In,munochm,,.wrs. Vol. 15. pp 7x7-790 1.1Pergamon Press Lfd 197X Pr~ntrd III Great Bntam
,KI,‘,-279,
:x
I ,l,,-,i7H-
\,,2
00 0
CONFORMATIONAL CHANGES OF DNA IN CHROMATIN AND THE NUCLEOSOME SUB-UNIT* ROBERT Department
of Biological Chemistry.
C. KRUEGER
University Cincinnati.
of C‘incinnatl OH 45167.
Medic;11 C’entcr. 231 Bcthc&
A\cnuc.
U.S.A.
Abstract--Conformation of the DNA in B salt-soluble chromatin and in the nucleosome sub-unit L\:L~ studied in a variety of ionic environments. Precipitation with Mg’ ’ wahused 2~sone kind of ;I probe to indicate the arrangement of the proteins in the complex Parallel circular dxhrox mcasurcment~ \\r‘~-c ;II\o made. Three types of DNA conformation have been described: (I ) an extended contiguration. (2) ;~n intermediate type as observed in the high mol. ut. salt-soluble chromutin and (3) the type \cen in Ihc nucleosome sub-unit. Both the chromatin and the nucleosomr iire pictured iis tlcx.ible complew\ thcitstructure being highly dependent on the Ionic enclronment.
INTRODUCTION
the DNA-protein complex which exists in the eukaryotic cell nucleus generally has been considered to be insoluble in salt solutions of ionic strength of the order of 0.15 M. In the past, the usual method of chromatin preparation has been to solubilize in H,O or medium of very low ionic strength (0.001-0.01 M) usually with the aid of some kind of shearing force (Zubay & Doty, 1959;Fredericq. 197 I). This chromatin is characterized by a high viscosity and the DNA in the complex exists in a rather extended configuration. The salt soluble type of chromatin studied in this laboratory is derived by way of an endogenous nucleolytic action on aggregated chromatin fibers (Rees & Krueger 1968; Carrel & Krueger, 1970; Krueger&Allison, 1973). As shown by hydrodynamic methods and by electron microscopic examination, the solubilized material is compact in nature and contains a very high mol. wt DNA component. In the previous studies from this laboratory, the production of salt soluble chromatin was carried out by incubation of rabbit thymus nuclear lysates for short periods of time (2C-30 min at 37°C). Under these conditions only large segments of the chromatin fiber were formed (l-5 x 1O6 daltons of DNA) and there was negligible formation of short pieces of DNA. The amount of DNA converted to the salt-soluble form was 2&40”,, while acid-soluble nucleotide production was less than 19,. In a recent study the endogenous reaction was allowed to proceed for 14 hr resulting in the formation of nucleosome monomers and oligomers (Krueger, 1978). The action of micrococcal nuclease on the salt-soluble chromatin was also examined; hydrolysis of the DNA into nucleosome sub-units was observed. According to the criterion of No11 et al. (1975). the salt-soluble complex displayed a native configuration for the sub-unit structure was clearly delineated and there was negligible background of heterogeneous DNA. The ‘trimming’ of the nucleosomal DNA to a core complex Chromatin,
* In honor of Michael Heidelberger
on his 90th birthday. 787
containing 145 base pairs of DNA wa:, also demonstrated. In the present report we have used ;I Mg- ’ precipitation assay and circular dichroic (CD) measurements to study changes of the conformation of DNA in chromatin and the nucleosome sub-unit ;I, a result of changes in the ionic environment. RIATERIAI.S
\\D
\lH’HODS
Rabtxt thymus nuclei were Iysed ;Ind \\ashed w\er;II tlmc\ In 0.10 .+I haCI-O.Ol .&I EDTA, pH7.J. After \r;i\hlng 111 H20 the chrom;ltln \liis solublllred b! \hearlng 111;L PotterElvejcm homogenizer :md clarltied b\ ccntrIIug:t~ton ;II 15.000 rwmin for 1 hr.
7xx Table 1. Circular Chromatin
dlchrolsm
of chromatin
type
preparations
Solvent
[W.’
Salt-soluble Salt-solLlhlc Nuclcoaome Nuclcosomc
0 04 .\f K,SO,-0.02
,M Tris
H:O 0.04 .\I K?SO,-0.02 H ,0
21 Tris
Nucleosome
0.03
‘: Number
in var~ouc solvents
\I K,SO,-0
of \cpa~-,~tc pl-cparatlon\
245&2910 (3)” 3433-35 IO (2) 1’3%174!, (6) I X6I-2996 (4) I0 I9
02 ‘&I Tr~s-0. I5 .\I KC‘1
(I)
,n I
and the chromatin huh-unit V,C h;l\e investigated changes that take place on changing the ionic environment. The study of the precipitation with MgL + may be a useful probe and we present here some of our preliminary results. The precipitation of the salt-soluble chromatin is shown in Fig. I. In 0.04 .M K,SO,-0.02 .%ITI-is SO, there h;th no change in the turbidity of the solution or an) precipitation up 10 0.003 A4 MgZ’. Beyond this concentration of Mg’ precipitation took place and was complete at 0.006 44 Mg’&. Since DNA itself binds Mg’ + but does not precipitate weassume that the interaction was with the protrlns in the complex. Transfer of the chromatin to H,O produced a change in the Mg” interactlon. Precipitation was now complete at 0.002 M Mg’ ’ Previously, Lewis cl/ ul. (1976) had shown that lowering the salt concentration caused an extension ol the DNA-protein complex. An unfolding ofchromatin fragments in low salt has also been observed h) Kenz PI ul. (I 977) and has been suggested by electron microscopic studies carried out in thi\ laborator\. We
chromatin For DNA analysis. samples were treated with I”,, sodium dodecyl sarcosinatr for I hr at 37 c‘and then clcctrophoresed on 2.5”,, polyacrylamide. DNA bands were visualized under u.\I. light after staining with ethldium bromide. The gels were calibrated with Hind II fragments ofSV40 DNA or Hint II fragments of(bX I74RF DNA. 7 ,2-Iurea denaturlng gels uere run according to Maniatlh CI rri. (197.5). tar hlstonc examination. samples ucrc diallred versus dilute phosphate buffer and heated in boiling H,O l’or 2 mln in I”,, wdlum dodecyl sulPate (SDS) and I”,, /i-mercnptoethanol and run on I5”,, gels in the presence of 0.7”,, SDS.
An aliquot of the chromatin or nucleoaome preparation was mixed with 0. I vol of MgSO, solution to give the desired final concentration. The mixture\ wre held at 20 C for 2 hr and centrifuged at 2000 rev mln for 20 min. The \upernatant solution was diluted 0.1-5.0 ml wth huffcr and read in the spectrophotometer. The amount prcclpltated did not appear to be dcpendcnt on tcmpcrature for It U;I\ unchanged h> incubation at 37 C’ or for 20 hr at 0 C’.
The measurement\ ut’rc run at 7&75 c‘ in a C‘arq spectropolarlmeter. In each ofthe two figures shown here the DNA concentrations acre set at the wme ~aluc so dlrcct comparicon\ could he made. In the experiment\ of Table I the DNA concentration \arlcd from one wlutlon to another and molar ellipticitie\ u,crc calculated from the follomlng formula:
The DNA conccntrat1on u\;!\ dctermlncd colorlmctrull) with diphenqlamlne after preupitation with cold TC‘A and extraction Uith hot T(A Mg.50; RESL LTS AND
In order to gain
further
FI#.
DISCLISSION
insight
on the structure
of
I.
The precipitation
MOLARITY
of salt-soluble chromatm
,I. 0.04 ,tl K>SO,-0.02 MTrts . . 0.0X :\I K,SO,-0.01
by Mg’ _.
SO,, pH 7.4: x 1 H,O, pH 7. I. :\I Trls SO,. pH 7.3
7%‘)
ii Fig
3. Thr CD spectra salt-soluble DNP
h-NANOMETERS
of DNA
H,O-retracted. and chromatm preparations. DNH 0.050 mr ml. tt111 ranye chromatln: DNA concentration
suggest that the extension has exposed protein molecules which become free to interact with the Mg’+ to cause precipitation. It has been known for some time that the HzO-soluble, sheared type of chromatin which is an extended structure, was insoluble in Mg’ + at 0.001 %I (Zubay & Doty. 1959; Itzhaki, 1966). An Interesting change m the course of the Mg’+ precipitation was seen with a salt-soluble chromatin preparation which had been dialyzed Lcrsus0.N M K.SO,-0.01 !MTrisSO,. A resistance to precipitation was evident. It IS suggested that the higher salt causes a compression of the chromatin complex and sequestering of the molecules that interact with ME’+. This observation is relevant when considering the ionic environment which obtains in ~ivo in the cell nucleus. The total ionic content of the
IC-
09-
nucleosomc sub-umt: concentration 0.01
100 base pairs of DNA: DNA mg ml. Full range 0.05 &gee\.
sheared chromarln. 0 03 JCPWL’\
is about 0.4 M while the ionic activity is at least 0.2 M and may be higher (Siebert & Langendorf. 1970). Some experiments with a nucleosome sub-unit are shown in Fig. 2. The absence of precipitation even at the level of 0.01 M Mg’ ’ ia a distinct departure from the behavior of the larger chromatin complex. It is not clear why the sub-unit does not precipitate with Mg’ + One possibility is that since the sub-unit does not contain histone HI. this histone may be the site for the interaction with Mg?‘. When the nucleosome subunit was transferred to HzO. interaction with Mg?+ became possible and precipitation followed, although in this case a higher of Mg’+ was necessary compared to that required for the salt-soluble chromatin in H .O solution. This alteration caused by the lower ionic strength may be related to a structure suggested by Lilley c’r al. (1977). The monomer sub-unit was pictured as two discrete disks and it is possible that in the low salt. these disks are displaced relative to each other allowing interaction with Mg’+ to occur. Parallel studieh of the CD spectra wcrc carried out. In Fig. 3, a comparison is made between the saltsoluble chromatin (DNP) and a water-soluble. sheared material (DNH). A change in the conformation of DNA caused bq its condensation into a compact structure is reflected in a decrease in the band strength at 275 nm. As shown in Fig. 4. a further depression of the band at 375nm is observed with the nucleosome sub-unit. This low value is in accord with studies from other laboratories (Mandel & Fasman, 1976; Whitlock & Simpson, 1976). The magnitude of the CD absorption for both the salt-soluble chromatin and the nuclcosome is highly dependent on the ionic environment as shown in Table I. Both types of preparations show increases on transfer to H,O and the nucleosome shows a decrease upon addifion of KC]. This could be a rellection of a flexibility existing in the high mol. wt complex and even in the nucleosome itself. The foregoing results taken together suggest that there are three distinct types of DNA conformation in these DNA-protein complexes. These are: (I) an extended conformation. represented by the H,Oextracted material and showing the highest CD nucleus
ahsorpt)on: (2) a compact type represented by the high mol. Mt. salt-voluble compleu this files ;I band strength of intermediate value: and (3) the type seen m the nucleosome with the lowest CD band. It remains to be determined to what type of base stacking and DNA helix structure this corresponds. Th,1nk., Arc‘ due III John I
,KI,‘,-279,
:x
I ,l,,-,i7H-
\,,2
00 0
CONFORMATIONAL CHANGES OF DNA IN CHROMATIN AND THE NUCLEOSOME SUB-UNIT* ROBERT Department
of Biological Chemistry.
C. KRUEGER
University Cincinnati.
of C‘incinnatl OH 45167.
Medic;11 C’entcr. 231 Bcthc&
A\cnuc.
U.S.A.
Abstract--Conformation of the DNA in B salt-soluble chromatin and in the nucleosome sub-unit L\:L~ studied in a variety of ionic environments. Precipitation with Mg’ ’ wahused 2~sone kind of ;I probe to indicate the arrangement of the proteins in the complex Parallel circular dxhrox mcasurcment~ \\r‘~-c ;II\o made. Three types of DNA conformation have been described: (I ) an extended contiguration. (2) ;~n intermediate type as observed in the high mol. ut. salt-soluble chromutin and (3) the type \cen in Ihc nucleosome sub-unit. Both the chromatin and the nucleosomr iire pictured iis tlcx.ible complew\ thcitstructure being highly dependent on the Ionic enclronment.
INTRODUCTION
the DNA-protein complex which exists in the eukaryotic cell nucleus generally has been considered to be insoluble in salt solutions of ionic strength of the order of 0.15 M. In the past, the usual method of chromatin preparation has been to solubilize in H,O or medium of very low ionic strength (0.001-0.01 M) usually with the aid of some kind of shearing force (Zubay & Doty, 1959;Fredericq. 197 I). This chromatin is characterized by a high viscosity and the DNA in the complex exists in a rather extended configuration. The salt soluble type of chromatin studied in this laboratory is derived by way of an endogenous nucleolytic action on aggregated chromatin fibers (Rees & Krueger 1968; Carrel & Krueger, 1970; Krueger&Allison, 1973). As shown by hydrodynamic methods and by electron microscopic examination, the solubilized material is compact in nature and contains a very high mol. wt DNA component. In the previous studies from this laboratory, the production of salt soluble chromatin was carried out by incubation of rabbit thymus nuclear lysates for short periods of time (2C-30 min at 37°C). Under these conditions only large segments of the chromatin fiber were formed (l-5 x 1O6 daltons of DNA) and there was negligible formation of short pieces of DNA. The amount of DNA converted to the salt-soluble form was 2&40”,, while acid-soluble nucleotide production was less than 19,. In a recent study the endogenous reaction was allowed to proceed for 14 hr resulting in the formation of nucleosome monomers and oligomers (Krueger, 1978). The action of micrococcal nuclease on the salt-soluble chromatin was also examined; hydrolysis of the DNA into nucleosome sub-units was observed. According to the criterion of No11 et al. (1975). the salt-soluble complex displayed a native configuration for the sub-unit structure was clearly delineated and there was negligible background of heterogeneous DNA. The ‘trimming’ of the nucleosomal DNA to a core complex Chromatin,
* In honor of Michael Heidelberger
on his 90th birthday. 787
containing 145 base pairs of DNA wa:, also demonstrated. In the present report we have used ;I Mg- ’ precipitation assay and circular dichroic (CD) measurements to study changes of the conformation of DNA in chromatin and the nucleosome sub-unit ;I, a result of changes in the ionic environment. RIATERIAI.S
\\D
\lH’HODS
Rabtxt thymus nuclei were Iysed ;Ind \\ashed w\er;II tlmc\ In 0.10 .+I haCI-O.Ol .&I EDTA, pH7.J. After \r;i\hlng 111 H20 the chrom;ltln \liis solublllred b! \hearlng 111;L PotterElvejcm homogenizer :md clarltied b\ ccntrIIug:t~ton ;II 15.000 rwmin for 1 hr.
7xx Table 1. Circular Chromatin
dlchrolsm
of chromatin
type
preparations
Solvent
[W.’
Salt-soluble Salt-solLlhlc Nuclcoaome Nuclcosomc
0 04 .\f K,SO,-0.02
,M Tris
H:O 0.04 .\I K?SO,-0.02 H ,0
21 Tris
Nucleosome
0.03
‘: Number
in var~ouc solvents
\I K,SO,-0
of \cpa~-,~tc pl-cparatlon\
245&2910 (3)” 3433-35 IO (2) 1’3%174!, (6) I X6I-2996 (4) I0 I9
02 ‘&I Tr~s-0. I5 .\I KC‘1
(I)
,n I
and the chromatin huh-unit V,C h;l\e investigated changes that take place on changing the ionic environment. The study of the precipitation with MgL + may be a useful probe and we present here some of our preliminary results. The precipitation of the salt-soluble chromatin is shown in Fig. I. In 0.04 .M K,SO,-0.02 .%ITI-is SO, there h;th no change in the turbidity of the solution or an) precipitation up 10 0.003 A4 MgZ’. Beyond this concentration of Mg’ precipitation took place and was complete at 0.006 44 Mg’&. Since DNA itself binds Mg’ + but does not precipitate weassume that the interaction was with the protrlns in the complex. Transfer of the chromatin to H,O produced a change in the Mg” interactlon. Precipitation was now complete at 0.002 M Mg’ ’ Previously, Lewis cl/ ul. (1976) had shown that lowering the salt concentration caused an extension ol the DNA-protein complex. An unfolding ofchromatin fragments in low salt has also been observed h) Kenz PI ul. (I 977) and has been suggested by electron microscopic studies carried out in thi\ laborator\. We
chromatin For DNA analysis. samples were treated with I”,, sodium dodecyl sarcosinatr for I hr at 37 c‘and then clcctrophoresed on 2.5”,, polyacrylamide. DNA bands were visualized under u.\I. light after staining with ethldium bromide. The gels were calibrated with Hind II fragments ofSV40 DNA or Hint II fragments of(bX I74RF DNA. 7 ,2-Iurea denaturlng gels uere run according to Maniatlh CI rri. (197.5). tar hlstonc examination. samples ucrc diallred versus dilute phosphate buffer and heated in boiling H,O l’or 2 mln in I”,, wdlum dodecyl sulPate (SDS) and I”,, /i-mercnptoethanol and run on I5”,, gels in the presence of 0.7”,, SDS.
An aliquot of the chromatin or nucleoaome preparation was mixed with 0. I vol of MgSO, solution to give the desired final concentration. The mixture\ wre held at 20 C for 2 hr and centrifuged at 2000 rev mln for 20 min. The \upernatant solution was diluted 0.1-5.0 ml wth huffcr and read in the spectrophotometer. The amount prcclpltated did not appear to be dcpendcnt on tcmpcrature for It U;I\ unchanged h> incubation at 37 C’ or for 20 hr at 0 C’.
The measurement\ ut’rc run at 7&75 c‘ in a C‘arq spectropolarlmeter. In each ofthe two figures shown here the DNA concentrations acre set at the wme ~aluc so dlrcct comparicon\ could he made. In the experiment\ of Table I the DNA concentration \arlcd from one wlutlon to another and molar ellipticitie\ u,crc calculated from the follomlng formula:
The DNA conccntrat1on u\;!\ dctermlncd colorlmctrull) with diphenqlamlne after preupitation with cold TC‘A and extraction Uith hot T(A Mg.50; RESL LTS AND
In order to gain
further
FI#.
DISCLISSION
insight
on the structure
of
I.
The precipitation
MOLARITY
of salt-soluble chromatm
,I. 0.04 ,tl K>SO,-0.02 MTrts . . 0.0X :\I K,SO,-0.01
by Mg’ _.
SO,, pH 7.4: x 1 H,O, pH 7. I. :\I Trls SO,. pH 7.3
7%‘)
ii Fig
3. Thr CD spectra salt-soluble DNP
h-NANOMETERS
of DNA
H,O-retracted. and chromatm preparations. DNH 0.050 mr ml. tt111 ranye chromatln: DNA concentration
suggest that the extension has exposed protein molecules which become free to interact with the Mg’+ to cause precipitation. It has been known for some time that the HzO-soluble, sheared type of chromatin which is an extended structure, was insoluble in Mg’ + at 0.001 %I (Zubay & Doty. 1959; Itzhaki, 1966). An Interesting change m the course of the Mg’+ precipitation was seen with a salt-soluble chromatin preparation which had been dialyzed Lcrsus0.N M K.SO,-0.01 !MTrisSO,. A resistance to precipitation was evident. It IS suggested that the higher salt causes a compression of the chromatin complex and sequestering of the molecules that interact with ME’+. This observation is relevant when considering the ionic environment which obtains in ~ivo in the cell nucleus. The total ionic content of the
IC-
09-
nucleosomc sub-umt: concentration 0.01
100 base pairs of DNA: DNA mg ml. Full range 0.05 &gee\.
sheared chromarln. 0 03 JCPWL’\
is about 0.4 M while the ionic activity is at least 0.2 M and may be higher (Siebert & Langendorf. 1970). Some experiments with a nucleosome sub-unit are shown in Fig. 2. The absence of precipitation even at the level of 0.01 M Mg’ ’ ia a distinct departure from the behavior of the larger chromatin complex. It is not clear why the sub-unit does not precipitate with Mg’ + One possibility is that since the sub-unit does not contain histone HI. this histone may be the site for the interaction with Mg?‘. When the nucleosome subunit was transferred to HzO. interaction with Mg?+ became possible and precipitation followed, although in this case a higher of Mg’+ was necessary compared to that required for the salt-soluble chromatin in H .O solution. This alteration caused by the lower ionic strength may be related to a structure suggested by Lilley c’r al. (1977). The monomer sub-unit was pictured as two discrete disks and it is possible that in the low salt. these disks are displaced relative to each other allowing interaction with Mg’+ to occur. Parallel studieh of the CD spectra wcrc carried out. In Fig. 3, a comparison is made between the saltsoluble chromatin (DNP) and a water-soluble. sheared material (DNH). A change in the conformation of DNA caused bq its condensation into a compact structure is reflected in a decrease in the band strength at 275 nm. As shown in Fig. 4. a further depression of the band at 375nm is observed with the nucleosome sub-unit. This low value is in accord with studies from other laboratories (Mandel & Fasman, 1976; Whitlock & Simpson, 1976). The magnitude of the CD absorption for both the salt-soluble chromatin and the nuclcosome is highly dependent on the ionic environment as shown in Table I. Both types of preparations show increases on transfer to H,O and the nucleosome shows a decrease upon addifion of KC]. This could be a rellection of a flexibility existing in the high mol. wt complex and even in the nucleosome itself. The foregoing results taken together suggest that there are three distinct types of DNA conformation in these DNA-protein complexes. These are: (I) an extended conformation. represented by the H,Oextracted material and showing the highest CD nucleus
ahsorpt)on: (2) a compact type represented by the high mol. Mt. salt-voluble compleu this files ;I band strength of intermediate value: and (3) the type seen m the nucleosome with the lowest CD band. It remains to be determined to what type of base stacking and DNA helix structure this corresponds. Th,1nk., Arc‘ due III John I
