PBAF loss leads to DNA damage-induced inflammatory signaling through defective G2/M checkpoint maintenance.

The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in ∼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer.

Repression of Transcription at DNA Breaks Requires Cohesin throughout Interphase and Prevents Genome Instability.

Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer.

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended ß-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin.

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity.

Influence of a Pre-throwing Protocol on Range of Motion and Strength in Baseball Athletes.

Though chronic overhand throwing is known to result in range of motion and strength adaptations, there is limited research regarding interventions for maintenance of these characteristics. Therefore, the purposes of this study were to explore the use of a pre-throwing protocol focusing on lumbopelvic-hip complex musculature in high school baseball players and examine its effects on shoulder and hip range of motion and isometric strength over the course of a season. Four exercises were added to the intervention team's warm-up routine. The control and intervention teams' passive bilateral hip and shoulder range of motion and isometric strength were assessed pre/post-season. A multivariate analysis of variance revealed significant differences in delta scores between the teams for multiple shoulder and hip range of motion and isometric strength variables. Key results were the intervention team lost significantly more stride hip external rotation but gained more isometric strength than the control team. The intervention team was also able to better maintain shoulder range of motion than the control team. Practitioners should use the results of this study and consider incorporating exercises that target lumbopelvic-hip complex musculature into their current training program.

Biceps Tendon Changes and Pitching Mechanics in Youth Softball Pitchers.

With the lack of pitch count regulation, youth softball pitchers are experiencing unremitting high stresses on the anterior shoulder. The purpose of this study was to examine the association of acute changes in the long head of the biceps tendon with pitching kinematics and kinetics in youth softball pitchers following an acute bout of pitching. Twenty-three softball pitchers (12.17±1.50 yrs.; 160.32±9.41 cm; 60.40±15.97 kg) participated. To investigate the association between biceps tendon changes and kinematic and kinetic changes from pre- to post-simulated game, each biceps tendon measure was split into those whose biceps tendon thickness, width, and/or area increased pre- to post-simulated game, and those whose did not. There were significant differences in biceps tendon longitudinal thickness (Z=- 2.739, p=0.006) and pitch speed; as well as a difference between groups in biceps tendon transverse thickness and the amount of change in trunk rotation at the start of the pitching motion (p=0.017) and the amount of change in trunk flexion at ball release (p=0.030). This study illustrates the association of trunk and lower extremity kinematics and shoulder kinetics with morphologic changes in the biceps tendon with an acute bout of windmill softball pitching.

Predicting synthetic lethal interactions using conserved patterns in protein interaction networks.

In response to a need for improved treatments, a number of promising novel targeted cancer therapies are being developed that exploit human synthetic lethal interactions. This is facilitating personalised medicine strategies in cancers where specific tumour suppressors have become inactivated. Mainly due to the constraints of the experimental procedures, relatively few human synthetic lethal interactions have been identified. Here we describe SLant (Synthetic Lethal analysis via Network topology), a computational systems approach to predicting human synthetic lethal interactions that works by identifying and exploiting conserved patterns in protein interaction network topology both within and across species. SLant out-performs previous attempts to classify human SSL interactions and experimental validation of the models predictions suggests it may provide useful guidance for future SSL screenings and ultimately aid targeted cancer therapy development.

Glenohumeral and Hip Range of Motion in Youth Softball Athletes.

The purpose of this study was to compare measures of bilateral shoulder and hip range of motion (ROM) between youth softball pitchers and position players. Fifty-two youth softball athletes (12.7±2.1 yrs.; 160.28±10.98 cm; 59.31±15.07 kg) participated. Bilateral hip and shoulder ROM were measured among pitchers (n=29) and position players (n=23). A 2 (pitcher/position player) × 2 (dominant/non-dominant) ANOVA was performed for both internal and external rotation ROM for the shoulder and hip. Paired sample t-tests were also conducted to examine side-to-side differences in total ROM. Data revealed a significant interaction between position and side dominance. Specifically, position players have significantly more non-dominant shoulder external rotation ROM (106.92°± 9.14°) than pitchers (100.12°± 11.21°). There were no significant differences between throwing and non-throwing sides. It is important that coaches and clinicians are aware of these adaptations between overhand and underhand throwing in order to properly develop conditioning and rehabilitation programs to alleviate injury susceptibility and cater to position-specific demands. Additionally, the current data suggest many of the functional adaptations seen within older populations are not fully developed until after youth.

Effects of a Simulated Game on Pitching Kinematics in Youth Softball Pitcher.

Despite evidence that overuse is the most common mechanism of injury, softball pitchers currently have no pitch count regulations. Pain has been associated with certain pitching pathomechanics, and some reports indicate increased pain following a single pitching bout. Therefore, the purpose of this study was to examine trunk and lower extremity kinematics during the first and last inning of a game, as well as last inning pitch volume in youth softball pitchers. Thirty-two youth softball pitchers (12.4±1.6 years, 159.4±8.9 cm, 62.0±13.6 kg) pitched a simulated game. Three fastballs were averaged and analyzed from the first and last inning. Kinematic data were collected at 100Hz using an electromagnetic system, synced with motion analysis software. A Wilcoxon signed rank test revealed pitchers exhibited less trunk rotation toward their pitching arm side in the last inning. A bivariate Pearson's correlation showed volume of pitches was correlated with stride length (r=0.367, p=0.039) and center of mass (r=0.364, p=0.041) at the start of the pitch, and trunk flexion at top of pitch (r=-0.392, p=0.026), foot contact (r=-0.413, p=0.019), and follow-through (r=-0.436, p=0.013). This study found that pitching a simulated game did result in altered pitching mechanics, meanwhile pitch volume was also correlated with pitching mechanics.

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.

Lumbopelvic-Hip Complex and Scapular Stabilizing Muscle Activations During Full-Body Exercises With and Without Resistance Bands.

Wasserberger, KW, Downs, JL, Barfield, JW, Williams, TK, and Oliver, GD. Lumbopelvic-hip complex and scapular stabilizing muscle activations during full-body exercises with and without resistance bands. J Strength Cond Res 34(10): 2840-2848, 2020-Inefficient sequencing in the kinetic chain has been linked to decreased performance and increased injury risk. Resistance band usage is very common in baseball/softball due to accessibility and low cost. However, resistance band use has primarily focused on the rotator cuff and surrounding shoulder musculature, thereby ignoring the rest of the kinetic chain. Currently, it is unclear whether resistance bands are effective tools for training musculature outside the throwing extremity. Thus, the purpose of this study was to examine muscle activations of the lumbopelvic-hip complex (LPHC) and scapular stabilizing musculature during 4 full-body exercises with and without the use of a resistance band. Twenty healthy, active individuals (174.39 ± 1.58 cm; 74.10 ± 1.75 kg; 21.85 ± 1.13 years) participated. Data were collected through surface electromyography for the LPHC and scapular stabilizing musculature during 4 full-body exercises with and without a resistance band. Statistical analyses were used to determine significant differences in activation levels between exercises and within exercises between conditions. Data showed a significant difference on muscle activation based on the interaction between resistance band usage and exercise choice (Λ = 0.276, F24, 136.92 = 3.19, p < 0.001). Adding a resistance band was effective in increasing muscle activation within the scapular stabilizing musculature. Conversely, the use of a resistance band was not as effective in increasing the activation of LPHC musculature. Future studies should investigate exercises that increase LPHC musculature activation. Coaches and clinicians can use these results to make more informed decisions when prescribing exercises to athletes for performance enhancement and rehabilitation.

Epigenetic changes in histone acetylation underpin resistance to the topoisomerase I inhibitor irinotecan.

The topoisomerase I (TOP1) inhibitor irinotecan triggers cell death by trapping TOP1 on DNA, generating cytotoxic protein-linked DNA breaks (PDBs). Despite its wide application in a variety of solid tumors, the mechanisms of cancer cell resistance to irinotecan remains poorly understood. Here, we generated colorectal cancer (CRC) cell models for irinotecan resistance and report that resistance is neither due to downregulation of the main cellular target of irinotecan TOP1 nor upregulation of the key TOP1 PDB repair factor TDP1. Instead, the faster repair of PDBs underlies resistance, which is associated with perturbed histone H4K16 acetylation. Subsequent treatment of irinotecan-resistant, but not parental, CRC cells with histone deacetylase (HDAC) inhibitors can effectively overcome resistance. Immunohistochemical analyses of CRC tissues further corroborate the importance of histone H4K16 acetylation in CRC. Finally, the resistant clones exhibit cross-resistance with oxaliplatin but not with ionising radiation or 5-fluoruracil, suggesting that the latter two could be employed following loss of irinotecan response. These findings identify perturbed chromatin acetylation in irinotecan resistance and establish HDAC inhibitors as potential therapeutic means to overcome resistance.

Defining Signatures of Arm-Wise Copy Number Change and Their Associated Drivers in Kidney Cancers.

Using pan-cancer data from The Cancer Genome Atlas (TCGA), we investigated how patterns in copy number alterations in cancer cells vary both by tissue type and as a function of genetic alteration. We find that patterns in both chromosomal ploidy and individual arm copy number are dependent on tumour type. We highlight for example, the significant losses in chromosome arm 3p and the gain of ploidy in 5q in kidney clear cell renal cell carcinoma tissue samples. We find that specific gene mutations are associated with genome-wide copy number changes. Using signatures derived from non-negative factorisation, we also find gene mutations that are associated with particular patterns of ploidy change. Finally, utilising a set of machine learning classifiers, we successfully predicted the presence of mutated genes in a sample using arm-wise copy number patterns as features. This demonstrates that mutations in specific genes are correlated and may lead to specific patterns of ploidy loss and gain across chromosome arms. Using these same classifiers, we highlight which arms are most predictive of commonly mutated genes in kidney renal clear cell carcinoma (KIRC).

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

Removal of H2A.Z by INO80 promotes homologous recombination.

The mammalian INO80 remodelling complex facilitates homologous recombination (HR), but the mechanism by which it does this is unclear. Budding yeast INO80 can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers. H2A.Z is actively incorporated at sites of damage in mammalian cells, raising the possibility that H2A.Z may need to be subsequently removed for resolution of repair. Here, we show that H2A.Z in human cells is indeed rapidly removed from chromatin flanking DNA damage by INO80. We also report that the histone chaperone ANP32E, which is implicated in removing H2AZ from chromatin, similarly promotes HR and appears to work on the same pathway as INO80 in these assays. Importantly, we demonstrate that the HR defect in cells depleted of INO80 or ANP32E can be rescued by H2A.Z co-depletion, suggesting that H2A.Z removal from chromatin is the primary function of INO80 and ANP32E in promoting homologous recombination.

Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases.

Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.

Roles of chromatin remodellers in DNA double strand break repair.

Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.

The BAH domain of Rsc2 is a histone H3 binding domain.

Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction.

A role for chromatin remodellers in replication of damaged DNA.

In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells.

Defects in DNA damage responses in SWI/SNF mutant cells and their impact on immune responses.

The mammalian SWI/SNF chromatin remodelling complexes are commonly dysregulated in cancer. These complexes contribute to maintaining genome stability through a variety of pathways. Recent research has highlighted an important interplay between genome instability and immune signalling, and evidence suggests that this interplay can modulate the response to immunotherapy. Here, we review emerging studies where direct evidence of this relationship has been uncovered in SWI/SNF deficient cells. We also highlight genome maintenance activities of SWI/SNF that could potentially shape immune responses and discuss potential therapeutic implications.