Chapter 5 - Single Molecule Studies of Chromatin Structure a(4)

S ingle Molecule Studies of Chromatin Structure and Dynamics 161

thi s m o d el ha s been c al c ulate d d ue to the three-d i m en s ional rotation of the o c ta m er with re s pe c t to the axi s of the DNA in the ? ber, it woul d be intere s ting to know how thi s c oul d o cc ur in a ? ber of nu c leo s o m e s an d how a d ja c ent nu c leo s o m e s m ay affe c t thi s pro c e ss .

F igure 5.9 d i s play s s o m e of the variou s a m ino a c i d s ite s of the hi s tone o c ta m er within the nu c leo s o m e that have been c onverte d into c y s teine s . Sin c e c y s teine s c an be c he m i c ally m o d i? e d with the m alei m i d e d erivative of a ? uore sc ent d ye, the s e s ite s repre s ent nu m erou s lo c ation s for labeling. Be c au s e s ingle-pair ? uore sc en c e re s onan c e energy tran s fer (s p F RET) i s e ss entially a s tu d y of the “ c onver s ation ”between two d ye s an d

there F igure 5.8: M odeling of a nucleosome transitioning from a wrapped state (far left panel) to an unwrapped state with applied force (far right panel). For description, see text and Kulic

and Schiessel (2004). From Schiessel (2006)

2.9 pN

2.6 pN

20 n m

10 s 2.1 pN

F igure 5.7: T wo states of a nucleosome as observed using external force. For description, see

text. Data from Mihardja et al. (2006)

162 C hapter 5

are nu m erou s lo c ation s to pla c e d ye s , the nu c leo s o m e pre s ent s a wealth of opportunitie s for future s tu d ie s .

C onclusion C ontrary to que s tion s about the s per m geno m e, inve s tigation s in s o m ati c c hro m atin s eek to un c over how the d yna m i cs within an d without nu c leo s o m e s lea ds to a cc e ss of the un d erlying DNA te m plate to biologi c al pro c e ss e s . It i s generally un d er s too d that thi s a cc e ss i s d yna m i c an d a point of regulation. How it o cc ur s c an be un c overe d by s ingle m ole c ule approa c he s . Many open que s tion s re m ain. F or exa m ple, we know that c ore hi s tone H3 variant H3.3 un d ergoe s repli c ation-in d epen d ent d epo s ition into c hro m atin in living c ell s ( A h m a d an d Henikoff, 2002 ). How d oe s that o cc ur?

P re s ently, there i s a boo m in c hro m atin re s ear c h. Previou s i d ea s have be c o m e “r e d i sc overe d ”an d the ? el d i s expan d ing d ra m ati c ally. There i s a nee d to un d er s tan d m e c hani s ti c ally what i s o cc urring at the DNA an d protein level s an d whi c h s ingle m ole c ule approa c he s c an provi d e H4

G13C S45C 3,4H4H2A A12C 88

6,7

H2B

T18C 2H2B T112C 5

H2A A45C 6,7,8H2A

L116C 3H4T71C H4 S47C 1H2A S113C 6,7H3

V35C K119C 9

F igure 5.9: L ocations of some of the amino acid residues in the nucleosome that have been converted to cysteines. Superscript numbers correspond to the following references:

(1) F laus et al. (1996) , (2) B runo et al. (2003) , (3) F laus et al. (2004) , (4) M uthurajan et al. (2004) , (5) P ark et al. (2004) , (6) K assabov et al. (2003) , (7) K assabov and Bartholomew (2004) , (8) Y ang and Hayes (2004) , and (9) L i and Widom (2004) . Figure was prepared in

PyMOL ( D eLano, 2002 ) from pdb ? le 1k x 5 (Davey et al., 2002)

S ingle Molecule Studies of Chromatin Structure and Dynamics 163 u s the s e novel un d er s tan d ing s. We hope that the rea d er ha s re c eive d a s en s e of c urrent

re s ear c h u s ing s ingle m ole c ule approa c he s in s per m an d s o m ati c c hro m atin.

A cknowledgments

W e thank Dr J. van Noort for s haring experi m ental re s ult s prior to publi c ation. We thank the m any people who have c ontribute d to the work we have d e sc ribe d, in c lu d ing Dr s Ro d Balhorn, Miro s lav To msc hik, K en van Hol d e, Hao c heng Zheng an d Jor d anka Zlatanova, an d we appre c iate a c riti c al rea d ing by Dr s Sya m P. Anan d an d Ri c har d Stein m an. Thi s work wa s s upporte d by NIH grant GM077872 (SHL) an d s tartup fun ds fro m Wa s hington State Univer s ity an d the Center for Repro d u c tive Biology (LRB).

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