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All studied eukaryotic genome are organized in a hierarchy as dynamic structures with the supercoiling of DNA around the histone octomer core, otherwise known as chromatin.
The densely packed chromatin structure is the structural basis of cellular control of gene expressions , dna replications, DNA repair as well as DNA recombination.
The chromatin is able to undergo regulation through covalent modification of the histone octomer core or nucleosome remodeling through ATP utilization to enable DNA accessibility to nucleosomal DNA, enabling or disenabling gene expression through regulatory subunits.
The fact that chromatin structure undeniably plays a crucial role in eukaryotic gene transcription has been proved throughout the decade, both in vivo and in vitro. The nucleosome, made up of histone octomer core, made up of protein spools , wrapped around with almost two turns of ~ 147bp of DNA , forms the building blocks of the chromatin structure. Nucleosomes, although previously was thought to have a structural function, in actual fact has crucial functional roles in maintaining transcriptional regulation where Chromatin remodeling factors function both in transcriptional activation as well as repression , but may have roles outside of transcriptional regulation as well.
The highly compact yet dynamic nucleoprotein complex, the nucleosome structure can be amended in 2 principle ways through the most widely characterized chromatin complexes
(i) ATP dependant complexes, which utilise energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA and introduce superhelical torsion into DNA , thus involving the movement of the histone octomers relative to DNA in order to make the DNA accessible.Doing so with the help of an ATPase motor, which through experiments was proved to have a 3' -5' translocase activity on DNA template, and initiates the generation of tortional strain in the presence of chromosomes (28,29,30) (ii) covalent modification of the N terminal Tails and occurs without the hydrolysis of ATP, as such occurng with the aid of histone acetyltransferases (HAT) and histone deacetylase (HDAC) complexes, which regulate the transcriptional activity of genes by determining the level of acetylation of the amino terminal domains of nucleosomal histones associated with them.
Although both remodeling methods have distinct mechanisms , in vivo they are interconnected functionally , within the cell. They are able to co-exist in the same complex, or they may exist in separate complexes that are both required for maximum opening of chromatin and activation of transcription, DNA replication and repair.
In this thesis we will be focusing on the ATP dependant chromatin factors and how they assist in chromosome remodeling affecting transcriptional regulation.
1.2 CHROMATIN AND NUCLEOSOME STRUCTURE
The Chromatin is an extremely compacted supercoiled structure of approximately 1.9 metres of DNA into a 9 µm diameter nucleus in most mammalian cells. It is wrapped around histone octomers by far reaching electrostatic interactions as well as through localized Hydrogen bonds and Van der Waals forces of attraction, forming a complex accredied as the nucleosome. These nucleosomes are joined by a linker DNA, forming a beads on a string appearance, upon observation under electron microscopy.
The nucleosome is made up of protein spools wrapped around with almost two turns of approximately 147 base pairs of DNA wrapped in superhelical turns around 2 copies of each of the histones, H2A,H2B,H3 and H4 as seen through X ray crystallography. (8)
Figure 1. Structure of the nucleosome core particle (Khorasanizadeh, 2004)
1.3 PACKAGING INTO HIGHER ORDER
From the beads on a string structure , the nucleosomes are further condensed and compacted and supercoiled in a highly organized fashion to form chromosomes, under normal resting stage in eukaryotic cells. The protein framework remains intact, even with the removal of the DNA. The exact mechanism and interactions for the folding as well as its precise framework has not been actuated. The overall packaging is summarized below.
1.4 CHROMATIN TERRITORIES (hetero / eu- chromatin)
Chromatin is subjected to various global remodeling processes, inclusive of the timely condensation and decondensation during cellular division processes such as mitosis and gamete formation.
Heterochromatin is the chromosome domain which remains in a condensed form in the interface stage of mitotic and meiotic divisions. Chromosomal stabilization, chromosomal segregation and the silencing of genes is its essential role in the nucleus. (Wallrath,1998; Henning,1999)
Euchromatin is the decondensed gene rich form and comprises the most active portion of the genome in which transcriptional processes occur actively at most instances, producing template mRNAs for translation into amino acid sequences. Certain remodeling enzymes such as ISWI has associations with euchromatic regions of Drosophila polytene chromosomes.
(13. Deuring R, Fanti L, Armstrong JA, Sarte M, Papoulas O, et al. (2000) The
ISWI chromatin-remodeling protein is required for gene expression and
the maintenance of higher order chromatin structure in vivo. Mol Cell 5:
FIGURE 3. The highly refractive and bright sections of the centromere region and the dot-like structures at the end of the chromosome depicts the densely packaged heterochromatic regions.
The darker , less refractive regions depicts decondensed chromatin and are euchromatic regions
Decondensation of the chromatin is vital for the cell, and ultimately the survival of the organism. This is so as the physical barrier of the densely condensed structure is no more, DNA becomes accessible to transcription factors, and production of needed protein goes on. This decondensation is effectively brought about with the assistance of ATP dependant chromatin remodeling factors, as well as histone acetyltransferases and deacetlyases.
Manisha Sinha1,2, Shinya Watanabe1, Aaron Johnson3, Danesh Moazed3,4, and Craig L.
Peterson1,5 Cell. 2009 September 18; 138(6): 1109-1121. doi:10.1016/j.cell.2009.07.013.
The existence of chromatin as certain territories may require ATP dependant chromatin remodellig for recombinational repair. Where heterochromatin takes on the responsibility of safeguarding the autosomal integrity and totality through the prevention of homologous recombination, unlike in other processes such as in meiosis where homologous recombination is required, the DNA exists as Euchromatin.
Sir2p, Sir3p, and Sir4p are structural components of heterochromatin of the yeast, Saccharomyces cerevisiae , and can be located along telomeric sites
Sir3p, together with Sir2p/Sir4p , when included to nucleosomal templates was found to be adequate to remove the Rad51p-catalyzed formation of joints . This suppression is only possible with the presence of histones, and are vital for gene silencing in vivo. Since the SWI/SNF chromatin remodeling machinery is necessary for the generation of the joint. thus the repression brought about by the Rad 51p, together with Rad54p is surmounted by ATP driven remodeling interactions.
1.5 POST TRANSLATIONAL MODIFICATION OF HISTONES
2.1 OVERVIEW ON THE MECHANISMS OF ACTION OF ATP DEPENDANT CHROMATIN REMODELLING
Studies and experimental findings to date has brought upon the revelation that the vital principle behind the remodeling process. The movement or translocation of DNA through an ATP utilization process. The fundamental mechanism however remains to be discovered.
There are 2 proposed mechanisms for nucleosome mobilization which is dependent upon ATP hydrolysis, through the Twist diffusion model of nucleosome sliding and through the Loop bulge model.
In the twist diffusion model of nucleosome sliding , base pairs are added in or removed from the linker DNA, forming a strain and a rotational effect, sliding the DNA across the histone , and has no , or minimal change on the DNA contacts.
Figure . shows the minor twist effect in the upper portion where a base pair has been taken in or released , forming a tortional strain on the DNA , resulting in the DNA rotating and ultimately translocating by 1 bp relative to the protein.
In the loop bulge model there is a conformational change , through the formation of intra nucleosomal DNA looping. The loop size , usually being more than 20 base pairs. As such in this model there is disruption of the nucleosome , resulting in loss or dispossession of exon content from the DNA.
The SWI/SNF Complex Creates Loop Domains in DNA and Polynucleosome Arrays and Can Disrupt DNA-Histone
Contacts within These Domains
DAVID P. BAZETT-JONES,1* JACQUES COˆTE´,2 CAROLYN C. LANDEL,3 CRAIG L. PETERSON,3 AND JERRY L. WORKMAN4
Depicts a hairpin loop formation on a linear plasmid DNA fragment, made with the assistance of ATP dependent SWI/SNF complex, and is believed to be part of the nucleosome remodeling process.
However there is a consideration to be made, since most assays performed are usually done on short templates of DNA , with an approximation of less than 300 base pairs. The end portion of DNA has an influence on nucleosome structure, rendering the experiments carried out generally inaccurate.
In addition DNA elastics also have to be taken into consideration, and how it may impinge each of the above mechanism of action.
In addition, to have a better understanding of the proposed models of nucleosome sliding ,the remodelling complexes must first be properly understood. Though the exact function of the remodelling complexes are yet to be fully comprehended, there are differences in their mechanism of action, which may give a better insight on remodeling nucleosomes, to enable gene expression in eukaryotes.
2.2 REMODELLING COMPLEXES RESPONSIBLE FOR THIS PROPOSED MECHANSISM OF ACTION
Are usually characterized based on the domain organization of their catalytic subunit.
The remodeling complexesATP dependant chromatin remodeling complexes all contain the ATPase subunit that belongs to the SNF2 superfamily of proteins. Based on the identity of these
proteins , they have been classified into 2 main categories. The SWI2/SNF2 group and the SWI(ISWI) group. The third classwhich shows the deacetylase activity is the CHD group.
Besides these eukaryotic cell homologues, there is also a prokaryotic counterpart which is believed to play a similar role as these ATP dependant complexes . Rap A , being a bacterial protein which consistently copurifies with DNA - dependant RNA polymerase and shows similarity in sequence homology to the SWI2/SNF2 ATPase.
Figure 7. The CHD / Mi-2 and Ino80 chromatin remodeling complexes (Eberharter and Becker, 2004) Various members of the ATP dependant remodeling complexes in eukaryotes
NUCLEOSOME REMODELLING COMPLEXES
2.2.1 SWI/SNF Family of ATPase group
The first SWI2/SNF2 ATPase group to be identified was the yeast SWI/SNF ATPase, which contained ~ 11 known subunit complex, including Swi2/Snf2.
The SWI /SNF complex was initially characterized as a matining type switching regulator, or as a requirement for growth on energy sources other than sucrose. The members of this larger remodeling families are multilobes structures and encompass a inner central trough or chamber, in which nucleosome binding is assumed to take place, as seen through electron microscopic reconstructions.
This group is inclusive of yeast SWI/SNF, yeast RSC, the Drosophila Brahma complex, the human BRM (hBRM) and BRG1(hBRG1) complexes. -(to include table). Most are mutisubunit complexes with a highly conserved ATPase as the catalytic centre. The ATPases contain an ATPase core domain which is surrounded by N- and C-terminal domains, which differ considerably between these ATPases famiies. The complexes differ in the number of subunits, ranging in from two in some in some ISWI complexes to 11 or more in the SWI/SNF complexes.( 7)
In S.cerevisiae, there are 2 similar versions (SWI/SNF and RSC) of the SWI/SNF complex.
RSC was found to have increased abundance in the cellular context and was crucial for cellular growth, while SWI/SNF was not essential for growth , or was it in abundance in the cells.
Both of these versions seemed to have totally distinct , non overlapping roles.
The Swi2 or Snf2 protein was the highly conserved catalytic subunit of the SWI/SNF complex in yeast, where its counterpart was Sth1 subunit in RSC.
In face RSC itself has been shown to exist in two functionally distince complexes that differ by containing either Rsc1 or 2.
As previously mentioned, in Drosophila there are 2 forms of SWI/SNF , which is known as BAP and PBAP, both containing the same catalytic Brahma catalytic subunit. They are however differentiated through the OSA subunit of the BAP and the Polybromo and the BAP70 subunits of the PBAF.
Once again as previously mentioned, though the characterization of the human SWI/SNF complex falls under two forms, being the BAF ( BRG1/hBRM - associated factors) and PBAF , there are indeed many forms of SWI/SNF that acquire tissue specific subunits or additional subcomplexes in which the SWI/SNF type remodelers are associated with other factors such as BRACA1 components of the deacetylase complex and histone methylase. Through mass spectomertr and by MudPIT, there was a newly identified complexRtt 102p.
In addition the the loss of RTT102 created similar phenotypes consistent with the
loss of other SWI/SNF subunits. The role of SWI/SNF by all indications is far reaching.
The SWI/SNF complex in mammalians plays important functions in many developmental programs such as muscle, heart, blood, skeletal, neuron, adipocyte, liver and immune system/Tcell development.
Yeast SWI/SNF has been shown to be involved in an early step in
homologous recombination (HR) while RSC promotes HR at the stage of strand invasion. RSC is involved in sister chromatid cohesion and chromosome segregation.
SWI/SNF has an impact on alternative splicing as BRM has been shown to regulate the
crosstalk of RNA polymerase II (Pol II) with RNA processing enzymes by reducing the rate
of Pol II elongation to promote splicing of less than optimal splice sites . Telomeric
silencing and silencing transcription of rRNA genes by RNA polymerase II also requires yeast
Many identifications of structural domains have been made for the subunits of SWI/SNF that have been indicated to have either DNA or histone binding activity and could conceivably help the SWI/SNF complex have affinity to the nucleosome, for its efficient restructuring. (Figure 3).
The ATPase domain consists of seven subdomains that structurally forms two lobes referred to as the DEXD and helicase motifs that form a cleft to which DNA binds based on X-ray crystal structure from the related Rad54 ATPase domain.
In addition the Swi2/Snf2 protein contains at its Cterminus
a bromo domain which has been shown to recognize specific acetylated lysines in
Figure 4. Snf2 family of ATPases
(A) The Snf2 family belongs to the DEAD/H superfamily of ATPases (Lusser and Kadonaga, 2003). (B) Domain
structure of the four major classes of the Snf2-like ATPases, which are subunits of chromatin remodeling complexes
ISWI family of complexes
The ATPase catalytic subunit in these complexes is the ISWI protein ( table 2 ). The ATPase
subunit of this group of chromatin remodeling enzymes has been named Imitation SWItch
(ISWI) because of its similarity to the SWI2 ATPase in the SNF2 subfamily.
d NURF and d CHRAC were the first members to be identified in this group, through biochemical methods of purificationbased on ability to disrupt and / or generate regularly spaced nucleosomal arrays from Drosophilia extracts embryo extracts using an in vitro assay for activities allowing transcription factor access to sites in nucleosomal arrays . All of these complexes contain the nucleosome-dependent ATPase ISWI, which has homology withSwi2/Snf2 exclusively over the region of the ATPase domain.
Years later identification of other remodelers that were characterized in this group were found in yeast, humans, mouse, and Xenopus .
Another widely studied member of these ISWI containing complexes is ACF, which had been studied through extracts from Drosophila .
The three distinct complexes, NURF,ACF and CHRAC are diminutive and have fewer subunits than its SWI/SNF counterparts.
Figure 6. The ISWI family of chromatin remodeling complexes (Eberharter and Becker, 2004)
The different members of the ISWI group of chromatin remodeling factors in yeast, Drosophila and mouse / human
are illustrated. The color code is indicated in the box.
The nucleosome revamping antecedent of ~500kDa, namely the NURF, is a four auxiliary segment comprising of BPTF/NURF301, Nurf-38, Nurf-55 and ISWI.
The ascertainment of NURF was paramount in its establishment as a requisite for producing hsp70 heatshock promoter easily reachable in the subsistence of the GAGA transcription factor and was passively exhibited to impel the fushi tarazu gene.
Nurf-38, being thought to be the smallest component of the NURF complex, was identified as being inorganic pyrophosphatase.
The purification of the NURF lobular heterogeneous molecular machinery and the integration of Nurf-38 have inorganic pyrophosphatase activity but the restraining of this ability does not influence the aptitude of NURF in nucleosome mobilization.
The ATPase activity of this complex is specifically stimulated by nucleosomes and not DNA, in contrast to the SWI/SNF complex where DNA and nucleosomes equally stimulate the ATPase activity. NURF interacts with the histone H4 N-terminal tail and this interaction is essential for its ATPase and nucleosome mobilization activity .
Using alanine scanning mutagenesis, residues in the N-terminal tail of histone H4 were shown to be important for nucleosome mobilization by NURF. NURF has been shown to activate transcription in vitro and in vivo. NURF also appears to have a role in X chromosome morphology and steroid signaling during larval to pupal metamorphosis. Transcriptional activation by NURF is brought about by mobilizing nucleosomes along the DNA which requires the largest subunit of NURF, NURF 301. The direction of nucleosome mobilization is modulated by transcription factor Gal4.
In comparison to the SWI/SNF remodeling subunit where nucleosomes and DNA correspondingly activate the ATPase movement, the activity of this complex is especially fostered by nucleosomes .
The residues in the N-terminal fag end of histone H4 were exhibited to be vital for nucleosome translocation of NURF by utilizing scanning mutagenesis. NURF has also passively exhibited transcription in vitro and vivo. In the process of larval to pupal metamorphosis, NURF has emanated to have a part to portray in X chromosome morphology and steroid signaling. The remodeling of NURF is chaperoned by the movement of nucleosomes accompanying the DNA likewise depending upon the greatest sub-segment of NURF, NURF 301. The regulation of the transcription element Gal4 is brought about by the inclination of the nucleosome.
CHRAC , chromatin accessibility complex, has an approximately 670 kDa molecular mass and contains five subunits.
Being able to generate regular spacing in nucleosome arrays the two isoforms of CHRAC,
CHRAC-14 and CHRAC-16 were passively exhibited to be part of the augmentation of early Drosophila.
A multiple segment complex known as ACF or ATP utilizing chromatin factor that can methodically precipitate histone octamers accompanying the DNA to articulate sustained sporadic layers of nucleosomes is formed by ISWI in Drosophila. The histone chaperone NAP1 is necessitated by the ACF mediated chromatin assembly.
In the interim, Acf1 portrays a distinguished role in the augmentation as the Acf1 void mutants were instituted to perish during the transition from the larval to pupal stage. ACF/CHRAC is a dominant chromatin assembly protein in Drosophila as depicted in recent observations in experiments. By virtue of the deprivation of counteraction from chromatin, cells deprived of ACF/CHRAC expeditiously progress through S phase.
Identification of two ISWI genes - ISW1 and ISW2 , in the yeast S.cerevisiae based on their extensive sequence homology with dISWI, the ATPase domain in Drosophila was done in 1999.
Isw1p forms two distinct complexes inside the cell - ISW1a (contains Isw1p, Ioc3p) and ISW1b (Isw1p, Ioc2p and Ioc4p). ISW1a shows a strong nucleosome spacing activity while ISW1b does not. Isw2p was found to be associated with a 140 kDa protein referred to as Itc1p which appears to be partially related to the Acf1 protein sharing the structural domains WAC, WAKZ, PHD fingers, DDT and bromodomain motifs. ISW2 also has two additional smaller subunits Dpb4 and Dls1 that have histone fold domains and are homologs respectively of the hCHRAC 15/17 and the dCHRAC 14/16 histone fold of protein pairs from the human and Drosophila CHRAC
complexes, respectively. ISW2 has a nucleosome spacing activity that is not as tightly regulated as ISW1a and ISW2 has no detectable nucleosome disruption activity . These similarities suggest that ISW2 may be viewed as a yeast CHRAC homolog underscoring the extensive organizational and functional conservation of chromatin remodeling complexes from divergent species.
Within the cell, two explicit complexes, namely ISW1a which comprises of Isw1p and loc3p, and ISW1b which contains Isw1p, loc2p and loc4p are formed by Isw1p. The distinctiveness between the two are evident as ISW1a displays a fortified and durable nucleosome spacing activity while ISW1b does not do so. A 140 kDa protein that is accredited as Itc1p and that resembles moderately to correspond to the Acf1 protein and allotting the structural divisions WAC, WAKZ, PHD fingers, DDT and bromodomain motifs have been detected to be correlated to ISw2p. In addition to that, ISW2 has two supplementary diminutive sub-segments Dpb4 and Dls1 which have histone fold domains and are homologs correspondingly of the hCHRAC 15/17 and the dCHRAC 14/16 histone fold of protein pairs from the human and Drosophila CHRAC complexes respectively. Since ISW!a and ISW2 have no visible nucleosome disruptive activity, ISW2 has a nucleosome spacing activity which is not rigidly controlled. Therefore these semblances connote that ISW2 would probably be contemplated as a yeast CHRAC homolog accentuating the capacious standardizational and operative preservation of chromatin remodeling complexes from unorthodox species.
2.3 Different outcomes of nucleosome mobilization - differences in step sizes
Despite the fact that both SWI/SNF and ISWI displayed the modulation of the translational setting of the nucleosomes, they appeared to have no resemblance in their aptitude to discompose the nucleosomes. Through the use of a restriction endonuclease accessibility assay, the discrepancies are apparent.
Through the inception of DNA loops on the exterior, SWI/SNF has appeared to accomplish nucleosomal DNA which are susceptible to the apparent intersection of endonuclease. The amplified susceptibility of nucleosomal DNA induced by SWI/SNF remodeling transpires without transposing the absolute nucleosome from the intrinsic DNA site to a contemporary distal translational bearing whereby the layout is situated in the connected DNA site. Since the absolute nucleosome is propelled as distant as possible to position the DNA site into the connected DNA region, ISWI complexes emerge not to beget nucleosomal DNA that are attainable as a consequence of the course of re-modeling. These are the variances which are most presumably resonated in their various roles in the cell as nucleosomal DNA sites are predominantly conceived by SWI/SNF that are susceptible to either repressors or activators of transcription, and subsequently ISWI primarily emerges to be convoluted in actuating nucleosomes so as to constitute a repressive chromatin environment.
These complexes are suggestive of moving nucleosomes by utilizing a spiral encaptured
type of mechanism. So on the exterior of the nucleosome, both of them emerge to generate DNA bulges. Due to the variations in the disparations in the sizes of the DNA bulge implanted by these complexes, the contrasts in remodeling outcomes will most presumably be contemplated in the step size of the DNA that is transposing through the nucleosome.
Two different reports suggest that ISWI complexes have a small DNA step size of about 10 bp which would likely cause the formation of a small bulge on the surface of the nucleosome that would not be readily cleaved by DNA endonucleases. One study mapped the translational positioning before and during remodeling by NURF with hydroxyl radical footprinting and found that NURF moved the nucleosome in ten base pair steps. Hydroxyl radical footprinting shows all the regions that are protected by the nucleosome, but it was possible to tract the location of the dyad axis of the nucleosome because the dyad had a rather distinctive footprint pattern. The one difficulty in this study was that nucleosomes were reconstituted on a DNA that had a high affinity for the histone octamer and that preferentially positioned the nucleosome to a single translational position. The DNA would then likely constrain the nucleosome to be offset from its original position in 10 bp increments in order to maintain the preferred rotational phasing of the nucleosome. Thus the 10 bp increments observed in these studies may not reflect the intrinsic step size of remodellign complexes such as NURF, but rather the thermodynamically preferred positioning of the nucleosome on this particular DNA sequence.
Another approach to map the step size of another ISWI complex ( i.e ISW2) was to use a DNA
that did not bind the nucleosome as tightly. Second, the movement of the nucleosome
was rapidly tracked such that it was possible to observe nucleosome movement after hydrolysis
of a single ATP by ISW2. Fortuitously, the new nucleosome position seen under these rapid
conditions was not a position on the DNA that was thermodynamically preferred to be bound
by the nucleosome, thus helping to avoid the potential confusion of the observed nucleosome
movement being due to the intrinsic property of the DNA template rather than that of ISW2.
Reaction conditions were slowed by lowering the temperature and the ATP concentration such
that ISW2 hydrolyzed 0.52 ATP per second making it possible to examine the early events of
ISW2 remodeling. ISW2 moved nucleosomes 9 and 11 bp in the time it took to hydrolyze one
ATP. These movements were found not to be thermodynamically preferred and would slip a
few more bp farther from the original position to move nucleosomes a total of 14 and 16 bp.
There was no evidence for single bp movements by ISW2 which is often considered to be a
trademark of the twist diffusion model.
Similar experiments were done with SWI/SNF in which the reaction was slowed down so that
SWI/SNF hydrolyzed 0.36 ATP per second. Using the same DNA template as for the
ISW2 experiments, SWI/SNF was found to move nucleosomes 52 bp from their original
position with no other intermediates evident. The approach used to map nucleosome
mobilization by SWI/SNF and ISW2 monitored the DNA contact point of residue 53 of histone
H2B. The site-directed mapping showed that for SWI/SNF there were two steps, the first being the loss of the H2B contact with DNA and then shortly afterwards its reappearance with DNA at a distance of 52 bp from it prior position.
A common unifying characteristic of ISW and SWI/ SNF remodeling.
There are two proposed mechanisms of ATP dependant nucleosome sliding.
The (a) twist diffusion and the (b) loop bulge propagation.
Recent single-molecule experiments indicate that, like helicases, many of these complexes use ATP to translocate on DNA. Despite sharing this fundamental property, two key classes of remodeling complexes, the ISWI class and the SWI/SNF class, generate distinct remodeled products. SWI/SNF complexes generate nucleosomes with altered positions, nucleosomes with DNA loops and nucleosomes that are capable of exchanging histone dimers or octamers. In
contrast, ISWI complexes generate nucleosomes with altered positions but in standard structures. Here, we draw analogies to monomeric and dimeric helicases and propose that ISWI and SWI/SNF complexes catalyze different outcomes in part because some ISWI complexes function as dimers while SWI/ SNF complexes function as monomers.
(a) The twist diffusion was an early idea as to how the proteinacious histone core could have been shifted along the DNA. This model suggests that the DNAmobilizes in 1basepair twists toward the nucleosome. DNA mobilization here does not require the amendment of location or torque of the core nucleosome structure. The twist effect due to the torsional strain is passed along the surface of the nucleosome , ultimately dispersing the strain.
Another variation to this model is that even though there is a twist defect, this could be accommodated by the nucleosome without the disruption of the histone - DNA contacts. where the histone octomer would shift along the DNA the size of the distortion.
Nucleosome crystallographic studies have found that the nucleosome can readily accommodate overtwisted DNA on its surface. However, data not consistent with this model has already been mentioned of nucleosome movement occurring in increments much larger than 1 bp. This model would also not be consistent with the ISW2 data mentioned earlier as the 1 nt gaps that interfere with remodeling were only in a ∼20 bp region encompassing the
internal contact site and 10 nts to one side of this site. If the 1 bp wave was required to propagate from the internal translocation site to the entry/exit site of the nucleosome then 1 nt gaps anywhere between these sites spanning a range of ∼60 bps should interfere rather than the observed highly localized region.
Notably, ATPdependent nucleosome sliding is not inhibited by physical barriers such as DNA hairpins and biotin crosslinks that should prevent rotation of the DNA duplex during sliding. In addition, chromatin remodelers have been reported to slide nucleosomes in increments of approximately 10 bp steps, maintaining the rotational phasing of nucleosomal DNA. Thus, while twist diffusion remains an attractive mechanism for shifting nucleosomes along DNA in response to thermal fluctuations, chromatin remodelers likely utilize a different mechanism for ATP-dependent sliding of nucleosomes.
(b) The other approach, being known as the loop bulge model is another proposed mechanism for nucleosome sliding. In this model it is suggested that the DNA from a linker shifts onto a nucleosome, creating a region of DNA similar to a hairpin loop. This transcient shifting causes the looping of the DNA around the proteinacious histone core, emerging from the other end through wave like propagation, by breaching contacts infront and reforming the contacts in its rear end. This energetically expensive approach, causes significant disruption of the DNA-histone contacts, however there is no distortion of rotational phasing, unlike the twist diffusion, which would require DNA rotation by approximately 35° with each base pair step.
The loop propagation model seems to also have its variations.
The first proposed mechanism of action is (a)
A method in which DNA mobilization occurs is by the action of the ATPase motor where it employs its function as a DNA translocase, drawing in DNA in a constant manner from the closest entry site toward the central dyad , resulting in a overhang in the form of a bulge. ( 32,47,48) the bulge would then be removed by the propagation into the far off exit site.backing up of this model is the single molecule experiments of the larger remodeling complexes- RSC and SWI/SNF, in which a similar mode of action was observed, the loop formation.(48,49)
These studies, put toether with the single molecule studies at the SHL2 region led to the presumption that this loop formation is at the domain of the dyad. The precise locality with respect to the octomer histones, currently however still remain vague.(48)
Yet this still remains as the winning model as it links DNA mobilization directly to the DNA translocation function of the chromatin remodeling complexes. The notion that SWI/SNF holds on to its position, encompassing around SHL2 , while trying to move away from the central dyad gives a possible explanation for the, obstruction of nucleosome mobilization due to the DNA gaps at the SHL2 region, when these multilobular complexes displaces the histones fifty bases pairs from the DNA.(32,50,51)
In instances where constant transcription may be necessary, the ATPase does not dissociate from the DNA during nucleosomal mobilization.
Though seemingly perfect, this proposed model has its drawbacks. The constant movement of the remodeling complexes along the nucleosomal DNA , would end up with supercoiling of DNA due to the change in the torsion as well as modification of the degree of rotational phasing.
Bringing one back to the twist diffusion model, in which the DNA rotation is of insignificant value during the energy utilizing process of ATP hydrolysis for nucleosomal mobilization.(10,11,12)
(b) BULGE LOOP MODEL 2
In the other suggested model, the loop formation presumably occurs in the SHL2 domain, with assistance of ATPase, hauling in sections of linker DNA.(12,43). The ATPase in this model remains established on the linker DNA as formation of a loop takes place, moving toward the central dyad on the DNA.
The accumulation of energy in the nuclosomal DNA due to tortional strain results in the movement of the loop formation around the nucleosome. The most effective DNA-histone interactions as previously mentions cluster along the dyad, the generation and the later mobilization of this loop from the distal areas from the dyad , past the dyad is an energetically expensive process due to the continuous dissociation and reformation of contacts, especially at the SHL2 region of strong contacts.(13-15)
A hypothesis toward this proposed model is that the generation of the loop in close proximity to the dyad, so as to reduce the energetic cost involved. Other questions such as if the non ATPase capability of the nucleosome remodelers are put to use as part of the remodeling mechanism ,and if they are, their interaction with the ATpase domains are also yet to be experimentally clarified.(12,43)
(c) the other proposed model is that the outer wrap of the DNA is removed easily in comparision with the inner wrap.
This was shown through FRET and AFM experiments as well as DNA unzipping .
There was found to be differences in strength of the DNA histone contacts. They were found to be energetically different at different regions. The central dyad had the most significance in energetic with the DNA. About 50 -60 base pairs away from either side of the central dyad were 2 other DNA histone contacts of significant energetic, though lesser than that of the central dyad with the DNA, as found with the unzipping technique. An energetically weak contact was found to be in the SHL 2.5 regionwhich was identified to undergo bulging or stretching under the crystal structure (6-9), and is also in correspondence with the area where ATP remodelling complexes are active.
DNA polymerase was let to run along a template nucleosome , the unzipping of the DNA till the central dyad did not shift the the histone core on the DNA.(23) . this was also in unision with the theory that the nucleosome is not shifted with the displacement of the outer wrap of the DNA. (19)
(d) disturbances of the contacts between the DNA and the histone around the central dyad initiates mobilization of the nucleosome.
Certain mutations were thought to seemingly 'nullify's the loss of the repositioning factor of the nucleosome.(26,27)
The identification of a category of SIN mutations in yeast , that were SWI/SNF independent, were found to be accumulated where histones H3 and H4 contact DNA at SHL+/- 0.5
Here there is a tremendous difference in theory as compared to the proposed mechanism of the unwrapping of the outer segment of DNA, suggesting that even small disturbances and perturbances of these points of contact at SHL+/- 0.5 allows easily mobilization of the histone core on DNA.
Nucleosome sliding without wave-like propagations along DNA
Another proposed mechanism is that the DNA transiently shifts , all at one go along the nucleosome, with the proteinacious histones functioning as reference points. To reduce the torsional strain, the shift corresponds to revolution of the histones in the nucleosome.
Moreover the exterior of the histone , containing charged residues -mainly argentine and lysine exert electrostatic forces of attraction on the DNA ,moving it in a manner similar to a chain reaction after the initiation of the movement - where the minor grooves of the double helix move in the middle of the strong SHL contacts of the histone. Experimental evidence also points that each of the strand interact in an individualistic fashion to the histone octomer. (23)
As such the turning of the histone core may result in a likened contacts being formed between itself and the DNA. An illustration of which; the turning of the histone octomer approximately by eighteen degrees results in a DNA strand being positioned on the previous location of the counterpart strand on the histone core. Continual rotation thus is believed to move the DNA along the nucleosome.
ATPase motor of chromatin remodelers
As previously mentioned, chromatin remodelers use an ATPase motor for nuclocosome mobilization, as it was shown through DNA gap experiments which pointed that this motor interacted with DNA at interior regions of the nucleosome. (12,31,32,33) through the accumulation of tortional strain in DNA, upsetting DNA - histone contacts . This motor is thought to be active in region SHL2 as shown through DNA gap experiments and crosslinking.(34,35)
Past this area, SHL2, its is now still yet to be discovererd how the remodeling complexes have interaction with the nucleosomes.
SWI/SNF and RSC, being the larger category of the multilobular remodelers, possess a centric cavity in which the nucleosome is thought to bind. (35-37)
DNA foot printing experiments have shed light that the 12 subunit SWI/SNF remodeler has a reasonable amount of synergy with the nucleosome, these iinteractions in addition to the fact that the nucleosome is encompassed by this remodeling machinery.(35)
The Iswi complex, being part of smaller category of remodelers have minimal nucleosome contacts but of a greater extent of interactions with the linker DNA instead. (31, 38-40)
As such the magnitude of the linker DNA , in terms of length as well as the assemblage of the Iswi complex on both sides of the nucleosome is crucial for nucleosome mobilization and the positioning of the histones on DNA, by Iswi-type remodelers.(11, 40-42)
Yet another factor to consider for the remodeling of chromatin is HISTONE VARIANTS.
As previously mentioned, histones are able to undergo a number of modifications, both ATP dependant as well as other non ATP methods such as methylations, acetylation, ubiquitination, etc.
However in addition to these, they can undergo histone substitutions by a histone variant. These variants provide specialised functions to the nucleosomes as they have a varied structure due to difference in their amino acid sequence, especially in the N- terminus(Henikoff et al., 2004).
Large numbers of this histone variants are embellished with precise locations of the chromosome. An illustration of which is the centromeric chromatin, which contains thee histone H3 variant CENP-A (centromer protein A) (Palmer et al., 1991).
Where CENP-A is crucial for structural and functional purposes of the centromer (Ahmad and Henikoff, 2001).
The other variant of the normally found histone H2A is the MacroH2A, which has an additional 25 kDa of the C-terminas and is found mainly in the nucleosomes of inactive X chromosomes
(Costanzi and Pehrson, 1998).
In nucleosomes with macroH2A , it was found that there was interference with the DNA binding of the transcription factor NF-kappa B and hinders the the action of the Swi/Snf remodelers (Angelov et al., 2003).
Other variants of the H2A for example H2A.X and H2A.Z form specialized chromatin structures that
Causes a difference in the DNA repair mechanism, chromatin remodeling as well as gene silencing (Dhillon and Kamakaka, 2002; Santisteban et al., 2000).
These variants are made throughout the cell cycle and are deposited independent of DNA replication.
Where on the other hand, the normal of histones is has expression Synthesis phase and they are depositioned during DNA replication.
As such in nucleosomes containing such variants , there are possibilities in which they function with slightly or totally varied mechanism of action than that of the two proposed for nucleosome sliding due to the difference in the features of these studied histone variants.
2 . Differences in energetics
Experiments involving nucleosome arrangement were carried out, in which temperatures had been varied. With an increament of temperature, it was identified that the SIN mutant histone octomers shifted at ease, in comparison to wild type, on DNA . This experiment thus explained how mutations could not reposition themselves during stress through crystal structures it was found that most molecular interactions were rather intact, though there were other disparities in positional octomer stability on DNA.
The largest of the ATP dependant remodelers, the SWI/SNF complex has greater contact over much of the nucleosome , as in comparison with the smaller ISWI families which make less contacts with the nucleosome , although the number of interactions outside of the nucleosome core is much more noteworthy.
This may be suggestive that the functions SWI/SNF complex perhaps might be more energetically expensive as compared to the ISWI family, and unlike the proposed likelyhood of the mechanisms the ISWI family of remodelers might play a more important role in vivo.
3. A new chromatin remodeling mechanism has also been suggested.
RAD 51, is an important recombinase in the metaphase in meiosis , in replication fork rescue , as well as during stages of DNA repair.
This recombinase ia known to form a hhelical filament through strand exchange reaction through the process of nucleation and filament extension on single stranded DNA.
Nevertheless it was also made known that this recombinase RAD 51 was also capable of polymerizing on double stranded DNA. RAd 51 has similar inclination toward double as well as single stranded DNA.
It has enhanced the effect of yet another recombinase, RAD 54 loop formation of DNA.
S. cerevisae RAD 51 had been experimented on various templates of chromatin , and best results had been observed with the presence of RAD 54, in an ATP dependant process.
Dupaigne P, Lavelle C, Justome A, Lafosse S, Mirambeau G, et al. (2008) Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism. PLoS
ONE 3(11): e3643. doi:10.1371/journal.pone.0003643
Figure . Rad51 polymerization on circular nucleosomal template. Rad51 unwinds DNA and destabilizes entire nucleosome
arrays in a partially reversible fashion. (a) Chromatin was reconstituted on the supercoiled plasmid, giving an array of 30 to 35 nucleosomes. Inset: enlarge image of nucleosomes (zoom 2.56); (b,c) when Rad51 is added, 2 to 3 filaments are generally formed (from 2 to 3 nucleation sites, probably starting in the linker DNA between nucleosomes), stretching over several hundred bp on straight nucleosome-free DNA and pushing nucleosomes into 2 to 3 dense arrays. . (red and white arrows show Rad51 filament and nucleosome clusters, respectively) Inset in (c): enlarge image of nucleosomes compacted by Rad51 filament (zoom 1.56) ; (d,e) subsequent addition of EDTA to a high concentration destabilizes Rad51 filaments, allowing supercoiled nucleosome arrays to relax; (f) further treatment at 40uC for 20 minutes leads to spontaneous nucleosome sliding, making it possible to check for nucleosome loss during the partially reversible remodeling process. Inset: enlarge image of nucleosomes after thermal redistribution (zoom 2.56). The scale bars represent 100 nm for all
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