The effects of postural support padding modifications to child restraints for children with disability on crash protection.

OBJECTIVE: Many children with physical disabilities need additional postural support when sitting and supplementary padding is used on standards approved child restraints to achieve this when traveling in a motor vehicle. However, the effect of this padding on crash protection for a child is unknown. This study aimed to investigate the effect of additional padding for postural support on crash protection for child occupants in forward facing child restraints. METHODS: Forty frontal sled tests at 49 km/h were conducted to compare Q1 anthropometric test device (ATD) responses in a forward-facing restraint, with and without additional padding in locations to increase recline of the restraint, and/or support the head, trunk and pelvis. Three padding materials were tested: cloth toweling, soft foam, and expanded polystyrene (EPS). The influence of padding on head excursion, peak 3 ms head acceleration, HIC15, peak 3 ms chest acceleration and chest deflection were analyzed. RESULTS: The influence of padding varied depending on the location of use. Padding used under the restraint to increase the recline angle increased head injury metrics. Toweling in multiple locations which included behind the head increased head excursion and chest injury metrics. There was minimal effect on injury risk measures with additional padding to support the sides of the head or the pelvis position. Rigid EPS foam, as recommended in Australian standards and guidelines, had minimal effect on injury metrics when used inside the restraint, as did tightly rolled or folded toweling secured to the restraint at single locations around the body of the child. CONCLUSIONS: This study does not support the use of postural support padding to increase recline of a forward-facing restraint or padding behind the head. Recommendations in published standards and guidelines to not use foam that is spongy, soft or easily compressed, with preference for secured firm foam or short-term use of tightly rolled or folded toweling under the child restraint cover is supported. This study also highlights the importance of considering the whole context of child occupant protection when using additional padding, particularly the change in the child's seated position when adding padding in relation to the standard safety features of the restraint.

DNA-bridging by an archaeal histone variant via a unique tetramerisation interface.

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into 'hypernucleosome' particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea.

Storage of halved strawberry fruits affects aroma, phytochemical content and gene expression, and is affected by pre-harvest factors.

Introduction: Strawberry fruit are highly valued for their aroma which develops during ripening. However, they have a short shelf-life. Low temperature storage is routinely used to extend shelf-life for transport and storage in the supply chain, however cold storage can also affect fruit aroma. Some fruit continue to ripen during chilled storage; however, strawberries are a non-climacteric fruit and hence ripening postharvest is limited. Although most strawberry fruit is sold whole, halved fruit is also used in ready to eat fresh fruit salads which are of increasing consumer demand and pose additional challenges to fresh fruit storage. Methods: To better understand the effects of cold storage, volatilomic and transcriptomic analyses were applied to halved Fragaria x ananassa cv. Elsanta fruit stored at 4 or 8°C for up to 12 days over two growing seasons. Results and discussion: The volatile organic compound (VOC) profile differed between 4 or 8°C on most days of storage. Major differences were detected between the two different years of harvest indicating that aroma change at harvest and during storage is highly dependent on environmental factors during growth. The major component of the aroma profile in both years was esters. Over 3000 genes changed in expression over 5 days of storage at 8°C in transcriptome analysis. Overall, phenylpropanoid metabolism, which may also affect VOCs, and starch metabolism were the most significantly affected pathways. Genes involved in autophagy were also differentially expressed. Expression of genes from 43 different transcription factor (TF) families changed in expression: mostly they were down-regulated but NAC and WRKY family genes were mainly up-regulated. Given the high ester representation amongst VOCs, the down-regulation of an alcohol acyl transferase (AAT) during storage is significant. A total of 113 differentially expressed genes were co-regulated with the AAT gene, including seven TFs. These may be potential AAT regulators.

Mitigating fuel tank syndrome pelvic injuries - is there potential for rider worn protectors?

OBJECTIVE: The aim of this study was to investigate the feasibility of rider-worn pelvis protection for mitigating injury risk when contacting the motorcycle fuel tank in a crash. METHODS: A newly developed test apparatus was designed and constructed to simulate the interaction between a rider's pelvis and the motorcycle fuel tank in a frontal crash. Impacts were performed at a velocity of 18 km/h into four motorcycle fuel tanks. Further testing used a rigid fuel tank surrogate and the pelvis surrogate in an unprotected condition and with a series of impact protector prototypes. A subset of prototype samples was also tested at varying tank angles (30°, 37.5°, 45°) and impact speeds (8.5 km/h, 13 km/h, 18 km/h). Analysis of variance was used to determine whether the protector prototypes reduced pelvis response compared to unprotected. RESULTS: Resultant peak pelvis acceleration was reduced by three pelvis impact protector prototypes compared to an unprotected condition. The reduction in peak acceleration occurred without a significant change in the peak pelvis rotational velocity. The pattern of protector performance was consistent at varying fuel tank angles but only reduced the pelvis response at the highest impact speed tested of 18 km/h. CONCLUSIONS: The results indicate that there may be potential for using pelvis impact protection to mitigate injury risk by absorbing and/or distributing impact energy that would otherwise be transmitted to the rider's pelvis. However, due to the current paucity in understanding of pelvis biomechanics to anteroposterior loading, it is unknown whether the pelvis acceleration reductions achieved would prevent injury.

Head excursion in frontal impacts is lower in high back booster seats than in forward facing child seats with internal harnesses designed for children up to 8 years of age.

OBJECTIVE: It is often assumed that a child restraint with a five or six-point internal harness provides greater protection for children in frontal crashes than a booster seat with a lap-sash seat belt. However, most research comparing these restraint types has focused on protection for children aged up to approximately 3-4 years of age. Recently, harnessed child restraints for older children up to approximately 8 years of age have become available, but there is little data on their performance compared to booster seats for children over 4 years of age. This study aimed to compare frontal crash performance of a series of harnessed child restraints for children aged 4-8 years to booster seats. METHODS: Four large harnessed child restraints (Type G in the Australian Standard, AS/NZS 1754:2013) and six high back booster seats (Type E in AS/NZS 1754:2013) were tested in frontal impact on a deceleration sled. Head and pelvis accelerations were recorded and head excursions were measured from high speed video. RESULTS: Head excursion was an average of 92 mm greater in the large harnessed child restraints than the high back booster seats. The initial position of the head in Type G restraints, an average of 58 mm further forward compared to Type E boosters, was the main contributor to the larger head excursion during impact. Conversely, peak head accelerations in the impact phase were, on average, 37.2 g lower in the large harnessed child restraints than the high back booster seats. CONCLUSIONS: These data suggest that recommendations for harnessed restraints and booster seats for children aged 4-8 years is not as obvious as is sometimes assumed. Harnessed restraints allow greater head excursion in frontal impacts, potentially increasing the chances of head impacts, especially in vehicles with limited clearance between the restraint and the seat in front. The likelihood, and types of, incorrect use that occur in each restraint type, the vehicle occupant space, and the restraint's crash performance under ideal conditions should be considered in recommending restraints for these older children.

Comparative performance of rearward and forward-facing child restraint systems with common use errors: Effect on crash injury risk for a 1-year-old occupant.

OBJECTIVE: To compare how errors in child restraint use influence crash injury risk in rearward and forward-facing restraints for a 1-year old occupant. METHODS: Three convertible child restraint systems (CRS) were subjected to frontal dynamic sled tests at 56 km/h in rearward-facing and forward-facing modes in a correct use (baseline) condition and in five incorrect use conditions: loose securing belt, loose harness, partial harness use, top tether slack, and three minor errors. Excursion, head, and chest 3 ms resultant acceleration, HIC15, and neck forces and moments of a Q1 anthropomorphic test device (ATD) seated in the restraints were measured. The effect of incorrect use on each outcome and restraint type was analyzed. RESULTS: The influence of errors varied across different outcome variables, the three restraints tested and orientation modes. Excursion increased in four of five incorrect use conditions in both rearward and forward-facing orientations. A very loose harness increased four of five outcome variables in at least one forward-facing restraint, whereas only excursion was increased when rearward-facing. Overall, there tended to be a more negative effect of incorrect use (demonstrated through increases in outcome variables compared to the baseline) in the forward-facing orientation. CONCLUSIONS: Overall, errors in use tended to have a larger negative impact on forward-facing restraints than rearward-facing restraints. Given the widespread nature of errors in use, this adds further weight to arguments to keep children rearward-facing to 12 months of age and older. The results also highlight a variation in response to errors across differently designed restraints, suggesting the influence of errors may be minimized by restraint design that is more resistant to errors.

Dynamic frontal crash performance of old and used child restraint systems.

OBJECTIVE: To examine the effect of age on the dynamic performance of child restraint systems in frontal crashes. METHODS: A sample of used (3-269 months from manufacture) and newly purchased child restraints were subjected to frontal crash simulations of more than 56 km/h and peak deceleration approximately 33 g on a deceleration sled. Restraints were monitored for evidence of damage before and after each impact. Anthropometric test device (ATD) head and chest responses and peak head excursions were recorded for rearward facing restraints using the Q1 ATD and for forward facing restraints and booster seats using the Q6 ATD. The influence of restraint age on peak 3 ms head acceleration, HIC15, head excursion, peak 3 ms chest acceleration and restraint damage were analyzed. RESULTS: In all impacts, the ATD remained within the restraint and secured to the test bench demonstrating the crash protection offered by the old and used restraints. There was no apparent relationship between ATD responses and restraint age for any restraint type. Older forward facing restraint systems had a very modest increase in forward head excursion (R2 = 0.59, p = 0.001) of 0.27 mm for each month of age (95% CI, 0.13 mm - 0.42 mm). This equates to a 0.7% increase in the minimum measured excursion per year of restraint age. There was also a small increased likelihood of critical damage to the restraints in the simulated crashes per month of restraint age (OR 1.031, 95% CI 1.010-1.069). CONCLUSIONS: Overall, degradation in restraint dynamic performance in older restraints, including some that are much older than the currently recommended 10-year lifetime, is minimal. However newer restraints may provide better protection due to marginal improvements in restraint design over time. Furthermore, the results of this study confirm previous recommendations that restraints should not be re-used after crash involvement.

Frontal crash seat belt restraint effectiveness and comfort accessories used by older occupants.

Objective: Around a quarter of older occupants use some type of comfort or orthopedic aftermarket accessory on the vehicle seat while traveling in a vehicle. The aim of this study was to investigate the effect of comfort accessories on the performance of the seat belt restraint system in a frontal crash in terms of potential injury implications for older occupants.Methods: Eight frontal sled tests (43 km/h, 32 g) were carried out on a deceleration sled fitted with a three-point lap-sash seat belt and a front passenger seat from a common Australian passenger car for each test. A 5th percentile Hybrid III anthropometric test device (ATD) was positioned in the seat and measurements were recorded for head center of gravity acceleration, chest acceleration, neck forces and moments and sternal deflection. Tests were carried out in a baseline condition and with seven comfort accessories. Each comfort accessory was inserted between the ATD and vehicle seat as it is intended to be used, with the ATD otherwise positioned as close as possible to the baseline test position.Results: Initial distance between the seat belt anchor and ATD hip was associated with a statistically significant decrease in Head Injury Criterion and increase in sternal deflection. Submarining was related to the ATD torso recline angle and angle of the lap belt from the seat belt anchor.Conclusions: Accessories placed between the seat back and the lumbar region of an occupant have the potential to increase the risk of submarining due to a change in posture and should be avoided if such a change in posture when seated with an accessory is excessive. Sitting on seat cushions resulted in the greatest increase in seat belt anchor to hip distances and hence largest increase in sternal deflection. Given the fragility, frailty and particular importance of chest injuries among older vehicle occupants, further investigation is needed to determine whether these changes in ATD sternal deflection observed with seat cushion use results in injury threshold limits being exceeded and whether pretensioners and load limiters would ameliorate these effects without causing other negative changes in occupant response or kinematics.

Cross-chest clips in child restraints: A crash testing study.

Objective: Cross-chest clips are widely used in North American child restraints but are less common in other countries, partially due to concerns over anterior neck contacts in frontal crashes. They have recently been reported to be associated with lower odds of injury in real-world crashes, but there is a paucity of crash test performance information. This study aimed to compare the dynamic performance of a small child occupant in frontal crash tests with and without cross-chest clips in place. Methods: Frontal sled tests at 49 km/h were conducted to compare 2 cross-chest clip designs to nonuse of a chest clip. Tests using a P3/4 anthropomorphic test device (ATD) to represent the smallest occupant in a forward-facing child restraint were conducted with the chest clips in the recommended position and also in an incorrect lower position and with and without additional harness slack present. Results: Though contacts were observed between the chest clips and the base of the ATD's neck, there was little difference observed in head excursion or ATD sensor loads in the presence of the chest clips. No detectable change in the neck forces or moments was detected at the time of the neck contacts. The position of the clips did not affect the results. Harness slack increased head excursion, as expected, but this effect did not differ between the tests with and without the clips. Conclusions: Cross-chest clips do not appear to greatly influence the dynamic performance of a forward-facing child restraint in a simulated frontal crash. Taken together with recent research suggesting a potential benefit in injury reduction from the clips in the real world, possibly due to maintaining the harness straps in place on a child's shoulders, it may be appropriate to re-evaluate safety standards that prevent their use.

Nucleosome dynamics of human iPSC during neural differentiation.

Nucleosome positioning is important for neurodevelopment, and genes mediating chromatin remodelling are strongly associated with human neurodevelopmental disorders. To investigate changes in nucleosome positioning during neural differentiation, we generate genome-wide nucleosome maps from an undifferentiated human-induced pluripotent stem cell (hiPSC) line and after its differentiation to the neural progenitor cell (NPC) stage. We find that nearly 3% of nucleosomes are highly positioned in NPC, but significantly, there are eightfold fewer positioned nucleosomes in pluripotent cells, indicating increased positioning during cell differentiation. Positioned nucleosomes do not strongly correlate with active chromatin marks or gene transcription. Unexpectedly, we find a small population of nucleosomes that occupy similar positions in pluripotent and neural progenitor cells and are found at binding sites of the key gene regulators NRSF/REST and CTCF Remarkably, the presence of these nucleosomes appears to be independent of the associated regulatory complexes. Together, these results present a scenario in human cells, where positioned nucleosomes are sparse and dynamic, but may act to alter gene expression at a distance via the structural conformation at sites of chromatin regulation.

Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis.

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5'UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes.

Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium.

Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states.

Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organisation.

Maintenance of the correct level and organisation of nucleosomes is crucial for genome function. Here, we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in the maintenance of nucleosome architecture in fission yeast. Cells lacking abo1(+) experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide.

The impact of the HIRA histone chaperone upon global nucleosome architecture.

HIRA is an evolutionarily conserved histone chaperone that mediates replication-independent nucleosome assembly and is important for a variety of processes such as cell cycle progression, development, and senescence. Here we have used a chromatin sequencing approach to determine the genome-wide contribution of HIRA to nucleosome organization in Schizosaccharomyces pombe. Cells lacking HIRA experience a global reduction in nucleosome occupancy at gene sequences, consistent with the proposed role for HIRA in chromatin reassembly behind elongating RNA polymerase II. In addition, we find that at its target promoters, HIRA commonly maintains the full occupancy of the -1 nucleosome. HIRA does not affect global chromatin structure at replication origins or in rDNA repeats but is required for nucleosome occupancy in silent regions of the genome. Nucleosome organization associated with the heterochromatic (dg-dh) repeats located at the centromere is perturbed by loss of HIRA function and furthermore HIRA is required for normal nucleosome occupancy at Tf2 LTR retrotransposons. Overall, our data indicate that HIRA plays an important role in maintaining nucleosome architecture at both euchromatic and heterochromatic loci.

An alternative beads-on-a-string chromatin architecture in Thermococcus kodakarensis.

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone-like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle-positioning code and are excluded from gene-regulatory DNA suggesting a functional organization. Beads-on-a-string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone-fold proteins in a variety of multimeric forms.

The INO80 chromatin remodeling complex prevents polyploidy and maintains normal chromatin structure at centromeres.

The INO80 chromatin remodeling complex functions in transcriptional regulation, DNA repair, and replication. Here we uncover a novel role for INO80 in regulating chromosome segregation. First, we show that the conserved Ies6 subunit is critical for INO80 function in vivo. Strikingly, we found that loss of either Ies6 or the Ino80 catalytic subunit results in rapid increase in ploidy. One route to polyploidy is through chromosome missegregation due to aberrant centromere structure, and we found that loss of either Ies6 or Ino80 leads to defective chromosome segregation. Importantly, we show that chromatin structure flanking centromeres is altered in cells lacking these subunits and that these alterations occur not in the Cse4-containing centromeric nucleosome, but in pericentric chromatin. We provide evidence that these effects are mediated through misincorporation of H2A.Z, and these findings indicate that H2A.Z-containing pericentric chromatin, as in higher eukaryotes with regional centromeres, is important for centromere function in budding yeast. These data reveal an important additional mechanism by which INO80 maintains genome stability.

SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae.

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

The two different isoforms of the RSC chromatin remodeling complex play distinct roles in DNA damage responses.

The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH domains.

Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing.

Microarray and next-generation sequencing techniques which allow whole genome analysis of chromatin structure and sequence-specific protein binding are revolutionizing our view of chromosome architecture and function. However, many current methods in this field rely on biochemical purification of highly specific fractions of DNA prepared from chromatin digested with either micrococcal nuclease or DNaseI and are restricted in the parameters they can measure. Here, we show that a broad size-range of genomic DNA species, produced by partial micrococcal nuclease digestion of chromatin, can be sequenced using paired-end mode next-generation technology. The paired sequence reads, rather than DNA molecules, can then be size-selected and mapped as particle classes to the target genome. Using budding yeast as a model, we show that this approach reveals position and structural information for a spectrum of nuclease resistant complexes ranging from transcription factor-bound DNA elements up to mono- and poly-nucleosomes. We illustrate the utility of this approach in visualizing the MNase digestion landscape of protein-coding gene transcriptional start sites, and demonstrate a comparative analysis which probes the function of the chromatin-remodelling transcription factor Cbf1p.

Repressive and non-repressive chromatin at native telomeres in Saccharomyces cerevisiae.

BACKGROUND: In Saccharomyces cerevisiae genes that are located close to a telomere can become transcriptionally repressed by an epigenetic process known as telomere position effect. There is large variation in the level of the telomere position effect among telomeres, with many native ends exhibiting little repression. RESULTS: Chromatin analysis, using microccocal nuclease and indirect end labelling, reveals distinct patterns for ends with different silencing states. Differences were observed in the promoter accessibility of a subtelomeric reporter gene and a characteristic array of phased nucleosomes was observed on the centromere proximal side of core X at a repressive end. The silent information regulator proteins 2 - 4, the yKu heterodimer and the subtelomeric core X element are all required for the maintenance of the chromatin structure of repressive ends. However, gene deletions of particular histone modification proteins can eliminate the silencing without the disruption of this chromatin structure. CONCLUSION: Our data identifies chromatin features that correlate with the silencing state and indicate that an array of phased nucleosomes is not sufficient for full repression.