the formation of nucleosomes is the first stage of dna packing into chromatin , followed by the assembly of the  beads - on - a - string  nucleosomal array into compact chromatin fibers ( e.g. ,   and references therein ) . histone is a key player in the formation of the 30-nm - thick fibers ( e.g. , ) ; however , interactions between the nucleosomal particles contribute to the assembly process as well . although the molecular mechanisms behind the formation of higher - order chromatin structures remain unclear , work employing model systems has shown that in vitro reconstituted nucleosome arrays containing only dna and core histone proteins undergo the same initial salt - dependent condensations as native chromatin [ 1 , 3 ] . in solutions containing physiological concentrations of mono- and divalent cations , nucleosome arrays spontaneously fold into structures with the same hydrodynamic shape as the 30-nm - diameter chromatin fiber [ 1 , 4 ] . this implies that the primary protein determinants defining these structures reside within the core histone proteins . indeed , in the early crystallography work of luger et al . , it was shown that the k16 to n25 segment of h4 makes extensive contacts with an h2a - h2b dimer of an adjacent particle . this h4 region , k16 to n25 , makes multiple hydrogen bonds and salt bridges between its basic side chains ( k16 , r19 , k20 , r23 ) and acidic side chains of h2a ( e56 , e61 , e64 , d90 , e91 , e92 ) and h2b ( e110 ) . these structure - based predictions were supported with studies [ 68 ] of recombinant core mutants . they showed that interaction between the h4 n - terminal domain ( ntd ) and a surface - exposed h2a region on a neighboring nucleosome is required for assembly of folded 30-nm secondary structures . more importantly , nucleosomal arrays lacking all core histone ntds are unable to fold into the 30-nm secondary chromatin structures [ 3 , 9 ] , even in the presence of bound linker histones . results indicate that all core histone n - terminal domains ( ntd ) participate in the oligomerization process and that the ntds function additively and independently . according to the monte carlo simulations , the h4 tails mediate the majority of the internucleosomal interactions , followed by the h3 , h2a , and h2b tails in decreasing order . the h4 histone role was also studied separately and was found that the n - terminal end of the h4 tail participates in intra- and internucleosomal interactions with protein and dna during folding and oligomerization of nucleosome arrays . atomic force microscopy ( afm ) was applied in   to study interactions between nucleosomes within the assembled    beads - on - a - string  nucleosomal array in the absence of h1 histone . the results led to the conclusion that attractive interactions between the nucleosomes existed which led to array compaction . in recent work , the interaction between nucleosomes was studied with the use of fret . with this technique , transiently formed dinucleosomes with the lifetime ~1 the acetylation of histones and the presence of divalent cations increased the stability of dinucleosomes .   here , we apply afm imaging to directly characterize the formation of tight dinucleosome complexes with the dinucleosome design . the data are in line with the dynamic nature of dinucleosomes , in which the distance between the nucleosomes varies in a broad range , enabling the formation of close nucleosome - nucleosome contacts . we demonstrate the importance of environmental conditions to these dynamics and characterize the role of the ionic detergent chaps . dna for nucleosome assembly was generated by pcr using the plasmid pgem3z-601 as a template , which codes for a high - affinity nucleosome positioning sequence . the pcr reaction ( 33 cycles of 94c/30  s , 54c/30  s , 72c/30  s ) was run in buffer containing 2.5  mm mgcl2 , 0.15  mm dntps , and 0.016  u/l of taq dna polymerase . two pcr reactions were run with forward and reverse primers , respectively , set 1 , primers cggccagtgaattgtaatacg and cggtacgctggg tatgtgatggaccctatacg , and set 2 , primers gccatccccagccggcaaggtcgctgttcaat and cgggatcctaatgaccaagg . these reactions created products , which incorporated nonpalindromic bseyi restriction site at the ends . after restriction digest with bseyi and ligation with t4 dna ligase , a dna template of 560  bp was obtained ( see figure s1 in supplementary material available online at doi:10.1155/2012/650840 ) , which contained two nucleosome positioning sequences with a 60  bp linker between them and arms of 127 and 79  bp in length . octamers were separated from tetramer and dimer fractions with size - exclusion chromatography ( sec ) with superdex 200 pc 3.2/30 column ( ge healthcare ) at 4c . fractions containing histones h2a , h2b , h3 , and h4 in approximately equal ratios were pooled and concentrated by centrifugation at 10,000  g. dinucleosome particle samples ( di - ncp ) were prepared as described earlier [ 18 , 19 ] . briefly , histone octamers and dna containing the nucleosome positioning sequence were mixed in equimolar concentrations in 2  m nacl and kept for 30  min at rt . a dilution series was prepared using 10  mm tris hcl to produce final concentrations of 1  m , 0.67  m , and 0.5  m nacl . diluted samples were kept at 4c for 1  h before dialysis against one change of volume of 0.2  m nacl overnight . nucleosomes were concentrated using microcon centrifugal filter devices , mwco 10,000 at 7,000  g for 10  min at 4c and dialyzed against one change of 200  ml of buffer containing 10  mm hepes - nacl , ph 7.5 , and 1  mm edta for 3  h at 4c . freshly cleaved mica was modified with a 167  m solution of 1-(3-aminopropyl)-silatrane ( aps ) for 30  min at room t to make aps - mica as described previously in [ 1921 ] . the nucleosome stock solution was diluted into 10  mm tris - hcl , ph 7.5 , 4  mm , mgcl2 buffer , and 5  l of the solution was deposited on aps - treated mica for 3 minutes , washed with deionized water , and dried under argon flow . afm images were collected on an mm afm / nanoscope iiid system ( bruker - nano / veeco , santa barbara , calif , usa ) as described in [ 19 , 20 , 22 ] . the following 5 initial parameters were measured : length of each dna arm , angle between arms ( interarm angle ) , height of the nucleosome core particle , and diameter as the width of the nucleosome core particle at half height . the length of dna was measured with femtoscan software using the  curve  parameter . the length of wrapped dna was measured by subtracting the sum of both dna arms and the linker from the length of free dna measured on the same sample ( see figure 1 ) . the length of the linker dna was measured from the center of one nucleosome along the linker to the center of another nucleosome minus 5  nm ( the size of the nucleosome particle itself ) . the di - ncp sample was imaged similarly to the mononucleosome sample described earlier [ 1822 ] . diluted samples ( 0.8  nm concentration in 10  mm tris - hcl , ph 7.5 , and 4  mm mgcl2 buffer ) were deposited on aps mica and after drying , imaged in air at ambient conditions . the nucleosomes appear as two bright globular features ( blobs ) separated with a dna region ( linker ) with two free dna arms at each nucleosome . images as shown in figure 1 were used for assessing the degree of nucleosome wrapping . this value was calculated from the measurements of the lengths of the dna arms and the linker dna as described in section 2 . the distribution is rather broad with a maximum for the gaussian = 107  nm . the expected length of wrapped dna for the di - ncp is 147 ( bp )  0.32 ( nm / bp )  2 = 94.08  nm . due to the tip convolution effect , this effect leads to a decrease of the length of the measured unwrapped dna by ~10  nm ; therefore , given the convolution effect , the measured mean value of wrapped dna is close to the expected value . at the same time , the distribution of the lengths is broad , ranging between ~86  nm and 127  nm ( mean values ) . this suggests that nucleosomes are dynamic , enabling the formation of particles with varied amounts of dna wrapping , which is in line with our previous study of mononucleosomes [ 18 , 19 , 22 ] . to address the question of whether nucleosome core particles interact with each other in the absence of h1 histone , we measured two parameters of dinucleosomes , the length of the linker , and the shortest internucleosome distance . the first parameter will answer the question of whether the interaction occurs via sliding of nucleosomes . a subset of three images of dinucleosomes with different relative positions is shown in figure 3 . the distance is considerably larger for the dinucleosome in figure 3(b ) ( ~17  nm ) and the last image ( figure 3(c ) ) corresponds to a linker length ~37  nm . in addition to the main peak at the maximum ~19  nm , there is a minor peak at ~35  nm . the appearance of the second peak suggests that there is a population of partially unwrapped nucleosomes . the most important finding of this analysis is that the measured linker length ( ~19  nm ) is very close to the expected value , 19.84  nm . the shortest distance between the nucleosomes was obtained by measuring the center - to - center distance . the histogram has a maximum at ~22  nm and a low percentage of nucleosomes with the internucleosomal distance of 10  nm . nucleosomes are unstable at low concentrations ( nanomolar range ) , and detergents are used to stabilize them ( e.g. , ) . we have shown recently that the zwitterionic detergent , chaps , considerably increases the stability of mononucleosomes at the nanomolar concentration and works in a broad range of ionic strengths . importantly , the sequence - specific positioning of the nucleosomes also changes in the presence of chaps . we analyzed the effect of chaps for the dinucleosome samples to understand whether chaps changes the stability and morphology of dinucleosomes . images in figure 5 are the typical images obtained for the dinucleosome sample incubated in tris - hcl buffer ( 10  mm tris - hcl , ph 7.5 , 4  mm mgcl2 , ph 7.5 ) at the concentration 0.8  nm for 30  min in the absence of chaps ( a ) and in the presence of 1.6  mm chaps ( b ) . the initial sample contained 85% of dinucleosomes and appeared under afm as the sample shown in figure 1 . incubation for 30  min ( figure 5(a ) ) leads to almost complete dissociation of the nucleosomes from the dna template ; therefore , only naked dna is seen under afm . in contrast , dinucleosomes remain intact if 1.6  mm chaps is present ( figure 5(b ) ) . a similar analysis was performed for other incubation times , and the data are assembled in figure s2 . this graph shows that in the absence of chaps , nucleosomes dissociate rapidly with a characteristic time ~7.5  min , whereas in the presence of chaps , dinucleosomes are stable after incubation for 48 hours . similar data were obtained for the mononucleosomes , suggesting that each nucleosome in the dinucleosome design behaves independently . we also measured the variation of the linker length over incubation time with 1.6  mm chaps . initially at zero incubation time , both samples were indistinguishable with a linker length ~20  nm . moreover , the distributions of the linker lengths for the control sample and the sample prepared in the presence of chaps were very close ( figure s3 ) . the time - dependent measurements of the linker lengths for the chaps containing samples showed dramatic changes in the width distributions over time . the values change in the order of 11.4  nm , 22.3  nm , 23.1  nm , and 18.1  nm for times 0  min , 5  min , 2 hours , and 48 hours , respectively . these findings suggest that the sequence specific formation of nucleosomes , in the presence of chaps , is less stringent . the data obtained in this paper show that the direct interaction of two nucleosomes separated by a linker in the absence of h1 histone can occur , but the probability of such events is rather low . we were able to identify by afm complexes with a small internucleosomal distance similar to the one shown in figure 3(a ) . however , the yield of such complexes according to the statistical analysis is low ( figure 4(b ) ) . at the same time , the afm analysis shows that nucleosomes with a broad range of internucleosomal distances are formed , suggesting that nucleosomal particles in the di - nucleosomal construct are rather dynamic , enabling the formation of systems with different distances between the nucleosomes . the highly dynamic feature of nucleosomes was directly proven in our early work with mononucleosomes , in which the dynamics were observed directly with time - lapse afm . therefore , nucleosomes can translocate along the dna , approach quite close to each other or move apart . the interaction between two nucleosomes was observed in , but these were free nucleosomes , and , in the complexes , the two nucleosomes were separated by approximately 2  nm . in addition , according to , the dinucleosome complexes are formed transiently , which is in line with our model of nucleosome dynamics . there are two models for the formation of tight dinucleosome complexes , translocation of nucleosomes , and their interaction in space via bending of the linker . the second mechanism requires substantial deformation of the linker .   to make a close contact , a u - turn deformation is needed , which is problematic for a linker as short as 60  bp . deformations of this kind require a segment with a random sequence 4 times larger in length . internucleosomal interactions were observed in , in which arrays of nucleosomes were used enabling the interaction of nucleosomes distantly located in the array . therefore , we assume that translocation of the nucleosome is the mechanism by which close contacts between the nucleosomes are made . there are two models for nucleosome dynamics , sliding and site transfer requiring unwrapping of both arms of the nucleosome [ 25 , 26 ] . indeed , the dynamics via unwrapping of both arms of the nucleosome was the only pathway imaged with time - lapse afm directly . however , recently we applied high - speed afm to directly image nucleosome dynamics . these studies enabled us to observe transient sliding of the nucleosome at the subsecond time scale . the nucleosome moved from the original site - specific position and returned back to the same position . note that this and our previous studies were performed with the use of the 601 sequence , characterized by a high affinity for   nucleosome formation . therefore , we assume that transient formation of tight di - nucleosomal complexes occurs via a sliding mechanism . according to the statistical analysis ( figure 4(a ) ) , the lengths of the linker for different nucleosomes vary over 15  nm . however , the range is two times wider if chaps is added ( figure s4 ) , suggesting that the dynamics of nucleosomes is much broader if chaps is present . we have shown recently ( see ) that chaps facilitates nucleosome dynamics and sliding away from the 601 motif into regions with a random sequence occurs with the formation of stable nonspecific nucleosomal particles . we hypothesized that in the presence of chaps , the stringency of the sequence specificity for nucleosome formation is lowered , enabling the formation of stably existing nucleosomes over the region away from the 601 motif . although chaps is not a natural product , it has similarity in structure with cholesterol . this suggests that the possible involvement of cholesterol , as a significant component of the nuclear membrane , in the regulation of chromatin dynamics should be taken into account . importantly , a modified water - soluble cholesterol , cholesteryl sulfate , is a minor constituent of various cell types including erythrocytes   and spermatozoa . we have shown   that cholesteryl sulfate also stabilizes nucleosomes and its participation in chromatin dynamics needs to be considered as well .
dynamics of nucleosomes and their interactions are important for understanding the mechanism of chromatin assembly . 
 internucleosomal interaction is required for the formation of higher - order chromatin structures . 
 although h1 histone is critically involved in the process of chromatin assembly , direct internucleosomal interactions contribute to this process as well . 
 to characterize the interactions of nucleosomes within the nucleosome array , we designed a dinucleosome and performed direct afm imaging . 
 the analysis of the afm data showed dinucleosomes are very dynamic systems , enabling the nucleosomes to move in a broad range along the dna template . 
 di - nucleosomes in close proximity were observed , but their population was low . 
 the use of the zwitterionic detergent , chaps , increased the dynamic range of the di - nucleosome , facilitating the formation of tight di - nucleosomes . 
 the role of chaps and similar natural products in chromatin structure and dynamics is also discussed .