'-	š-
E	-
3 C	Lu
V	-
U	^
	v _
	£
	0
BO                 SO
Cannels (FL2-A)
NaBt 24h GO/1   - 71.1%
S        = 15.6% G2-M= 13.3%
1ZQ               150
Cannels (FL2-A)
NaBt72h GO/1  =  83. S       =4, G2/M =   12
0% 4% ,6%
1EQ
Cannels (FL2-A)
59
■Jifl
I
HSA 8 and related structures
Harničarová et al., 2006
6 h                 24 h	48 h                 72 h
	
»••:. i	■ usr|
	
*~«m   ^-*   fc^ai   &-..A	
c-Myc
- (67kDa)
Total protein level
C    NaBt     C    NaBt     C    NaBt    C    NaBt
Oi
'+-č
k
tr-
C
A)     chromosome-to-centre of gravity/R
0.03
Ě      0.02
0.111
0.00 *=-*-
Control
NaBt 24h
kfc
Jl
NaBt 72h
II,   .III
n
0 20 40 60 BO 100 0 20 40 60 »0 10« 0 20 40 60 80 100
Distance in [%] of nuclear radius
B)    centromere-to-centre of gravity/R
Control
NaBt 24h
NaBt 72h
-I
lit
0  20 40 60 SO 100 0  20 41) 60 W   1(H) i\    2(1 40 60 SO 100
Distance in [%] of nuclear radius
!
I
C)     c-myc-to-centre of gravity/R
0.03
0.02
0.01
0.00 4--*>-
Control
■JL
ik
NaBt 24h
J
í
NaBt 72h
\—-p-
0    20  40   60   80 100 9    20   40   60   SO 10« 0    20   40   60   80  100 Distance in [%] of nuclear radius
Chromoso me-to-cente r of gravity/R; mean±SE in % of radius
				
		Normal		Derivative
A Control (n	= 149)	67.7±1.6		75.1 ±1.6*
NaBt 24 h	(n = 132)	67.2±1.5		71.9 ±1.5
NaBt 72 h	(n = 153)	67.6±1.6		73.6 ±1.4*
		Centromere-to-		center of gravity/R;
		mean±SE		in % of radius
		Normal		Derivative
B Control (n	= 512)	63.4 ±1.0		74.7±0.9*
NaBt 24 h	(rl = 536)	64.5 ±1.1		76.4±0.8*
NaBt 72 h	(íi-575)	70.5 ±1.0*		83.1±0.7 '
		c-myc-	to-center of gravity/R;	
		mean±SE		in % of radius
		Normal		Derivative
C Control (n	= 149)	71.5 ±1.4		78.2 ±0.7*
NaBt 24 h	(n = 132)	69.1 ±1.2		76.9 ±0.9
NaBt 72 h	(n = 153)	69.0 ±1.7		76.0 ±0.8*
		c-myc	to-center of gravity of chromosome/n	
		mean±SE in % of radius of		
		chromosome territory		
		Normal		Derivative
D Control (n	= 149)	63.9 ±1.1		58.5 ±0.7*
NaBt 24 h	(n = 132)	56.3 ±1.0*		57.8±0.7
NaBt 72 h	(n = 153)	57.2 ±1.0*		59.9 ±0.8
				
Harničarová et al., 2006
Human c-myc gene and two resultant protein products
Sequences of oligoprobes were used according to Singer group and are follows
5' —	t r*P0    r+P1r*P2 -L-Jb			ttenuation sits	-Or						
							1		H		J— r
							1				
		Exon 1 y   ,' „c-Myc V*''         Exon 2			Exon 2   y          rr*'' £''                        Eior				E*Oll 3	3	------C02H c-Myc 2 c-Myc 1
	CI HZN —	only  1 G        AUG l                j.			1						
				Transact řváti o n domain		NL		BR	HLH	ĽZ	
											
											
											
Ryan and Birnie, 1996
(1)     5'-TCG T*AG TCG AGG T*CA TAG TTC CT*G TTG GTG AAG CT*A ACG TT*G AGG GGC AT-3'
T)     5'-CCA CAT* ACA GTC CTG GAT* GAT GAT TTT T*TG ATG AAG GT*C TCG TCG T*CC G-3'
i)     5'-TGA CCT* TTT GCC AGG AGC CT*G CCT CTT TT*C CAC AGA AAC AAC AT*C GAT* TT-3'
Í4)     5'-CTG GT*G CAT TTT CGG T*TG TTG CTG AT*C TGT CTC AGG ACT* CTG ACA CT*G TC-3'
^)     5'-GGC CTT* TTC ATT* GTT TT*C CAA CTC CGG GAT* CTG GT*C ACG CAG GGC AAA AA-3'
JAGGAACAAGAAGATGAC
EXON1
5'CCCCCGAGCTGTGCTGCTCGCGGCCGCCAC
CGCCGGGCCCCGGCCGTCCCTGGCTCCCCTCC
TGCCTCGAGAAGGGCAGGGCTTCTCAGAGGC
TTGGCGGGAAAAAGAACGGAGGGAGGGATC
GCGCTGAGTATAAAAGCCGGTTTTCGGGGCT
TTATCTAACTCGCTGTAGTAATTCCAGCGAGA
GGCAGAGGGAGCGAGCGGGCGGCCGGCTAG
GGTGGAAGAGCCGGGCGAGCAGAGCTGCGC
TGCGGGCGTCCTGGGAAGGGAGATCCGGAGC
GAATAGGGGGCTTCGCCTCTGGCCCAGCCCT
CCCGCTGATCCCCCAGCCAGCGGTCCGCAAC
CCTTGCCGCATCCACGAAACTTTGCCCATAGC
AGCGGGCGGGCACTTTGCACTGGAACTTACA
ACACCCGAGCAAGGACGCGACTCTCCCGACG
CGGGGAGGCTATTCTGCCCATTTGGGGACAC
TTCCCCGCCGCTGCCAGGACCCGCTTCTCTGA
AAGGCTCTCCTTGCAGCTGCTTAGACGCTGG
ATTTTTTTCGGGTAGTGGAAAACCAG 3'
CTACGACTCGGTGCAGCCGTATTTCTACTGCGACGAGGAGGAGAi TCTACCAGCAGCAGCAGCAGAGCGAGCTGCAGCCCCCGGCGCCC CGAGGATATCTGGAAGAAATTCGAGCTGCTGCCCACCCCGCCCC: CCCCTAGCCGCCGCTCCGGGCTCTGCTCGCCCTCCTACGTTGCGG, ACACCCTTCTCCCTTCGGGGAGACAACGACGGCGGTGGCGGGAG'
GACATGGTGAACi (2)CGGACGAC&1 GTATGTGG
^CCTTCATCAAAAACATC.
3GCCACAGCGTCTGCTCCACCTCCAGCTTGTACCTGCAGGATCTC ľGCCGCCGCCTCAGAGTGCATCGACCCCTCGGTGGTCTTCCCCTA ľTCTCAACGACAGCAGCTCGCCCAAGTCCTGCGCCTCGCAAGAC' \GCGCCTTCTCTCCGTCCTCGGATTCTCTGCTCTCCTCGACGGAG, rCCCCGCAGGGCAGCCCCGAGCCCCTGGTGCTCCATGAGGAGAC
(3)AAATCG. CA
:AA'i'cci'AU'i'Ai'A'i'AU'i'Acc'i'AU'i'A'i'i'Ai'Auu'i'A(_

c-myc gene-to-C
HT29
70.5±1.5%
Distances in % of radius
~
~
~
~
0.04 0.03 0.02 0.01 0.00
A549
71.8±0.6%
0       20      40      60      80     100

_d.
Distances in % of radius
T
T
T
0      20      40      60      80     100
HT29-APC
38.7±1.4%
Distances in % of radius
0       20      40      60      80     100
I 20
I 40
60      80     100
0.04
0.03
HT29-APC-ZnCl2 41.1±1.3%
stances in % of radius
I                               I                              I                              I
20     40     60     80    100
0.04
0.03 -
4549
43.2 ±2.1%
0      20     40     60     80     100
c-myc /DNA
Aa	Ab	Ac	Ad
	t  1		1^.                     a
B
c-myc 7 DNA
I
c-myc /pKi-67
GO
early Gl     mid Gl      late Gl
G2       metaphase
SC35/c-mvcT
\lav projvťtiun
B
PML/c-myc1
Max, projection
11» li« i!
5 £ 21)
	I	1	1
XII
g _ 60
IS
z 9
a ^ 4(1
v    —
s « ig
IM     2
c S  211
_u
A549
HT29
A549
HT29
n
s*
o
n
i
3
n H


03
% of c-myc RNA signals associated with nucleoli
% of c-myc RNA signals co-localized with RNAP II
m
c
» c


x
>
h-»
SSaSSSňiíÄSiííaÄSa^^
**^******$***SSS*Sa5SK*íK
> 'Jí
-U
vO

í*í»íSa*****í5*»*5*í*N*5
^^^^^^^
Western blot analysis
ON
H
m
u
<
I
H
es
u
a.
<q
H
CS. TT


■■
APC
(310 kDa)
c-MYC (67 kDa)
Total protein level
Number of c-myc RNA signals
100
80 ,60
40
20
0
100
so
;60
40 20 0 1
VZ
HT29
n=102
Vy
VÄ___Ežžä___.
HT29-APC-ZnCl;
n=128'
L
VZ
HT29-APC n=114

_^_
f
V77?\
AS49 n=75
B
12           3                         12
Number of c-myc transcription sites
J U 25		U a. <	* Ü s	
20		Cfvl	■ S	
^  15		LE	N   I	
	OS		la	
10			1 dm	1   >r,
	= 1	i	1	"—
5	1	l	1=1	■
0	1	U	■J	1      ■
Cells with c-myc transcription site
cap
JiiiGppp
pofyA denyfation
##u***    pofyA tail
spliceosomes
RNA splicing
messenger RNA
1. Differentiation of mouse emryonic cells (ES and EC)
division
=>
Zygote (0cl4 positive)
Morula (Qct4 positive)
Embryoidni tělíska (EBs)
4>v
4P
Diiferenliated cells (Ocl4 negative)
Cultured ES cells (Oc*4 positive)
Inner Cell Mass (1CM, Oct4 positive)
.Trophoblast (Oci4 negative)
Základ extra-em ryon
Blastocyst        tkání extraction
If
1CM cells (Oct4 positive)
ES: Embryonální kmenové buňky (ES) jsou imortalizované buněčné linie derivované z vnitřní buněčné masy 3.5 denní blastocysty. Tyto buňky se mohou  rozmnožovat v nevydiferencovaném stavu za přítomnosti LIF faktoru. ES buňky mají schopnost diferencovat in   vitro   v   progenitorová   stádia.   ES buňky tvoří tak zvaná emryoidní tělíska  (EBs),  což jsou  třídimenzionální struktury se schopností diferencovat do různých buněčných typů, například: hematopoietických,           myogenických,
neurálních   a  jiných.   Ovlivnění  těchto buněk  například  RA  vede   k  indukci neurální diferenciace.                                       27
Neurální diferenciace ES / EC buněk
■  spontánně v EB
■  spontánně v monovrstvě
■ v EB v přítomnosti kyseliny retinové (RA)
■ v monovrstvě bez přítomnosti séra
■ v monovrstvě bez přítomnosti séra + RA ?
bezsérové médium
doplňky média: ITS, N2, B27
inhibitory jiných diferenciací: Noggin, Chordin, Follistatin
33
Release of RXR, PML, PLZF, ate.
Release of co-repressars
ATRA
DIFFERENTIATION
▼   *<"
^T
^-repressors                S                 M%                     ,
-—^            v.    Str "*"'■* S                f
V      *                        . PWlL-RARfj
APOPTOSiS
s:
Restored retinoid signaling
+
PWIL-RARíí
/                                    •••
Release effetts an
other pathways
i
•\
i
PML- RARalphe degradation
■  »
» * *
• ■
■ ■ 4 * * * ■ w
•   •
Up- regulation of RAR alpha
Nuclear Bodies reappeared
with relocalization of PMLand c-Eher MB proteins
M E F                      h ES cell
a                    b •	c
MEFs /hES cells (OCT4)
MEF/OCT4         hES cell/OCT4
	d
MEFs/hES cells - RA (OCT4)
Bone marrow *—**
MSCICpŕHge
■'■•;
**»
Myeloblast
Promyelocyt
Rrtmüoctfei
Eosinophil x   / \   \^ ^wiutrophll^    X
&
BsUdiacylsil
. V
' i; } ReHukxym N
ÖyBuiOCylO
•      DoSopNI §   9
Myocyte          Monoblaft
•É-ié-4
Metamyelocyte
é é i
Band form Segmented form
F^öheralujgod
•%  Degenerate form
Lymphatic sjůiew
Thymus
To
9 ô B
niťKuwiirwiŕvl^Et              i"-._.-._i-j__t
ö iťľtmvTottesr       COTrtriDä
PtomibĽHf    ■     Cwottyla \
T Tn-tfCtiiB
€H
l:JL i.i<. I
•
Hi"*:"
BjHraghetyií      Ti^rpUocyw
Peripheral blood
řU I.OiYDlchf LrfHrfldtftb.ikUf^TWni ■"J *"l»rimrtil Ulf-
Bone marrow *—**
MSCICpŕHge
■'■•;
**»
Myeloblast
Promyelocyt
Rrtmüoctfei
Eosinophil x   / \   \^ ^wiutrophll^    X
&
BsUdiacylsil
. V
' i; } ReHukxym N
ÖyBuiOCylO
•      DoSopNI §   9
Myocyte          Monoblaft
•É-ié-4
Metamyelocyte
é é i
Band form Segmented form
F^öheralujgod
•%  Degenerate form
Lymphatic sjůiew
Thvmus
To
9 ô B
niťKuwiirwiŕvl^Et              i"-._.-._i-j__t
ö iťľtmvTottesr       COTrtriDä
PtomibĽHf    ■     Cwottyla \
T --miiinoHaH
€H
PÍM '.!<• i
•
Hi"*:"
BjHraghetyií      Ti^rpUocyw
Peripheral blood
řU I.Oirmwiiftkxricťlh.ibHfiirixin ■"J *"l»rimrtil Ulf-
Beta-like globin gene cluster
PAC 4396 (180 Kb)  I—//-BAC 4396-44 (100 Kb)          ,_
5'HS:    5  4 3 2  1                                                               ,
-18   -10,9                                                                                       5 H^:   1
-214    -14,7   -6,1                                                                                                    +21,8
1     < 1   ,     I      £                  GY AT    f P        5        P                              I
T7-*-h«-SP8
V                             s-
Arrayed on chromosome 11, encodes one embryonic (e) and two fetal (Gy, Ay) and two adult (8, ß) globin chains. Expression of ß-like genes undergoes a developmental related switching mechanism: s: expressed in early embryo fetal y: fetal life. o, ß: adulthood.
Chiiuvbz hi b-iikb vbiib
bryihuiü ssii anibrbsiťiaťiuú.
Probability density
Probability density
Probability density
Probability density
Probability density
o      o      o      o
Is
> a
a"
O w
3    §	s	I   I  I  1
]		O K M "* » P! ís ^ i-i h- t"1 H-  «#
		
		
\\ -'! v"-1      *x		
		
		
		
\V-  '••* ■    \\\\\1		
.WWWWWN   Ö		
A
B
0.030
0.02?
3    0.020
■g   0.015
.o
2   o.oio
0.00? 0.000
K562 / CONTROL
C- BCR - ABL/R = 70.8±0.6%
0.030 0.02? 0.020 0.01? 0.010 0.00? 0.000
K562 / PMA
C- BCR- ABL/R = 74.7*0.7%*
H
n.
D
	0.030
	0.02?
&	0.020 0.015 0.010
	0.00?
	0.000
HL60 / CONTROL C-BCR/R = 50.4± 0.8 %
H
0.030 0.02? 0.020 0.01? 0.010 0.00? 0.000
HL60 / PMA C-BCR/R = 49.4±0.6 %
n_
F
0.030 ! HL60 / CONTROL ;    C-ABL/R = 55.3 ± 1.8'
0.020 0.01? 0.010 0.00? 0.000
_Q
o
o
5 o
•f o
2 o P-, o
o
o
o
o
040 035 030 023
020
015 -010 00? 000
Ph+ CML
C-BCR/R = 52.5 ± 1.3 %
C-ABL/R = 53.5 ± 1.3 %
Ô        20        40        60        80       100
Distance m % of radius
0.030 0.02? 0.020 0.01? 0.010 0.00? 0.000
HL60 / PMA C-ABL/R = 53.8 ± 0.6 %
n
20       40        60        80       100
Distance m ° o of radius
Bártova et al., Figure 4
MICRONUCLEI
a.
b.
Bártova et al., Figure 6
210 kDa 135 kDa
65 kDa 45 kDa
1       2	3	4	5	6
—   —	•	«i	•	m\
«—»	«	•»	-•	«•
w
H O *o O
C/5
3
n

o
X!
>
Bcr-Abl c-Abl
Lamin B
Cleavage "***      fragment
Total protein level
				E			
				Acetyl H3 (K9)		—	s 1
			Lamin B m       Cleavage fragment				
							
				di-meH3(K9)	• —•		
							
«M	<WWI*	3	Total protein				
							
o	-d > as	*3		Total protein level			
		>			n	*0 > &5	H H O ■o O in O
Bártova et al., Figure 7
ZAVMi
Diferenciace je charakteristická nejenom specifickými změnami na úrovni morfologie buněk, ale významně se mění i struktura chromatinu. Tyto strukturální změny v genomu   mají velký význam z hlediska transkripční aktivity genů.