Temperature regulates negative supercoils to modulate meiotic crossovers and chromosome organization.

Crossover recombination is a hallmark of meiosis that holds the paternal and maternal chromosomes (homologs) together for their faithful segregation, while promoting genetic diversity of the progeny. The pattern of crossover is mainly controlled by the architecture of the meiotic chromosomes. Environmental factors, especially temperature, also play an important role in modulating crossovers. However, it is unclear how temperature affects crossovers. Here, we examined the distribution of budding yeast axis components (Red1, Hop1, and Rec8) and the crossover-associated Zip3 foci in detail at different temperatures, and found that both increased and decreased temperatures result in shorter meiotic chromosome axes and more crossovers. Further investigations showed that temperature changes coordinately enhanced the hyperabundant accumulation of Hop1 and Red1 on chromosomes and the number of Zip3 foci. Most importantly, temperature-induced changes in the distribution of axis proteins and Zip3 foci depend on changes in DNA negative supercoils. These results suggest that yeast meiosis senses temperature changes by increasing the level of negative supercoils to increase crossovers and modulate chromosome organization. These findings provide a new perspective on understanding the effect and mechanism of temperature on meiotic recombination and chromosome organization, with important implications for evolution and breeding.

RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in hepatocellular carcinoma.

BACKGROUND: As a small G protein of Ras family, Ras-like-without-CAAX-1 (RIT1) plays a critical role in various tumors. Our previous study has demonstrated the involvement of RIT1 in promoting malignant progression of hepatocellular carcinoma (HCC). However, its underlying mechanism remains unclear. METHODS: Gene set enrichment analysis (GSEA) was conducted in the TCGA LIHC cohort to investigate the underlying biological mechanism of RIT1. Live cell imaging, immunofluorescence (IF) and flow cytometry assays were used to verify biological function of RIT1 in HCC mitosis. Subcutaneous xenografting of human HCC cells in BALB/c nude mice was utilized to assess tumor proliferation in vivo. RNA-seq, co-immunoprecipitation (Co-IP), mass spectrometry analyses, western blot and IF assays were employed to elucidate the mechanisms by which RIT1 regulates mitosis and promotes proliferation in HCC. RESULTS: Our findings demonstrate that RIT1 plays a crucial role in regulating mitosis in HCC. Knockdown of RIT1 disrupts cell division, leading to G2/M phase arrest, mitotic catastrophe, and apoptosis in HCC cells. SMC3 is found to interact with RIT1 and knockdown of SMC3 attenuates the proliferative effects mediated by RIT1 both in vitro and in vivo. Mechanistically, RIT1 protects and maintains SMC3 acetylation by binding to SMC3 and PDS5 during mitosis, thereby promoting rapid cell division and proliferation in HCC. Notably, we have observed an upregulation of SMC3 expression in HCC tissues, which is associated with poor patient survival and promotion of HCC cell proliferation. Furthermore, there is a significant positive correlation between the expression levels of RIT1, SMC3, and PDS5. Importantly, HCC patients with high expression of both RIT1 and SMC3 exhibit worse prognosis compared to those with high RIT1 but low SMC3 expression. CONCLUSIONS: Our findings underscore the crucial role of RIT1 in regulating mitosis in HCC and further demonstrate its potential as a promising therapeutic target for HCC treatment.

Regulation of cohesin-mediated chromosome folding by PDS5 in mammals.

Cohesin regulates sister chromatid cohesion but also contributes to chromosome folding by promoting the formation of chromatin loops, a process mediated by loop extrusion. Although PDS5 regulates cohesin dynamics on chromatin, the exact function of PDS5 in cohesin-mediated chromatin looping remains unclear. Two paralogs of PDS5 exist in vertebrates, PDS5A and PDS5B. Here we show that PDS5A and PDS5B co-localize with RAD21 and CTCF at loop anchors. Rapid PDS5A or PDS5B degradation in liver cancer cells using an inducible degron system reduces chromatin loops and increases loop size. RAD21 enrichment at loop anchors is decreased upon depletion of PDS5A or PDS5B. PDS5B loss also reduces CTCF signals at loop anchors and has a stronger effect on loop enlargement compared with PDS5A. Co-depletion of PDS5A and PDS5B reduces RAD21 levels at loop anchors although the amount of cohesin on chromatin is increased. Our study provides insight into how PDS5 proteins regulate cohesin-mediated chromatin looping.

PDS5A and PDS5B differentially affect gene expression without altering cohesin localization across the genome.

BACKGROUND: Cohesin is an important structural regulator of the genome, regulating both three-dimensional genome organization and gene expression. The core cohesin trimer interacts with various HEAT repeat accessory subunits, yielding cohesin complexes of distinct compositions and potentially distinct functions. The roles of the two mutually exclusive HEAT repeat subunits PDS5A and PDS5B are not well understood. RESULTS: Here, we determine that PDS5A and PDS5B have highly similar localization patterns across the mouse embryonic stem cell (mESC) genome and they show a strong overlap with other cohesin HEAT repeat accessory subunits, STAG1 and STAG2. Using CRISPR/Cas9 genome editing to generate individual stable knockout lines for PDS5A and PDS5B, we find that loss of one PDS5 subunit does not alter the distribution of the other PDS5 subunit, nor the core cohesin complex. Both PDS5A and PDS5B are required for proper gene expression, yet they display only partially overlapping effects on gene targets. Remarkably, gene expression following dual depletion of the PDS5 HEAT repeat proteins does not completely overlap the gene expression changes caused by dual depletion of the STAG HEAT repeat proteins, despite the overlapping genomic distribution of all four proteins. Furthermore, dual loss of PDS5A and PDS5B decreases cohesin association with NIPBL and WAPL, reduces SMC3 acetylation, and does not alter overall levels of cohesin on the genome. CONCLUSIONS: This work reveals the importance of PDS5A and PDS5B for proper cohesin function. Loss of either subunit has little effect on cohesin localization across the genome yet PDS5A and PDS5B are differentially required for gene expression.

Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

Single-cell proteomics has the potential to decipher tumor heterogeneity, and a method like single-cell proteomics by mass spectrometry (SCoPE-MS) allows profiling several tens of single cells for >1,000 proteins per cell. This method, however, cannot link the proteome of individual cells with phenotypes of interest. Here, we developed a microscopy-based functional single-cell proteomic-profiling technology, called FUNpro, to address this. FUNpro enables screening, identification, and isolation of single cells of interest in a real-time fashion, even if the phenotypes are dynamic or the cells of interest are rare. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified the PDS5A protein contributing to the abnormal DDR dynamics and helping the cells survive after IR.

S. cerevisiae Cells Can Grow without the Pds5 Cohesin Subunit.

During DNA replication, the newly created sister chromatids are held together until their separation at anaphase. The cohesin complex is in charge of creating and maintaining sister chromatid cohesion (SCC) in all eukaryotes. In Saccharomyces cerevisiae cells, cohesin is composed of two elongated proteins, Smc1 and Smc3, bridged by the kleisin Mcd1/Scc1. The latter also acts as a scaffold for three additional proteins, Scc3/Irr1, Wpl1/Rad61, and Pds5. Although the HEAT-repeat protein Pds5 is essential for cohesion, its precise function is still debated. Deletion of the ELG1 gene, encoding a PCNA unloader, can partially suppress the temperature-sensitive pds5-1 allele, but not a complete deletion of PDS5. We carried out a genetic screen for high-copy-number suppressors and another for spontaneously arising mutants, allowing the survival of a pds5Δ elg1Δ strain. Our results show that cells remain viable in the absence of Pds5 provided that there is both an elevation in the level of Mcd1 (which can be due to mutations in the CLN2 gene, encoding a G1 cyclin), and an increase in the level of SUMO-modified PCNA on chromatin (caused by lack of PCNA unloading in elg1Δ mutants). The elevated SUMO-PCNA levels increase the recruitment of the Srs2 helicase, which evicts Rad51 molecules from the moving fork, creating single-stranded DNA (ssDNA) regions that serve as sites for increased cohesin loading and SCC establishment. Thus, our results delineate a double role for Pds5 in protecting the cohesin ring and interacting with the DNA replication machinery. IMPORTANCE Sister chromatid cohesion is vital for faithful chromosome segregation, chromosome folding into loops, and gene expression. A multisubunit protein complex known as cohesin holds the sister chromatids from S phase until the anaphase stage. In this study, we explore the function of the essential cohesin subunit Pds5 in the regulation of sister chromatid cohesion. We performed two independent genetic screens to bypass the function of the Pds5 protein. We observe that Pds5 protein is a cohesin stabilizer, and elevating the levels of Mcd1 protein along with SUMO-PCNA accumulation on chromatin can compensate for the loss of the PDS5 gene. In addition, Pds5 plays a role in coordinating the DNA replication and sister chromatid cohesion establishment. This work elucidates the function of cohesin subunit Pds5, the G1 cyclin Cln2, and replication factors PCNA, Elg1, and Srs2 in the proper regulation of sister chromatid cohesion.

Knockdown of CDCA5 suppresses malignant progression of breast cancer cells by regulating PDS5A.

Breast cancer is one of the most common malignant tumors in women. Cell division cycle­associated 5 (CDCA5) is closely associated with the behavior of various cancer types. The aim of the present study was to explore the effect of CDCA5 on breast cancer. Western blot analysis and reverse transcription­quantitative PCR were used to detect the expression level of CDCA5 in human normal mammary cells and human breast cancer cell lines. To determine its function in MDA­MB­231 cells, CDCA5 was silenced in MDA­MB­231 cells by transient short hairpin RNA transfection. Cell Counting Kit­8 and clonogenicity assays were used to evaluate cell proliferation. Wound healing and Transwell assays were used to detect cell invasion and migration. Western blot analysis was used to detect the protein expressions of Ki67 and PCNA associated with proliferation, MMP2 and MMP9 associated with migration. CDCA5 was found to be markedly increased in breast cancer cell lines. CDCA5 knockdown was able to suppress cell proliferation, invasion and migration. CDCA5 inhibition downregulated PDS5 cohesin­associated factor A (PDS5A) expression in breast cancer cells. PDS5A overexpression was found to reverse the effect of CDCA5 inhibition on breast cancer cell proliferation and migration. CDCA5 knockdown was shown to suppress the malignant progression of breast cancer cells by regulating PDS5A. The present findings may provide new potential targets for breast cancer therapy.

The ubiquitin-proteasome system regulates meiotic chromosome organization.

Meiotic crossover (CO) recombination is tightly regulated by chromosome architecture to ensure faithful chromosome segregation and to reshuffle alleles between parental chromosomes for genetic diversity of progeny. However, regulation of the meiotic chromosome loop/axis organization is poorly understood. Here, we identify a molecular pathway for axis length regulation. We show that the cohesin regulator Pds5 can interact with proteasomes. Meiosis-specific depletion of proteasomes and/or Pds5 results in a similarly shortened chromosome axis, suggesting proteasomes and Pds5 regulate axis length in the same pathway. Protein ubiquitination is accumulated in pds5 and proteasome mutants. Moreover, decreased chromosome axis length in these mutants can be largely rescued by decreasing ubiquitin availability and thus decreasing protein ubiquitination. Further investigation reveals that two ubiquitin E3 ligases, SCF (Skp­Cullin­F-box) and Ufd4, are involved in this Pds5­ubiquitin/proteasome pathway to cooperatively control chromosome axis length. These results support the hypothesis that ubiquitination of chromosome proteins results in a shortened chromosome axis, and cohesin­Pds5 recruits proteasomes onto chromosomes to regulate ubiquitination level and thus axis length. These findings reveal an unexpected role of the ubiquitin­proteasome system in meiosis and contribute to our knowledge of how Pds5 regulates meiotic chromosome organization. A conserved regulatory mechanism probably exists in higher eukaryotes.

Meiotic prophase roles of Pds5 in recombination and chromosome condensation in budding yeast.

Genetic variation in eukaryotes is mediated during meiosis by the exchange of genetic material between homologous chromosomes to produce recombinant chromosomes. Cohesin is essential to promote proper chromosome segregation, chromosome morphogenesis, and recombination in meiotic cells. Cohesin consists of three main subunits-Smc1, Smc3, and the kleisin subunit Mcd1/Scc1 (Rec8 in meiosis)-and cohesin accessory factors. In Saccharomyces cerevisiae, the cohesin regulatory subunit Pds5 plays a role in homolog pairing, meiotic axis formation, and interhomolog recombination. In this study, we examine the prophase functions of Pds5 by performing physical analysis of recombination and three-dimensional high-resolution microscopy analysis to identify its roles in meiosis-specific recombination and chromosome morphogenesis. To investigate whether Pds5 plays a role in mitotic-like recombination, we inhibited Mek1 kinase activity, which resulted in switching to sister template bias by Rad51-dependent recombination. Reductions in double-strand breaks and crossover products and defective interhomolog recombination occurred in the absence of Pds5. Furthermore, recombination intermediates, including single-end invasion and double-Holliday junction, were reduced in the absence of Pds5 with Mek1 kinase inactivation compared to Mek1 kinase inactivation cells. Interestingly, the absence of Pds5 resulted in increasing numbers of chromosomes with hypercompaction of the chromosome axis. Thus, we suggest that Pds5 plays an essential role in recombination by suppressing the pairing of sister chromatids and abnormal compaction of the chromosome axis.

Rec8 Cohesin: A Structural Platform for Shaping the Meiotic Chromosomes.

Meiosis is critically different from mitosis in that during meiosis, pairing and segregation of homologous chromosomes occur. During meiosis, the morphology of sister chromatids changes drastically, forming a prominent axial structure in the synaptonemal complex. The meiosis-specific cohesin complex plays a central role in the regulation of the processes required for recombination. In particular, the Rec8 subunit of the meiotic cohesin complex, which is conserved in a wide range of eukaryotes, has been analyzed for its function in modulating chromosomal architecture during the pairing and recombination of homologous chromosomes in meiosis. Here, we review the current understanding of Rec8 cohesin as a structural platform for meiotic chromosomes.

PRR11 unveiled as a top candidate biomarker within the RBM3-regulated transcriptome in pancreatic cancer.

The outlook for patients with pancreatic cancer remains dismal. Treatment options are limited and chemotherapy remains standard of care, leading to only modest survival benefits. Hence, there is a great need to further explore the mechanistic basis for the intrinsic therapeutic resistance of this disease, and to identify novel predictive biomarkers. RNA-binding motif protein 3 (RBM3) has emerged as a promising biomarker of disease severity and chemotherapy response in several types of cancer, including pancreatic cancer. The aim of this study was to unearth RBM3-regulated genes and proteins in pancreatic cancer cells in vitro, and to examine their expression and prognostic significance in human tumours. Next-generation RNA sequencing was applied to compare transcriptomes of MIAPaCa-2 cells with and without RBM3 knockdown. The prognostic value of differentially expressed genes (DEGs) was examined in The Cancer Genome Atlas (TCGA). Top deregulated genes were selected for further studies in vitro and for immunohistochemical analysis of corresponding protein expression in tumours from a clinically well-annotated consecutive cohort of 46 patients with resected pancreatic cancer. In total, 19 DEGs (p < 0.01) were revealed, among which some with functions in cell cycle and cell division stood out; PDS5A (PDS cohesin associated factor A) as the top downregulated gene, CCND3 (cyclin D3) as the top upregulated gene, and PRR11 (proline rich 11) as being highly prognostic in TCGA. Silencing of RBM3 in MiaPaCa-2 cells led to congruent alterations of PDS5A, cyclin D3, and PRR11 levels. High protein expression of PRR11 was associated with adverse clinicopathological features and shorter overall survival. Neither PDS5A nor cyclin D3 protein expression was prognostic. This study unveils several RBM3-regulated genes with potential clinical relevance in pancreatic cancer, among which PRR11 shows the most consistent association with disease severity, at both transcriptome and protein levels.

Psychometric properties and validity of the German version of the Post-Traumatic Diagnostic Scale for DSM-5 (PDS-5).

Background: The availability of psychometrically sound instruments for the assessment of Post-Traumatic Stress Disorder (PTSD) is indispensable for clinical and scientific work with individuals suffering from trauma-related distress. Objective: The aim of the present study was to translate the Post-Traumatic Diagnostic Scale for DSM-5 (PDS-5) into German and to evaluate its psychometric properties as well as convergent, discriminant, and factorial validity. Method: The authorized German translation of the PDS-5 was completed by 270 patients admitted to specialized outpatient trauma clinics. Of these, 57.8% completed the PDS for a second time (mean time between assessments was 12.0 days). In order to examine convergent and discriminant validity of the PDS-5, the Post-traumatic Stress Disorder Checklist for DSM-5 as well as Patient Health Questionnaire subscales assessing depression (PHQ-9), somatization (PHQ-15), and Generalized Anxiety Disorder (GAD-7) were applied. Results: The PDS-5 total score showed excellent internal consistency (α = .91) and re-test reliability (rho = .84). Convergent validity was supported by a strong correlation with the total score of the Post-traumatic Stress Disorder Checklist for DSM-5 (PCL-5; rho = .91). Correlations with Patient Health Questionnaire subscales of depression (rho = .81), anxiety (rho = .72), and somatization (rho = .65) were significantly lower (all p < .001) indicating discriminant validity of the PDS-5. Confirmative Factor Analysis did not result in a clear preference for one of the tested models. Defining a diagnostic cut-off value of ≥36 based on ROC analysis resulted in high sensitivity (.92) and specificity (.96) compared to a probable PTSD diagnosis according to the PCL-5. Conclusions: In summary, our results indicate that the German PDS-5 translation provides valid and reliable information concerning both PTSD severity and diagnosis.

Antecedentes: La disponibilidad de instrumentos psicométricamente sólidos para la evaluación del trastorno de estrés postraumático (TEPT) es indispensable para el trabajo clínico y científico con personas que sufren angustia relacionada con el trauma.Objetivo: El objetivo del presente estudio fue traducir la Escala de Diagnóstico Postraumático del DSM-5 (PDS-5) al alemán y evaluar sus propiedades psicométricas, así como su validez convergente, discriminante y factorial.Método: La traducción al alemán autorizada del PDS-5 fue completada por 270 pacientes ingresados en clínicas de trauma ambulatorias especializadas. De estos, el 57,8% completó la PDS por segunda vez (el tiempo medio entre evaluaciones fue de 12,0 días). Con el fin de examinar la validez convergente y discriminante del PDS-5, la lista de verificación del trastorno de estrés postraumático para el DSM-5, así como las subescalas de depresión del Cuestionario de salud del paciente (PHQ-9), de somatización (PHQ-15) y de trastorno de ansiedad generalizada (GAD-7) fueron aplicadas.Resultados: La puntuación total del PDS-5 mostró una excelente consistencia interna (α = .91) y confiabilidad al reaplicar (rho = .84). La validez convergente fue apoyada por una fuerte correlación con la puntuación total de la lista de verificación de trastorno de estrés postraumático para el DSM-5 (PCL-5; rho = .91). Las correlaciones con las subescalas del Cuestionario de Salud del Paciente de depresión (rho = .81), ansiedad (rho = .72) y somatización (rho = .65) fueron significativamente más bajas (todas p <.001) lindicando validez discriminante del PDS-5. El Análisis Factorial Confirmativo no resultó en una preferencia clara por uno de los modelos probados. La definición de un valour de corte de diagnóstico de ≥36 basado en el análisis ROC resultó en una alta sensibilidad (.92) y especificidad (.96) en comparación con un diagnóstico de TEPT probable según el PCL-5.Conclusiones: En resumen, nuestros resultados indican que la traducción al alemán PDS-5 proporciona información válida y confiable sobre la severidad y diagnóstico del TEPT.

SMC complexes are guarded by the SUMO protease Ulp2 against SUMO-chain-mediated turnover.

Structural maintenance of chromosomes (SMCs) complexes, cohesin, condensin, and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability of PDS5 null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality of PDS5-deleted cells can also be bypassed by simultaneous loss of the proliferating cell nuclear antigen (PCNA) unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO chain assembly, which we propose to time the availability of SMC complexes on chromatin.

PDS5A and PDS5B in Cohesin Function and Human Disease.

Precocious dissociation of sisters 5 (PDS5) is an associate protein of cohesin that is conserved from yeast to humans. It acts as a regulator of the cohesin complex and plays important roles in various cellular processes, such as sister chromatid cohesion, DNA damage repair, gene transcription, and DNA replication. Vertebrates have two paralogs of PDS5, PDS5A and PDS5B, which have redundant and unique roles in regulating cohesin functions. Herein, we discuss the molecular characteristics and functions of PDS5, as well as the effects of its mutations in the development of diseases and their relevance for novel therapeutic strategies.

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.

Downregulation of APRIN expression increases cancer cell proliferation via an interleukin-6/STAT3/cyclin D axis.

APRIN is a putative tumor suppressor whose expression is low in a variety of cancer cells. While decreased expression of APRIN leads to increased cell proliferation, unfavorable diagnosis or metastases in various cancer types, there is limited knowledge on the cellular mechanism of APRIN in cellular responses. The effect of APRIN depletion on cancer cell proliferation was examined in the present study, and the IL-6/STAT3/cyclin D axis was identified as a novel regulatory mechanism. Stable depletion of APRIN in cancer cells resulted in increased cell proliferation. Cytokine array analysis of the cells revealed that downregulation of APRIN induced secretion of interleukin-6 (IL-6) with corresponding activation of STAT3, a downstream intracellular mediator. Levels of cyclin D1 were increased in cells with APRIN depletion and cyclin D1 expression was associated with increased STAT3 binding on cyclin D1 promoter sequence; assessed by chromatin immunoprecipitation assay. The addition of an IL-6 neutralizing antibody P620 to the cell culture attenuated STAT3 activation and cyclin D1 expression in APRIN-depleted cells with corresponding decrease in cell proliferation. These experiments suggest that APRIN regulates cancer cell proliferation via an IL-6/STAT3/cyclin D axis and that targeting this axis in APRIN-associated cancer might provide a novel therapeutic approach.

Cohesin residency determines chromatin loop patterns.

The organization of chromatin into higher order structures is essential for chromosome segregation, the repair of DNA-damage, and the regulation of gene expression. Using Micro-C XL to detect chromosomal interactions, we observed the pervasive presence of cohesin-dependent loops with defined positions throughout the genome of budding yeast, as seen in mammalian cells. In early S phase, cohesin stably binds to cohesin associated regions (CARs) genome-wide. Subsequently, positioned loops accumulate with CARs at the bases of the loops. Cohesin regulators Wpl1 and Pds5 alter the levels and distribution of cohesin at CARs, changing the pattern of positioned loops. From these observations, we propose that cohesin with loop extrusion activity is stopped by preexisting CAR-bound cohesins, generating positioned loops. The patterns of loops observed in a population of wild-type and mutant cells can be explained by this mechanism, coupled with a heterogeneous residency of cohesin at CARs in individual cells.

Pds5A and Pds5B Display Non-redundant Functions in Mitosis and Their Loss Triggers Chk1 Activation.

BACKGROUND: Pds5 is an abundant HEAT-repeat-containing protein that binds to cohesin and mediates sister chromatid cohesion. In vertebrates, Pds5A and Pds5B are known to protect DNA replication fork, as their loss leads to DNA damage. Pds5 interacts directly with Wapl, to remove cohesin during mitosis. AIM: To analyze the effects of the loss of Pds5 proteins-mediated DNA damage on the cell cycle checkpoints and to examine the possibility that Pds5 proteins have an overlapping function. METHODS: We first analyzed the cell cycle regulation of Pds5 proteins and defects in S-phase; DNA damage was confirmed after Pds5A/B knockdown. The activation of cell cycle checkpoints and apoptosis were examined by the level of p-Chk1S317, MAD2 localization, and the level of pro-apoptotic markers, respectively. RESULTS: Pds5 proteins dissociated from chromatin in a stepwise manner, and their loss led to activation of pro-apoptotic markers associated with the phosphorylation of Chk1S317 due to DNA damage. Depletion of either Pds5A or Pds5B alone increased Smc3 acetylation in perturbed cell cycle, while depletion of both proteins severely impaired Smc3 acetylation. Moreover, the loss of Pds5A/Pds5B activated the SAC in an ATR-Chk1-dependent manner and stabilized Wapl on chromatin. The depletion of Chk1 rescued the S-phase delay associated with Pds5 depletion and significantly increased mitotic catastrophe. CONCLUSION: Pds5A and Pds5B display overlapping functions in facilitating Smc3 acetylation. Somewhat paradoxically, they also have non-redundant functions in terms of cohesin removal due to the activated surveillance mechanism that leads to phosphorylation of Chk1S317.

CTCF as a boundary factor for cohesin-mediated loop extrusion: evidence for a multi-step mechanism.

Mammalian genome structure is closely linked to function. At the scale of kilobases to megabases, CTCF and cohesin organize the genome into chromatin loops. Mechanistically, cohesin is proposed to extrude chromatin loops bidirectionally until it encounters occupied CTCF DNA-binding sites. Curiously, loops form predominantly between CTCF binding sites in a convergent orientation. How CTCF interacts with and blocks cohesin extrusion in an orientation-specific manner has remained a mechanistic mystery. Here, we review recent papers that have shed light on these processes and suggest a multi-step interaction between CTCF and cohesin. This interaction may first involve a pausing step, where CTCF halts cohesin extrusion, followed by a stabilization step of the CTCF-cohesin complex, resulting in a chromatin loop. Finally, we discuss our own recent studies on an internal RNA-Binding Region (RBRi) in CTCF to elucidate its role in regulating CTCF clustering, target search mechanisms and chromatin loop formation and future challenges.

Dimensionality of DSM-5 PTSD symptoms: Validation of the Chinese version of the posttraumatic diagnostic scale for DSM-5 across multiple trauma samples.

The Posttraumatic Diagnostic Scale for DSM-5 (PDS-5) is an updated DSM-5 version of the PDS, a widely used measure for PTSD. The PDS-5 has recently been shown to possess sound psychometric properties and awaits cross-cultural validation. The present study aimed first, to evaluate the psychometric properties of the Chinese version of the PDS-5; second, to evaluate alternative factor models of DSM-5 PTSD symptoms with multiple trauma samples. Data were collected from five samples of Taiwanese trauma-exposed individuals (total N = 903): 138 burn injury survivors, 403 earthquake survivors, 181 trauma-exposed young adults, 91 trauma-exposed undergraduates, and 90 female domestic violence survivors. The Chinese PDS-5 possessed excellent internal consistency (α s = .94-.95) and satisfactory five-week (r = .80) and one-year temporal stability (r = 0.76). Convergent, concurrent, and discriminant validity were also established. Consistent with recent studies, confirmatory factor analyses demonstrated the best fit of a seven-factor Hybrid model, followed by a six-factor Anhedonia model across multiple trauma samples.