Chromatin movement in the maintenance of genome stability.

Mechanistic analyses based on improved imaging techniques have begun to explore the biological implications of chromatin movement within the nucleus. Studies in both prokaryotes and eukaryotes have shed light on what regulates the mobility of DNA over long distances. Interestingly, in eukaryotes, genomic loci increase their movement in response to double-strand break induction. Break mobility, in turn, correlates with the efficiency of repair by homologous recombination. We review here the source and regulation of DNA mobility and discuss how it can both contribute to and jeopardize genome stability.

Expanded CAG/CTG repeats resist gene silencing mediated by targeted epigenome editing.

Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

SWR1 and INO80 chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice.

Persistent DNA double-strand breaks (DSBs) are recruited to the nuclear periphery in budding yeast. Both the Nup84 pore subcomplex and Mps3, an inner nuclear membrane (INM) SUN domain protein, have been implicated in DSB binding. It was unclear what, if anything, distinguishes the two potential sites of repair. Here, we characterize and distinguish the two binding sites. First, DSB-pore interaction occurs independently of cell-cycle phase and requires neither the chromatin remodeler INO80 nor recombinase Rad51 activity. In contrast, Mps3 binding is S and G2 phase specific and requires both factors. SWR1-dependent incorporation of Htz1 (H2A.Z) is necessary for break relocation to either site in both G1- and S-phase cells. Importantly, functional assays indicate that mutations in the two sites have additive repair defects, arguing that the two perinuclear anchorage sites define distinct survival pathways.

Checkpoint kinases and the INO80 nucleosome remodeling complex enhance global chromatin mobility in response to DNA damage.

Double-strand break repair by recombination requires a homology search. In yeast, induced breaks move significantly more than undamaged loci. To examine whether DNA damage provokes an increase in chromatin mobility generally, we tracked undamaged loci under DNA-damaging conditions. We found that the yeast checkpoint factors Mec1, Rad9, and Rad53 are required for genome-wide increases in chromatin mobility, but not the repair protein Rad51. Mec1 activation by targeted Ddc1/Ddc2 enhances chromatin mobility even in the absence of damage. Finally, the INO80 chromatin remodeler is shown to act downstream from Mec1 to increase chromatin mobility, highlighting an additional damage-related role of this nucleosome remodeling complex.

Targeted INO80 enhances subnuclear chromatin movement and ectopic homologous recombination.

Chromatin in the interphase nucleus moves in a constrained random walk. Despite extensive study, the molecular causes of such movement and its impact on DNA-based reactions are unclear. Using high-precision live fluorescence microscopy in budding yeast, we quantified the movement of tagged chromosomal loci to which transcriptional activators or nucleosome remodeling complexes were targeted. We found that local binding of the transcriptional activator VP16, but not of the Gal4 acidic domain, enhances chromatin mobility. The increase in movement did not correlate strictly with RNA polymerase II (PolII) elongation, but could be phenocopied by targeting the INO80 remodeler to the locus. Enhanced chromatin mobility required Ino80's ATPase activity. Consistently, the INO80-dependent remodeling of nucleosomes upon transcriptional activation of the endogenous PHO5 promoter enhanced chromatin movement locally. Finally, increased mobility at a double-strand break was also shown to depend in part on the INO80 complex. This correlated with increased rates of spontaneous gene conversion. We propose that local chromatin remodeling and nucleosome eviction increase large-scale chromatin movements by enhancing the flexibility of the chromatin fiber.

Tissue specificity in DNA repair: lessons from trinucleotide repeat instability.

DNA must constantly be repaired to maintain genome stability. Although it is clear that DNA repair reactions depend on cell type and developmental stage, we know surprisingly little about the mechanisms that underlie this tissue specificity. This is due, in part, to the lack of adequate study systems. This review discusses recent progress toward understanding the mechanism leading to varying rates of instability at expanded trinucleotide repeats (TNRs) in different tissues. Although they are not DNA lesions, TNRs are hotspots for genome instability because normal DNA repair activities cause changes in repeat length. The rates of expansions and contractions are readily detectable and depend on cell identity, making TNR instability a particularly convenient model system. A better understanding of this type of genome instability will provide a foundation for studying tissue-specific DNA repair more generally, which has implications in cancer and other diseases caused by mutations in the caretakers of the genome.

Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability.

Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.

Cohesin and the nucleolus constrain the mobility of spontaneous repair foci.

The regulation of chromatin mobility in response to DNA damage is important for homologous recombination in yeast. Anchorage reduces rates of recombination, whereas increased chromatin mobility correlates with more efficient homology search. Here we tracked the mobility and localization of spontaneous S-phase lesions bound by Rad52, and find that these foci have reduced movement, unlike enzymatically induced double-strand breaks. Moreover, spontaneous repair foci are positioned in the nuclear core, abutting the nucleolus. We show that cohesin and nucleolar integrity constrain the mobility of these foci, consistent with the notion that spontaneous, S-phase damage is preferentially repaired from the sister chromatid.

Developing small Cas9 hybrids using molecular modeling.

The contraction of CAG/CTG repeats is an attractive approach to correct the mutation that causes at least 15 neuromuscular and neurodegenerative diseases, including Huntington's disease and Myotonic Dystrophy type 1. Contractions can be achieved in vivo using the Cas9 D10A nickase from Streptococcus pyogenes (SpCas9) using a single guide RNA (sgRNA) against the repeat tract. One hurdle on the path to the clinic is that SpCas9 is too large to be packaged together with its sgRNA into a single adeno-associated virus. Here we aimed to circumvent this problem using the smaller Cas9 orthologue, SlugCas9, and the Cas9 ancestor OgeuIscB. We found them to be ineffective in inducing contractions, despite their advertised PAM sequences being compatible with CAG/CTG repeats. Thus, we further developed smaller Cas9 hybrids, made of the PAM interacting domain of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues. We also designed the cognate sgRNA hybrids using molecular dynamic simulations and binding energy calculations. We found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit their target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing larger scale complexes of Cas9/sgRNA hybrids.

Xpa deficiency reduces CAG trinucleotide repeat instability in neuronal tissues in a mouse model of SCA1.

Expansion of trinucleotide repeats (TNRs) is responsible for a number of human neurodegenerative disorders. The molecular mechanisms that underlie TNR instability in humans are not clear. Based on results from model systems, several mechanisms for instability have been proposed, all of which focus on the ability of TNRs to form alternative structures during normal DNA transactions, including replication, DNA repair and transcription. These abnormal structures are thought to trigger changes in TNR length. We have previously shown that transcription-induced TNR instability in cultured human cells depends on several genes known to be involved in transcription-coupled nucleotide excision repair (NER). We hypothesized that NER normally functions to destabilize expanded TNRs. To test this hypothesis, we bred an Xpa null allele, which eliminates NER, into the TNR mouse model for spinocerebellar ataxia type 1 (SCA1), which carries an expanded CAG repeat tract at the endogenous mouse Sca1 locus. We find that Xpa deficiency does not substantially affect TNR instability in either the male or female germline; however, it dramatically reduces CAG repeat instability in neuronal tissues-striatum, hippocampus and cerebral cortex-but does not alter CAG instability in kidney or liver. The tissue-specific effect of Xpa deficiency represents a novel finding; it suggests that tissue-to-tissue variation in CAG repeat instability arises, in part, by different underlying mechanisms. These results validate our original findings in cultured human cells and suggest that transcription may induce NER-dependent TNR instability in neuronal tissues in humans.

Quality of Narratives in Assessment: Piloting a List of Evidence-Based Quality Indicators.

Background & Need for Innovation: Appraising the quality of narratives used in assessment is challenging for educators and administrators. Although some quality indicators for writing narratives exist in the literature, they remain context specific and not always sufficiently operational to be easily used. Creating a tool that gathers applicable quality indicators and ensuring its standardized use would equip assessors to appraise the quality of narratives. Steps taken for Development and Implementation of innovation: We used DeVellis' framework to develop a checklist of evidence-informed indicators for quality narratives. Two team members independently piloted the checklist using four series of narratives coming from three different sources. After each series, team members documented their agreement and achieved a consensus. We calculated frequencies of occurrence for each quality indicator as well as the interrater agreement to assess the standardized application of the checklist. Outcomes of Innovation: We identified seven quality indicators and applied them on narratives. Frequencies of quality indicators ranged from 0% to 100%. Interrater agreement ranged from 88.7% to 100% for the four series. Critical Reflection: Although we were able to achieve a standardized application of a list of quality indicators for narratives used in health sciences education, it does not exclude the fact that users would need training to be able to write good quality narratives. We also noted that some quality indicators were less frequent than others and we suggested a few reflections on this.

Assessment Practices in Continuing Professional Development Activities in Health Professions: A Scoping Review.

CONTEXT: In continuing professional development (CPD), educators face the need to develop and implement innovative assessment strategies to adhere to accreditation standards and support lifelong learning. However, little is known about the development and validation of these assessment practices. We aimed to document the breadth and depth of what is known about the development and implementation of assessment practices within CPD activities. METHODS: We conducted a scoping review using the framework proposed by Arksey and O'Malley (2005) and updated in 2020. We examined five databases and identified 1733 abstracts. Two team members screened titles and abstracts for inclusion/exclusion. After data extraction, we conducted a descriptive analysis of quantitative data and a thematic analysis of qualitative data. RESULTS: A total of 130 studies were retained for the full review. Most reported assessments are written assessments (n = 100), such as multiple-choice items (n = 79). In 99 studies, authors developed an assessment for research purpose rather than for the CPD activity itself. The assessment validation process was detailed in 105 articles. In most cases, the authors examined the content with experts (n = 57) or pilot-tested the assessment (n = 50). We identified three themes: 1-satisfaction with assessment choices; 2-difficulties experienced during the administration of the assessment; and 3-complexity of the validation process. CONCLUSION: Building on the adage "assessment drives learning," it is imperative that the CPD practices contribute to the intended learning and limit the unintended negative consequences of assessment. Our results suggest that validation processes must be considered and adapted within CPD contexts.

Instability and chromatin structure of expanded trinucleotide repeats.

Trinucleotide repeat expansion underlies at least 17 neurological diseases. In affected individuals, the expanded locus is characterized by dramatic changes in chromatin structure and in repeat tract length. Interestingly, recent studies show that several chromatin modifiers, including a histone acetyltransferase, a DNA methyltransferase and the chromatin insulator CTCF can modulate repeat instability. Here, we propose that the unusual chromatin structure of expanded repeats directly impacts their instability. We discuss several potential models for how this might occur, including a role for DNA repair-dependent epigenetic reprogramming in increasing repeat instability, and the capacity of epigenetic marks to alter sense and antisense transcription, thereby affecting repeat instability.

Written-Based Progress Testing: A Scoping Review.

PURPOSE: Progress testing is an increasingly popular form of assessment in which a comprehensive test is administered to learners repeatedly over time. To inform potential users, this scoping review aimed to document barriers, facilitators, and potential outcomes of the use of written progress tests in higher education. METHOD: The authors followed Arksey and O'Malley's scoping review methodology to identify and summarize the literature on progress testing. They searched 6 databases (Academic Search Complete, CINAHL, ERIC, Education Source, MEDLINE, and PsycINFO) on 2 occasions (May 22, 2018, and April 21, 2020) and included articles written in English or French and pertaining to written progress tests in higher education. Two authors screened articles for the inclusion criteria (90% agreement), then data extraction was performed by pairs of authors. Using a snowball approach, the authors also screened additional articles identified from the included reference lists. They completed a thematic analysis through an iterative process. RESULTS: A total of 104 articles were included. The majority of progress tests used a multiple-choice and/or true-or-false question format (95, 91.3%) and were administered 4 times a year (38, 36.5%). The most documented source of validity evidence was internal consistency (38, 36.5%). Four major themes were identified: (1) barriers and challenges to the implementation of progress testing (e.g., need for additional resources); (2) established collaboration as a facilitator of progress testing implementation; (3) factors that increase the acceptance of progress testing (e.g., formative use); and (4) outcomes and consequences of progress test use (e.g., progress testing contributes to an increase in knowledge). CONCLUSIONS: Progress testing appears to have a positive impact on learning, and there is significant validity evidence to support its use. Although progress testing is resource- and time-intensive, strategies such as collaboration with other institutions may facilitate its use.

Narrative Assessments in Higher Education: A Scoping Review to Identify Evidence-Based Quality Indicators.

PURPOSE: Narrative comments are increasingly used in assessment to document trainees' performance and to make important decisions about academic progress. However, little is known about how to document the quality of narrative comments, since traditional psychometric analysis cannot be applied. The authors aimed to generate a list of quality indicators for narrative comments, to identify recommendations for writing high-quality narrative comments, and to document factors that influence the quality of narrative comments used in assessments in higher education. METHOD: The authors conducted a scoping review according to Arksey & O'Malley's framework. The search strategy yielded 690 articles from 6 databases. Team members screened abstracts for inclusion and exclusion, then extracted numerical and qualitative data based on predetermined categories. Numerical data were used for descriptive analysis. The authors completed the thematic analysis of qualitative data with iterative discussions until they achieved consensus for the interpretation of the results. RESULTS: After the full-text review of 213 selected articles, 47 were included. Through the thematic analysis, the authors identified 7 quality indicators, 12 recommendations for writing quality narratives, and 3 factors that influence the quality of narrative comments used in assessment. The 7 quality indicators are (1) describes performance with a focus on particular elements (attitudes, knowledge, skills); (2) provides a balanced message between positive elements and elements needing improvement; (3) provides recommendations to learners on how to improve their performance; (4) compares the observed performance with an expected standard of performance; (5) provides justification for the mark/score given; (6) uses language that is clear and easily understood; and (7) uses a nonjudgmental style. CONCLUSIONS: Assessors can use these quality indicators and recommendations to write high-quality narrative comments, thus reinforcing the appropriate documentation of trainees' performance, facilitating solid decision making about trainees' progression, and enhancing the impact of narrative feedback for both learners and programs.

Repeat Detector: versatile sizing of expanded tandem repeats and identification of interrupted alleles from targeted DNA sequencing.

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and preclinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntington's disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntington's disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and in the development of novel therapies.

Transcription promotes contraction of CAG repeat tracts in human cells.

Induced transcription through CAG repeats in human cells increases repeat contraction approximately 15-fold in both confluent and proliferating cells. Repeats are stabilized against contraction by siRNA knockdown of MSH2, MSH3 or XPA, but not by knockdown of MSH6, XPC or FEN1. These results define a pathway for CAG.CTG repeat contraction that is initiated by transcription, depends on elements of mismatch and nucleotide-excision repair and does not require DNA replication.

Modifiers of CAG/CTG Repeat Instability: Insights from Mammalian Models.

At fifteen different genomic locations, the expansion of a CAG/CTG repeat causes a neurodegenerative or neuromuscular disease, the most common being Huntington's disease and myotonic dystrophy type 1. These disorders are characterized by germline and somatic instability of the causative CAG/CTG repeat mutations. Repeat lengthening, or expansion, in the germline leads to an earlier age of onset or more severe symptoms in the next generation. In somatic cells, repeat expansion is thought to precipitate the rate of disease. The mechanisms underlying repeat instability are not well understood. Here we review the mammalian model systems that have been used to study CAG/CTG repeat instability, and the modifiers identified in these systems. Mouse models have demonstrated prominent roles for proteins in the mismatch repair pathway as critical drivers of CAG/CTG instability, which is also suggested by recent genome-wide association studies in humans. We draw attention to a network of connections between modifiers identified across several systems that might indicate pathway crosstalk in the context of repeat instability, and which could provide hypotheses for further validation or discovery. Overall, the data indicate that repeat dynamics might be modulated by altering the levels of DNA metabolic proteins, their regulation, their interaction with chromatin, or by direct perturbation of the repeat tract. Applying novel methodologies and technologies to this exciting area of research will be needed to gain deeper mechanistic insight that can be harnessed for therapies aimed at preventing repeat expansion or promoting repeat contraction.

Dnmt1 deficiency promotes CAG repeat expansion in the mouse germline.

Expanded CAG repeat tracts are the cause of at least a dozen neurodegenerative disorders. In humans, long CAG repeats tend to expand during transmissions from parent to offspring, leading to an earlier age of disease onset and more severe symptoms in subsequent generations. Here, we show that the maintenance DNA methyltransferase Dnmt1, which preserves the patterns of CpG methylation, plays a key role in CAG repeat instability in human cells and in the male and female mouse germlines. SiRNA knockdown of Dnmt1 in human cells destabilized CAG triplet repeats, and Dnmt1 deficiency in mice promoted intergenerational expansion of CAG repeats at the murine spinocerebellar ataxia type 1 (Sca1) locus. Importantly, Dnmt1(+/-) SCA1 mice, unlike their Dnmt1(+/+) SCA1 counterparts, closely reproduced the intergenerational instability patterns observed in human SCA1 patients. In addition, we found aberrant DNA and histone methylation at sites within the CpG island that abuts the expanded repeat tract in Dnmt1-deficient mice. These studies suggest that local chromatin structure may play a role in triplet repeat instability. These results are consistent with normal epigenetic changes during germline development contributing to intergenerational instability of CAG repeats in mice and in humans.