SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs.

Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A's AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Centromere chromatin structure - Lessons from neocentromeres.

Centromeres are highly specialized genomic loci that function during mitosis to maintain genome stability. Formed primarily on repetitive α-satellite DNA sequence characterisation of native centromeric chromatin structure has remained challenging. Fortuitously, neocentromeres are formed on a unique DNA sequence and represent an excellent model to interrogate centromeric chromatin structure. This review uncovers the specific findings from independent neocentromere studies that have advanced our understanding of canonical centromere chromatin structure.

Analysis of active and inactive X chromosome architecture reveals the independent organization of 30 nm and large-scale chromatin structures.

Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytological appearance of large-scale chromatin structures.

Autophagy supports PDGFRA-dependent brain tumor development by enhancing oncogenic signaling.

Autophagy is a conserved cellular degradation process. While autophagy-related proteins were shown to influence the signaling and trafficking of some receptor tyrosine kinases, the relevance of this during cancer development is unclear. Here, we identify a role for autophagy in regulating platelet-derived growth factor receptor alpha (PDGFRA) signaling and levels. We find that PDGFRA can be targeted for autophagic degradation through the activity of the autophagy cargo receptor p62. As a result, short-term autophagy inhibition leads to elevated levels of PDGFRA but an unexpected defect in PDGFA-mediated signaling due to perturbed receptor trafficking. Defective PDGFRA signaling led to its reduced levels during prolonged autophagy inhibition, suggesting a mechanism of adaptation. Importantly, PDGFA-driven gliomagenesis in mice was disrupted when autophagy was inhibited in a manner dependent on Pten status, thus highlighting a genotype-specific role for autophagy during tumorigenesis. In summary, our data provide a mechanism by which cells require autophagy to drive tumor formation.

The Greentown Project: Building Evidence to inform Intervention Design for Juveniles Caught-up in Local Criminal Networks.

A small minority of juveniles are responsible for the majority of detected juvenile crime in Ireland. This situation presents significant policy concerns. The current paper, based on findings from a comparative analysis builds on a multi-step research design process to provide evidence-based knowledge to inform the design of a new targeted intervention. An initial social network analysis of national crime and intelligence data produced localized basic criminal network maps illustrating co-offending and intelligence relationships between adults and juveniles in specific Police sub-districts (Part 1). These network maps then provided an enquiry frame for interviews with members of the police forces in three case study locations (Part 2). A comparative analysis of the three studies (Part 3) identified diversity in network structure and inherent resilience. The analysis also identifies core similarities in juveniles' vulnerabilities and risks to recruitment. These factors are important considerations for an intervention seeking to disrupt networks and create safe "exit" environments for juveniles.

Human centromere repositioning activates transcription and opens chromatin fibre structure.

Human centromeres appear as constrictions on mitotic chromosomes and form a platform for kinetochore assembly in mitosis. Biophysical experiments led to a suggestion that repetitive DNA at centromeric regions form a compact scaffold necessary for function, but this was revised when neocentromeres were discovered on non-repetitive DNA. To test whether centromeres have a special chromatin structure we have analysed the architecture of a neocentromere. Centromere repositioning is accompanied by RNA polymerase II recruitment and active transcription to form a decompacted, negatively supercoiled domain enriched in 'open' chromatin fibres. In contrast, centromerisation causes a spreading of repressive epigenetic marks to surrounding regions, delimited by H3K27me3 polycomb boundaries and divergent genes. This flanking domain is transcriptionally silent and partially remodelled to form 'compact' chromatin, similar to satellite-containing DNA sequences, and exhibits genomic instability. We suggest transcription disrupts chromatin to provide a foundation for kinetochore formation whilst compact pericentromeric heterochromatin generates mechanical rigidity.

Acute depletion of the ARID1A subunit of SWI/SNF complexes reveals distinct pathways for activation and repression of transcription.

The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300. Co-incident EP300 dissociation and lost chromatin accessibility at enhancer elements are highly enriched adjacent to rapidly downregulated genes. In contrast, sites of gained EP300 occupancy are linked to genes that are transcriptionally upregulated. These chromatin changes are associated with a small number of genes that are differentially expressed in the first hours following loss of ARID1A. Indirect or adaptive changes dominate the transcriptome following growth for days after loss of ARID1A and result in strong engagement with cancer pathways. The identification of this hierarchy suggests sites for intervention in ARID1A-driven diseases.

Exposure to Domestic Violence and Abuse: Evidence of Distinct Physical and Psychological Dimensions.

Recent literature on exposure to domestic violence (DV) highlights the need for increased understanding of the dynamics of domestic violence and abuse (DVA). The current aims were to explore whether two separate dimensions, physical and psychological DVA, were evident in adult children's reports of their exposure to DVA in their family of origin, and whether these dimensions affected psychological well-being and perceived satisfaction with emotional support (hereafter referred to as social support satisfaction). Young adults (N = 465, aged 17-25, 70% female) reported their experiences of DVA as perpetrated by their parents/caregivers, as well as psychological well-being and social support satisfaction, in an online survey. Using confirmatory factor analysis (CFA), we verified the presence of a two-factor model (physical and psychological DVA). Hierarchical linear regression analysis demonstrated the differing impact of these two factors: Specifically, although exposure to psychological DVA (domestic abuse [DA]) was related to reduced psychological well-being, there was no significant effect of exposure to physical DVA (DV). However, mediation analysis suggested the presence of a suppression effect; there was a magnification of the negative relationship between exposure to psychological DA and social support satisfaction when exposure to physical DV was accounted for. Although findings are preliminary, they provide strong evidence to support theoretical arguments regarding the need for future research to conceptualize exposure to DVA in terms of both physical and psychological dimensions. Our findings also highlight that to improve service response and provide effective interventions, it is essential to include exposure to psychological DA in risk assessments of such young adults.

Common Fragile Sites Are Characterized by Faulty Condensin Loading after Replication Stress.

Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability.

Progressive loss of estrogen receptor alpha cofactor recruitment in endocrine resistance.

Differential expression of estrogen receptor-alpha (ERalpha) cofactors has been implicated in endocrine resistance in breast cancer. Using a three-stage MCF-7 cell-based model that emulates the clinical manifestation of acquired endocrine resistant breast cancer we now show, using a combination of chromatin immunoprecipitation and RNA interference, that there is a progressive loss of ERalpha cofactor recruitment to the estrogen-dependent pS2 gene and reduced requirement for cofactor expression. Maximal estrogen induced pS2 induction requires ERalpha and cofactor recruitment in MCF-7 cells, but in the progression to endocrine resistance these requirements are altered and expression has become less dependent on cofactors. Additionally, in estrogen-resistant MCF-7 cells there is a global loss of requirement of individual cofactors for proliferative cell growth indicating that other genes have lost the need for transcriptional cofactors. This loss of the requirement for cofactors may represent an important mechanism for gene misregulation in cancer.

Restricted reproductive rights and risky sexual behaviour: How political disenfranchisement relates to women's sense of control, well-being and sexual health.

Few studies have investigated the role of disenfranchisement and denial of agency in women's sexual health. To address this, a cross-sectional study of disenfranchisement, control (general and reproductive control) and health was conducted in Ireland, where abortion is severely restricted. Multiple mediation models ( N = 513 women) indicated that general but not reproductive control mediates the association between disenfranchisement and psychological well-being. Additionally, serial mediation shows disenfranchisement is associated with lower sense of control, which is linked to poorer well-being and risky sexual behaviour. Disenfranchisement arising from socio-political contexts may have important implications for women's sexual health.

Endocrine therapy resistance can be associated with high estrogen receptor alpha (ERalpha) expression and reduced ERalpha phosphorylation in breast cancer models.

Hormone-dependent estrogen receptor (ER)-positive breast cancer cells may adapt to low estrogen environments such as produced by aromatase inhibitors. In many instances, cells become insensitive to the effects of estrogen but may still retain dependence on ER. We have investigated the expression, function, and activation of ERalpha in two endocrine-resistant MCF-7 models to identify mechanisms that could contribute to resistance. While MCF-7/LCC1 cells are partially estrogen dependent, MCF-7/LCC9 cells are fully estrogen insensitive and fulvestrant and tamoxifen resistant. In both MCF-7/LCC1 and MCF-7/LCC9 cell lines, high expression of ERalpha was associated with enhanced binding to the trefoil factor 1 (TFF1) promoter in the absence of estrogen and increased transcription of TFF1 and progesterone receptor. In contrast to the observations derived from hypersensitive and supersensitive models, these cells were truly estrogen independent; nevertheless, removal of ERalpha by siRNA, or fulvestrant, a specific ER downregulator, inhibited growth indicating dependence on ERalpha. In the absence of estrogen, neither ERalpha Ser118 nor Ser167 were phosphorylated as frequently found in other ligand-independent cell line models. Addition of estrogen activated ERalpha Ser118 in MCF-7 and LCC1 cells but not in LCC9 cells. We suggest that the estrogen-independent growth within these cell lines is accounted for by high levels of ERalpha expression driving transcription and full estrogen independence explained by lack of ERalpha activation through Ser118.

Inactivation of Apc perturbs mammary development, but only directly results in acanthoma in the context of Tcf-1 deficiency.

Apc (adenomatous polyposis coli) encodes a tumour suppressor gene that is mutated in the majority of colorectal cancers. Recent evidence has also implicated Apc mutations in the aetiology of breast tumours. Apc is a component of the canonical Wnt signal transduction pathway, of which one target is Tcf-1. In the mouse, mutations of both Apc and Tcf-1 have been implicated in mammary tumorigenesis. We have conditionally inactivated Apc in both the presence and absence of Tcf-1 to examine the function of these genes in both normal and neoplastic development. Mice harbouring mammary-specific mutations in Apc show markedly delayed development of the mammary ductal network. During lactation, the mice develop multiple metaplastic growths which, surprisingly, do not spontaneously progress to neoplasia up to a year following their induction. However, additional deficiency of Tcf-1 completely blocks normal mammary development and results in acanthoma.

Family identification: a beneficial process for young adults who grow up in homes affected by parental intimate partner violence.

Exposure to parental intimate partner violence (parental IPV) is a complex trauma. Research within social psychology establishes that identification with social groups impacts positively on how we appraise, respond to and recover from traumatic events. IPV is also a highly stigmatized social phenomenon and social isolation is a major factor for families affected by IPV, yet strong identification with the family group may act as a beneficial psychological resource to young people who grew up in homes affected by IPV. The current study, an online survey of 355 students (M age = 20, 70% female), investigated if a psychosocial process, specifically identification with the family, may influence the relationship between the predictor, exposure to parental IPV, and outcomes, global self-esteem and state anxiety. Mediation analysis suggests that identification with the family has a positive influence on the relationship between exposure to parental IPV and psychological outcomes; exposure to parental IPV results in reduced family identification, but when family identification is strong it results in both reduced anxiety and increased self-esteem for young people. The findings highlight the importance of having a strong sense of belonging to the extended family for young people who were exposed to parental IPV, thus has implications for prevention, intervention, and social policy.

Profiling DNA supercoiling domains in vivo.

Transitions in DNA structure have the capacity to regulate genes, but have been poorly characterised in eukaryotes due to a lack of appropriate techniques. One important example is DNA supercoiling, which can directly regulate transcription initiation, elongation and coordinated expression of neighbouring genes. DNA supercoiling is the over- or under-winding of the DNA double helix, which occurs as a consequence of polymerase activity and is modulated by topoisomerase activity [5]. To map the distribution of DNA supercoiling in nuclei, we developed biotinylated 4,5,8-trimethylpsoralen (bTMP) pull-down to preferentially enrich for under-wound DNA. Here we describe in detail the experimental design, quality controls and analyses associated with the study by Naughton et al. [13] that characterised for the first time the large-scale distribution of DNA supercoiling in human cells (GEO: GSE43488 and GSE43450GSE43488GSE43450).

Divergent RNA transcription: a role in promoter unwinding?

New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance.

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures.

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling, we used biotinylated trimethylpsoralen as a DNA structure probe to show that the human genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF insulator protein-binding sites. Underwound domains are transcriptionally active and enriched in topoisomerase I, 'open' chromatin fibers and DNase I sites, but they are depleted of topoisomerase II. Furthermore, DNA supercoiling affects additional levels of chromatin compaction as underwound domains are cytologically decondensed, topologically constrained and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation, providing an evolutionary purpose for clustering genes along chromosomes.