Chapter 5 - Single Molecule Studies of Chromatin Structure a

CHAPTER 5 S ingle Molecule Studies of Chromatin

Structure and Dynamics

S anford H. L euba *

D epartment of Cell Biology and Physiology, University of Pittsburgh School of Medicine,

Petersen Institute of NanoScience and Engineering, Department of Bioengineering, Swanson School of Engineering,

2.26g Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213-1863, USA

L aurence R. B rewer

S chool of Chemical Engineering and Bioengineering, Center for Reproductive Biology,

Washington State University, Pullman, WA 99164-2710, USA

S ummary

I n the eukaryoti c c ell nu c leu s, DNA i s pa c kage d into c hro m atin. In m o s t c ell s, the DNA i s wrappe d aroun d c ore hi s tone s to for m nu c leo s o m e s. In the s per m c ell, however, prota m ine s repla c e the hi s tone s. Un d er s tan d ing how hi s tone s in s o m ati c c ell s pa c kage c hro m atin an d regulate a cc e ss for 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 to the un d erlying DNA te m plate i s of c urrent intere s t. Deter m ining how prota m ine repa c kage s c hro m atin in the s per m c ell i s i m portant for un d er s tan d ing how the geno m e i s ina c tivate d

an d prote c te d prior to fertilization of the egg. Single m ole c ule approa c he s in whi c h one

m ole c ule i s s tu d ie d at a ti m e are provi d ing new infor m ation about c hro m atin. The s e a ss ay s are able to a sc ertain new d ata about s tru c ture an d d yna m i cs that i s not po ss ible to d i sc ern

fro m the average m ilieu of bulk m ea s ure m ent s. Thi s c hapter provi d e s an overview of the

? el d in c lu d ing the author s’own re s ear c h.

K ey Words

c hro m atin; nu c leo s o m e; hi s tone; prota m ine; toroi d; opti c al tweezer s; s ingle-pair

? uore sc en c e re s onan c e energy tran s fer

*C orre s pon d ing Author

143

144 C hapter 5

I ntroduction

I n thi s c hapter, we d i sc u ss two d i s tin c t s ubunit s of eukaryoti c c hro m atin: the nu c leo s o m e in s o m ati c c ell s an d the toroi d in the s per m c ell. Although they are of s i m ilar d i m en s ion s, approxi m ately ten s of nano m eter s, they fun c tion in d ra m ati c ally d ifferent way s. The

nu c leo s o m e c ontain s 147 ba s e pair s (bp) of DNA that i s wrappe d 1.65 turn s aroun d a

hi s tone o c ta m er. Un d er s tan d ing how the s e protein s regulate a cc e ss to the DNA they are in c onta c t with for fun d a m ental c ellular pro c e ss e s i s ju s t beginning to be un d er s too d. Toroi ds, on the other han d, c ontain 50 k iloba s e pair s (kb) of DNA that ha s been pa c kage d by s per m nu c lear protein s into s u c h a d en s e for m (the DNA loop s that for m the toroi d are s eparate d by approxi m ately 2.4 n m, for m ing a hexagonal latti c e) that a cc e ss to the DNA by an enzy m e s u c h a s RNA poly m era s e i s i m po ss ible. The geno m e i s“a s leep ”

an d ina c tive until it i s“r eawakene d”following fertilization. In the following page s,

we d e sc ribe what ha s been re c ently learne d about the s e two re m arkable s ubunit s of eukaryoti c c hro m atin u s ing experi m ental te c hnique s that allow in s ight into m e c hani sms that govern their fun c tion at the s ingle m ole c ule level.

S perm Chromatin

S per m c hro m atin in m a mm al s i s d en s ely pa c kage d, approa c hing that of DNA in viru s e s, to prote c t it fro m d a m aging nu c lea s e s prior to fertilizing the egg. In 1953 Wilkin s an d Ran d all obtaine d the ? r s t X-ray sc attering m ea s ure m ent s fro m s per m hea ds s howing that the DNA within wa s c ry s talline in for m. Although the s e m ea s ure m ent s e s tabli s he d the m ole c ular s tru c ture of DNA, for whi c h Wilkin s, Cri c k, an d Wat s on won the Nobel Prize for Me d i c ine, we are only ju s t beginning to learn how s per m c hro m atin i s pa c kage d into thi s re m arkably c o m pa c t for m. U s ing kno c kout m i c e an d opti c al m i c ro sc opy,

repro d u c tive biologi s t s have d eter m ine d that the DNA i s pa c kage d by s per m nu c lear protein s, a s part of a pro c e ss known a s s per m iogene s i s( G agnon, 1999 )( F igure 5.1 ), to prote c t it fro m d a m aging biologi c al agent s an d to c au s e the c e ss ation of tran sc ription, ina c tivating the geno m e. Hi s tone s are d i s pla c e d fro m nu c leo s o m e s, an d the DNA i s organize d by protein s c alle d prota m ine s into sm all d onut-s hape d“t oroi ds”( H u d et al., 1993 )( F igure 5.2 )c oin c i d ent with a 40-fol d re d u c tion in the c ell’s volu m e. Toroi ds are the pri m ary s ubunit of s per m c hro m atin ( H u d et al., 1993 ) an d c ontain approxi m ately

50 k b of DNA. In hu m an s per m, approxi m ately 85% of the geno m e i s repa c kage d a s toroi ds an d 15% re m ain s in the for m of nu c leo s o m e s.

T o un d er s tan d ju s t how tightly c o m pre ss e d the s per m geno m e i s, we c an c al c ulate it s

fra c tional volu m e in the s per m nu c leu s. The s per m geno m e c an be m o d ele d a s a c ylin d er

S ingle Molecule Studies of Chromatin Structure and Dynamics 145

that i s 1 n m in ra d iu s , with length equal to 1.5 G bp ti m e s the d i s tan c e between ba s e pair s

(0.34 n m /bp) giving a volu m e of 1.6 μ m 3

. The volu m e of the hy d rate d m ou s e s per m nu c leu s i s 12 μ m 3 ( A llen et al., 1996 ). The fra c tional volu m e taken up by the s per m geno m e in the nu c leu s i s then 13%, c o m parable to the fra c tional volu m e of DNA in viru s e s ( P urohit et al., F igure 5.1: S teps

o f spermiogenesis in the mouse. Both the changes in shape of the cell and chromatin density are shown as well as the order of appearance of the sperm nuclear

proteins ( Z hao et al., 2004b )

F igure 5.2: D NA – p rotamine toroids imaged using electron microscopy. The scale bar is

100 n m ( H ud et al., 1993 )

Histones

Protamine 1

preProtamine 2

Protamine 2Round spermatids Step 678910111213141516

Elongating Condensing Condensed spermatids

TP1

TP2

146 C hapter 5

2005 ). W hat is the purpose of packaging the sperm genome in such a compact state?The

? r s t rea s on i s to prote c t the geno m e fro m har m ful biologi c al agent s s u c h a s nu c lea s e s an d free ra d i c al s prior to fertilization of the egg. Se c on d, the volu m e taken up by the geno m e

i s re d u c e d to allow the s per m hea d to be c o m pre ss e d. Thi s allow s the s per m to a ss u m e a

hy d ro d yna m i c s hape an d s wi m a s qui c kly a s po ss ible to m axi m ize it s c han c e s of fertilizing the egg. Thir d, the pa c kaging of the DNA into toroi ds c au s e s the c e ss ation of tran sc ription an d ina c tivate s the geno m e until it c an be unpa c kage d an d rea c tivate d following fertilization. In the following s e c tion s, we will d e sc ribe the d ifferent s tep s of s per m iogene s i s, what ha s been learne d about the m ole c ular m e c hani sms that o cc ur d uring ea c h s tep u s ing s ingle

m ole c ule experi m ent s an d what intere s ting proble ms re m ain to be s olve d.

S permiogenesis

S per m iogene s i s( F igure 5.1 ) o cc ur s after m eio s i s, an d for Eutherian (pla c ental)

m a mm al s, it i s initiate d by the s ynthe s i s an d bin d ing of the tran s ition protein s (TP1 an d TP2) to DNA, c oin c i d ent with the ? r s t pha s e of s per m c hro m atin c on d en s ation an d the

d i s pla c

e m ent o

f hi s tone s fro m nu c leo s o m e s( O ko et al., 1996 ). The tran s ition protein s are then repla c e d by the prota m ine s, P1 an d P2, whi c h are highly ba s i c arginine-ri c h protein s c ontainin

g 21 po s itive c harge s that bin d to DNA, c o m pletely neutralizing the negatively c harge d pho s pho d ie s ter ba c kbone an d for m ing DNA –p rota m ine toroi ds( F igure 5.2 ). The toroi d i s a d onut-s hape d s tru c ture c ontaining 50 k b of DNA in an extre m ely c o m pa c t for m that i s50 –100 n m in d ia m eter ( H u d et al., 1993 ). The po s ition of c hro m o s o m e s an d the higher-or d er s tru c ture of toroi ds for m e d fro m the s e c hro m o s o m e s within the s per m nu c leu s are unknown.

P revious Studies of Toroid Structure

H u d an d Balhorn (1993) were the ? r s t to i d entify the toroi d a s the fun d a m ental unit

of s per m c hro m atin in m a mm al s u s ing both ato m i c for c e m i c ro sc opy an d ele c tron

m i c ro sc opy. H u d an d Downing (2001)were able to ob s erve the s tru c ture of DNA

within toroi ds for m e d u s ingλ-phage DNA an d c obalt-hexa m ine (3 ?)an d i m age d u s ing c ryoele c tron m i c ro sc opy. Thi s te c hnique, while provi d ing exqui s ite d etail of DNA in the interior of the s e toroi ds, i s not a m enable to un d er s tan d ing the kineti cs of toroi d for m ation. S ingle Molecule Experiments

W e have s tu d ie d the for m ation of DNA –p rota m ine toroi ds in real ti m e u s ing s ingle DNA m ole c ule s to un d er s tan d how the s per m geno m e i s repa c kage d d uring s per m iogene s i s. Our

S ingle Molecule Studies of Chromatin Structure and Dynamics 147

m etho d avoi ds the proble ms of aggregation an d pre c ipitation of DNA – p rotein c o m plexe s that are inherent to bulk bio c he m i s try approa c he s . An opti c al trap (la s er-tweezer s ) wa s u s e d to m ove a s ingle DNA m ole c ule fro m one c hannel of a m i c ro? ui d i c ? ow c ell into another ( F igure 5.3 )c ontaining a s pe c i? c s per m nu c lear protein. The DNA m ole c ule

DNA

Protamine (A)

(B)In ports

Flow

Oil Out port

??1.047?m

??

488 nm