别再提起Experi m ent s c on d u c te d by our group s howe d that the bin d ing rate of prota m ine 2 to
s ingle DNA m ole c ule s in the pre s en c e of zin c in c rea s e d by threefol d( B rewer et al., 2002 )
152 C hapter 5
c o m pare
d to th
e bin d ing rate without zin c. The s e re s ult s were c on s i s tent with previou s
s tu d ie s s howing that when zin c bin ds to prota m ine 2, it m ay c au s e a s tru c tural c hange in the protein, s u c h a s a zin c ? nger, that help s the protein bin d to DNA (B ian c hi et al., 1992, 1994;G atewoo d et al., 1990). K un d u an d Rao (1996)an d M eetei et al. (2000)have al s o s hown that TP2 c ontain s zin c ? nger m otif s.
C onclusions: Sperm Chromatin
U n d er s tan d ing how s per m c hro m atin i s repa c kage d an d c o m pa c te d d uring
s per m iogene s i s i s i m portant for d eter m ining the organization of the s per m geno m e, an
ar c hite c ture that i s c ru c ial for prote c ting it s geneti c integrity an d fertility. Chro m atin
c o m pa c tion m ay al s o play a role in re
d u c ing th
e volu m e o
f the s per m nu c leu s an d
s haping the c ell. It ha s been d if?c ult to s tu d y how hi s tone s are d i s pla c e d d uring
s per m iogene s i s u s ing m ou s e kno c kout m o d el s. Single m ole c ule te c hnique s s houl d be parti c ularly a m enable to d eter m ining the m e c hani sm of d i s pla c e m ent an d the role that
po s ttran sc riptional m o d i?c ation s of c hro m atin play in thi s pro c e ss. Many fun d a m ental proble ms re m ain to be s tu d ie d to un d er s tan d how the s per m geno m e i s organize d an d the s per m c ell i s refor m e d d uring s per m iogene s i s. Single m ole c ule te c hnique s, whi c h allow kineti c an d s tru c tural infor m ation to be elu c i d ate d without the proble ms of inter m ole c ular aggregation an d pre c ipitation, will be in s tru m ental in s olving the m.
S omatic Chromatin
T he nu c leo s o m e i s the fun d a m ental portion of protein –D NA pa c kaging in the s o m ati c eukaryoti c c ell nu c leu s (T s anev et al., 1992;T urner, 2002;v an Hol d e, 1988;W olffe, 1998;Z latanova an d Leuba, 2004b). In a nu c leo s o m e, there are about two wrap s of DNA aroun d a hi s tone o c ta m er of the c ore hi s tone s H3, H4, H2B, an d H2A. A ? fth hi s tone, linker hi s tone H1, bin ds to the DNA between a d ja c ent nu c leo s o m e s. Although we have ha d a general un d er s tan d ing of nu c leo s o m e s tru c ture for over three d e c a d e s, que s tion s
re m ain how biologi c al pro c e ss e s s u c h a s tran sc ription, repli c ation, re c o m bination, an d repair a cc e ss the un d erlying DNA te m plate in the c hro m atin m ilieu. Regulation of a cc e ss to the s e biologi c al pro c e ss e s i s thought to lie in the c o m plexitie s of (i) po s ttran s lational m o d i?c ation s to the hi s tone s an d the DNA, (ii) the hi s tone variant s, an d (iii) external protein s that re m o d el c hro m atin ( Z latanova an d Leuba, 2004b ).
R e c ently, a nu m ber of s ingle m ole c ule approa c he s( L euba an d Zlatanova, 2001 ) have been d evelope d to probe d yna m i c c hange s o cc urring in in d ivi d ual nu c leo s o m e s, ? ber s
of nu c leo s o m e s, or in d ivi d ual c hro m o s o m e s in s olution (reviewe d in C airn s, 2007;
S ingle Molecule Studies of Chromatin Structure and Dynamics 153 C lau d et an d Be d nar, 2006;L angow s ki an d S c hie ss el, 2004;L euba an d Zlatanova, 2002; L euba et al., 2004;M arko an d Poirier, 2003;P oirier an d Marko, 2002, 2003;S c hie ss el, 2006;v an Hol d e an d Zlatanova, 2006;Z latanova, 2003;Z latanova an d Leuba, 2002, 2003, 2004a;Z latanova an d van Hol d e, 2006).
A great d eal of re s ear c h ha s inve s tigate d the o cc upan c y of nu c leo s o m e s at s pe c i?c lo c i on a DNA m ole c ule or c hro m o s o m e. Re c ently, re s ear c her s have s y s te m ati c ally probe d thi s
o cc upan c y in entire geno m e s (e.g., I o s hikhe s et al., 2006;L ee et al., 2004;S egal et al., 2006;Y uan et al., 2005). However, the lo c alization of a nu c leo s o m e at a s pe c i?c s ite d oe s not m ean that it re m ain s s tati c. The nu c leo s o m e it s elf c oul d open up. Perhap s, it i s not
s urpri s ing that while evolution ha s d evelope d a pa c kaging d evi c e to prote c t the DNA, it ha s al s o d evelope d m etho ds to open nu c leo s o m e s. F or exa m ple, we un d er s tan d that after repli c ation, H3/H4 tetra m er s bin d to the DNA followe d by H2A/H2B d i m er s an d linker hi s tone ( W or c el et al., 1978 ); tran sc ription c oul d be viewe d a s a rever s al of that pro c e ss (e.g., v an Hol d e et al.,1992).
O riginally, nu c leo s o m e s were c on s i d ere d s olely a s pa c kaging d evi c e s to ?t the 2 m of
DNA within the c on? ne s of the c ell nu c leu s, a c o m pa c tion of ?10 000 fol d. The general working d og m a wa s that they were i mm ovable obje c t s, i.e., like “r o c k s”that s eque s tere d the DNA for pa c kaging. With the d i sc overy of nu c leo s o m e s li d ing ( B ear d, 1978; reviewe d in v an Hol d e, 1988), we un d er s too d that at lea s t the DNA portion i s m obile. The s tru c ture of the nu c leo s o m e ( L uger et al., 1997 )s upporte d that notion be c au s e the B-fa c tor s, whi c h in d i c ate ther m al ? u c tuation s in the c ry s tal s tru c ture, were s igni?c antly higher for the DNA portion than for the c ore hi s tone fol d region. The hi s tone c ore portion wa s s till like a “r o c k. ”Pioneering work of Prunell ( H a m i c he et al., 1996 )d e m on s trate d internal rearrange m ent s of the hi s tone fol d d o m ain c ore upon tor s ion applie d by the DNA m ole c ule.
N u c leo s o m e ? u c tuation s ha s lea d to the ob s ervation s of m ultiple s tate s other than the
c anoni c al 147 bp nu c leo s o m al c ore parti c le (Table 5.1). The m otivation for the c reation of thi s table wa s to
d
e sc ribe s o m e o
f the re s ear c h that ha s le d to the un d er s tan d in
g of nu c leo s o m e s
a s m obile entitie s. The table i s by no m ean s exhau s tive, but it d oe s exhibit a wi d e d i s play of work. Single m ole c ule work i s d i s playe d along s i d e en s e m ble s tu d ie s. We have atte m pte d
to d e sc ribe the d ata in the table in ter ms of two s tate s; however, not all of the paper s c an be ea s ily d e sc ribe d that way. One c an s ee that the s tu d y of nu c leo s o m e s ha s expan d e d fro m the c o m pari s on s of nu c leo s o m e s with their nake d DNA m oietie s at high ioni c c on d ition s to the s tu d y of internal rever s ible d yna m i cs o cc urring in in d ivi d ual nu c leo s o m e s in real ti m e.
A c ouple of re c ent exa m ple s of two or m ore s tate s o cc urring in a s ingle nu c leo s o m e are d i s playe d in F igure s 5.6 an d5.7.U s ing a nu c leo s o m e c ontaining a d onor an d an a cc eptor
154 T able 5.1: S ome examples of chromatin and of nucleosomes in multiple states Array
C
hapter
5
S ingle Molecule Studies of Chromatin Structure and Dynamics 155
(Continued )
T able 5.1: C ontinued
156 Array
C
hapter
5
S ingle Molecule Studies of Chromatin Structure and Dynamics
1
57
Continued
158 C hapter
5 T able 5.1: C ontinued
S ingle Molecule Studies of Chromatin Structure and Dynamics 1
59
160 C hapter 5
d y
e pair, we ob s erve d that an in d ivi d ual nu c leo s o m e c an rever s ibly an d d yna m i c ally ? u c tuate between the c anoni c al c lo s e d s tate an d a m ore open s tate where the d ye s are too far apart for energy tran s fer ( F igure 5.6 ) ( T o msc hik et al., 2005 ). Rever s ible ? u c tuation s in a s ingle nu c leo s o m e were later ob s erve d in opti c al tweezer experi m ent s ( F igure
5.7 )( M ihar d ja et al., 2006 ). U s ing a long linear DNA m ole c ule with a high af? nity for bin d ing one nu c leo s o m e at a s ingle nu c leo s o m al po s itioning s equen c e, re s ear c her s were able to rea c h for c e s where the nu c leo s o m e woul d “h op ”ba c k an d forth between two s tate s . At 2.1 p N applie d for c e, the nu c leo s o m e woul d pre d o m inantly re m ain in the “c anoni c al ”s tate an d only o cc a s ionally hop to a s tate with one unwrapping of the DNA gyre s . At 2.9 p N, the nu c leo s o m e woul d pre d o m inantly re m ain with only one wrap of the DNA gyre s an d o cc a s ionally hop ba c k into the fully wrappe d c anoni c al s tate.
A n exa m ple of m o d eling of the unraveling of a s ingle nu c leo s o m e d ue to applie d for c e i s d i s playe d in F igure 5.8( K uli c an d S c hie ss el, 2004 ; S c hie ss el, 2006 ). One ob s erve s that the hi s tone o c ta m er un d ergoe s a 180 ° rotation a s the m o d el nu c leo s o m e progre ss e s fro m ~1.5 wrap s of DNA aroun d the o c ta m er to a s tate where the o c ta m er i s only tou c hing one s urfa c e of the DNA m ole c ule. Sin c e the eventual unwrapping of the nu c leo s o m e in
F l u o r e s c e n c e i n t e n s i t y Time (s) F igure 5.6: T wo states of a nucleosome as observed by single-pair ? uorescence resonance energy transfer (spFRET) time trajectory. Dark gray curve
r epresents acceptor signal and light gray curve is donor dye signal. The anticorrelation of the two independent signals con? rms that the two dyes are on the same DNA molecule. Reversible ? ipping between a high-FRET state and a low-FRET state occurs before acceptor dye photobleaching at ～19 s . As the baseline of intensities of the photobleached state of the acceptor dye is lower than the low nucleosomal state intensities (see the two different dashed lines), the low nucleosomal state is not due to blinking of the acceptor dye. Adapted from Tomschik et al. (2005)
