Something silent this way forms: the functional organization of the repressive nuclear compartment.

The repressive compartment of the nucleus is comprised primarily of telomeric and centromeric regions, the silent portion of ribosomal RNA genes, the majority of transposable element repeats, and facultatively repressed genes specific to different cell types. This compartment localizes into three main regions: the peripheral heterochromatin, perinucleolar heterochromatin, and pericentromeric heterochromatin. Both chromatin remodeling proteins and transcription of noncoding RNAs are involved in maintenance of repression in these compartments. Global reorganization of the repressive compartment occurs at each cell division, during early development, and during terminal differentiation. Differential action of chromatin remodeling complexes and boundary element looping activities are involved in mediating these organizational changes. We discuss the evidence that heterochromatin formation and compartmentalization may drive nuclear organization.

Impact of Caffeine Ingestion on the Driving Performance of Anesthesiology Residents After 6 Consecutive Overnight Work Shifts.

BACKGROUND: Residency training in anesthesiology involves care of hospitalized patients and necessitates overnight work, resulting in altered sleep patterns and sleep deprivation. Caffeine consumption is commonly used to improve alertness when fatigued after overnight work, in preparation for the commute home. METHODS: We studied the impact of drinking a caffeinated energy drink (160 mg of caffeine) on driving performance in a high-fidelity, virtual reality driving simulator (Virginia Driving Safety Laboratory using the Driver Guidance System) in anesthesiology resident physicians immediately after 6 consecutive night-float shifts. Twenty-six residents participated and were randomized to either consume a caffeinated or noncaffeinated energy drink 60 minutes before the driving simulation session. After a subsequent week of night-float work, residents performed the same driving session (in a crossover fashion) with the opposite intervention. Psychomotor vigilance task (PVT) testing was used to evaluate reaction time and lapses in attention. RESULTS: After 6 consecutive night-float shifts, anesthesiology residents who consumed a caffeinated energy drink had increased variability in driving for throttle, steering, and speed during the first 10 minutes of open-road driving but proceeded to demonstrate improved driving performance with fewer obstacle collisions (epoch 2: 0.65 vs 0.87; epoch 3: 0.47 vs 0.95; P = .03) in the final 30 minutes of driving as compared to driving performance after consumption of a noncaffeinated energy drink. Improved driving performance was most apparent during the last 30 minutes of the simulated drive in the caffeinated condition. Mean reaction time between the caffeine and noncaffeine states differed significantly (278.9 ± 29.1 vs 294.0 ± 36.3 milliseconds; P = .021), while the number of major lapses (0.09 ± 0.43 vs 0.27 ± 0.55; P = .257) and minor lapses (1.05 ± 1.39 vs 2.05 ± 3.06; P = .197) was not significantly different. CONCLUSIONS: After consuming a caffeinated energy drink on conclusion of 6 shifts of night-float work, anesthesiology residents had improved control of driving performance variables in a high-fidelity driving simulator, including a significant reduction in collisions as well as slightly faster reaction times.

Driving Performance of Residents after Six Consecutive Overnight Work Shifts.

BACKGROUND: Residency training requires work in clinical settings for extended periods of time, resulting in altered sleep patterns, sleep deprivation, and potentially deleterious effects on safe performance of daily activities, including driving a motor vehicle. METHODS: Twenty-nine anesthesiology resident physicians in postgraduate year 2 to 4 drove for 55 min in the Virginia Driving Safety Laboratory using the Driver Guidance System (MBFARR, LLC, USA). Two driving simulator sessions were conducted, one experimental session immediately after the final shift of six consecutive night shifts and one control session at the beginning of a normal day shift (not after call). Both sessions were conducted at 8:00 AM. Psychomotor vigilance task testing was employed to evaluate reaction time and lapses in attention. RESULTS: After six consecutive night shifts, residents experienced significantly impaired control of all the driving variables including speed, lane position, throttle, and steering. They were also more likely to be involved in collisions. After six consecutive night shifts, residents had a significant increase in reaction times (281.1 vs. 298.5 ms; P = 0.001) and had a significant increase in the number of both minor (0.85 vs. 1.88; P = 0.01) and major lapses (0.00 vs. 0.31; P = 0.008) in attention. CONCLUSIONS: Resident physicians have greater difficulty controlling speed and driving performance in the driving simulator after six consecutive night shifts. Reaction times are also increased with emphasis on increases in minor and major lapses in attention after six consecutive night shifts.

Ventricular assist devices and increased blood product utilization for cardiac transplantation.

BACKGROUND AND AIM OF STUDY: The purpose of this study was to examine whether blood product utilization, one-year cell-mediated rejection rates, and mid-term survival significantly differ for ventricular assist device (VAD patients compared to non-VAD (NVAD) patients following cardiac transplantation. METHODS: From July 2004 to August 2011, 79 patients underwent cardiac transplantation at a single institution. Following exclusion of patients bridged to transplantation with VADs other than the HeartMate II® LVAD (n = 10), patients were stratified by VAD presence at transplantation: VAD patients (n = 35, age: 54.0 [48.0-59.0] years) vs. NVAD patients (n = 34, age: 52.5 [42.8-59.3] years). The primary outcomes of interest were blood product transfusion requirements, one-year cell-mediated rejection rates, and mid-term survival post-transplantation. RESULTS: Preoperative patient characteristics were similar for VAD and NVAD patients. NVAD patients presented with higher median preoperative creatinine levels compared to VAD patients (1.3 [1.1-1.6] vs. 1.1 [0.9-1.4], p = 0.004). VAD patients accrued higher intraoperative transfusion of all blood products (all p ≤ 0.001) compared to NVAD patients. The incidence of clinically significant cell-mediated rejection within the first posttransplant year was higher in VAD compared to NVAD patients (66.7% vs. 33.3%, p = 0.02). During a median follow-up period of 3.2 (2.0, 6.3) years, VAD patients demonstrated an increased postoperative mortality that did not reach statistical significance (20.0% vs. 8.8%, p = 0.20). CONCLUSIONS: During the initial era as a bridge to transplantation, the HeartMate II® LVAD significantly increased blood product utilization and one-year cell-mediated rejection rates for cardiac transplantation. Further study is warranted to optimize anticoagulation strategies and to define causal relationships between these factors for the current era of cardiac transplantation.

Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing.

Replication of the genome before mitotic cell division is a highly regulated process that ensures the fidelity of DNA duplication. DNA replication initiates at specific locations, termed origins of replication, and progresses in a defined temporal order during the S phase of the cell cycle. The relationship between replication timing and gene expression has been the subject of some speculation. A recent genome-wide analysis in Saccharomyces cerevisiae showed no association between replication timing and gene expression. In higher eukaryotes, the limited number of genomic loci analyzed has not permitted a firm conclusion regarding this association. To explore the relationship between DNA replication and gene expression in higher eukaryotes, we developed a strategy to measure the timing of DNA replication for thousands of genes in a single DNA array hybridization experiment. Using this approach, we generated a genome-wide map of replication timing for Drosophila melanogaster. Moreover, by surveying over 40% of all D. melanogaster genes, we found a strong correlation between DNA replication early in S phase and transcriptional activity. As this correlation does not exist in S. cerevisiae, this interplay between DNA replication and transcription may be a unique characteristic of higher eukaryotes.

The emergence of lineage-specific chromosomal topologies from coordinate gene regulation.

Although the importance of chromosome organization during mitosis is clear, it remains to be determined whether the nucleus assumes other functionally relevant chromosomal topologies. We have previously shown that homologous chromosomes have a tendency to associate during hematopoiesis according to their distribution of coregulated genes, suggesting cell-specific nuclear organization. Here, using the mathematical approaches of distance matrices and coupled oscillators, we model the dynamic relationship between gene expression and chromosomal associations during the differentiation of a multipotential hematopoietic progenitor. Our analysis reveals dramatic changes in total genomic order: Commitment of the progenitor results in an initial increase in entropy at both the level of gene coregulation and chromosomal organization, which we suggest represents a phase transition, followed by a progressive decline in entropy during differentiation. The stabilization of a highly ordered state in the differentiated cell types results in lineage-specific chromosomal topologies and is related to the emergence of coherence-or self-organization-between chromosomal associations and coordinate gene regulation. We discuss how these observations may be generally relevant to cell fate decisions encountered by progenitor/stem cells.

Networking the nucleus.

The nuclei of differentiating cells exhibit several fundamental principles of self-organization. They are composed of many dynamical units connected physically and functionally to each other--a complex network--and the different parts of the system are mutually adapted and produce a characteristic end state. A unique cell-specific signature emerges over time from complex interactions among constituent elements that delineate coordinate gene expression and chromosome topology. Each element itself consists of many interacting components, all dynamical in nature. Self-organizing systems can be simplified while retaining complex information using approaches that examine the relationship between elements, such as spatial relationships and transcriptional information. These relationships can be represented using well-defined networks. We hypothesize that during the process of differentiation, networks within the cell nucleus rewire according to simple rules, from which a higher level of order emerges. Studying the interaction within and among networks provides a useful framework for investigating the complex organization and dynamic function of the nucleus.

HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Coordinate gene regulation during hematopoiesis is related to genomic organization.

Gene loci are found in nuclear subcompartments that are related to their expression status. For instance, silent genes are often localized to heterochromatin and the nuclear periphery, whereas active genes tend to be found in the nuclear center. Evidence also suggests that chromosomes may be specifically positioned within the nucleus; however, the nature of this organization and how it is achieved are not yet fully understood. To examine whether gene regulation is related to a discernible pattern of genomic organization, we analyzed the linear arrangement of co-regulated genes along chromosomes and determined the organization of chromosomes during the differentiation of a hematopoietic progenitor to erythroid and neutrophil cell types. Our analysis reveals that there is a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. We suggest that proximity in the form of chromosomal gene distribution and homolog association may be the basis for organizing the genome for coordinate gene regulation during cellular differentiation.

Speed, accuracy, and confidence in Google, Ovid, PubMed, and UpToDate: results of a randomised trial.

BACKGROUND: The explosion of biomedical information has led to an 'information paradox'-the volume of biomedical information available has made it increasingly difficult to find relevant information when needed. It is thus increasingly critical for physicians to acquire a working knowledge of biomedical informatics. AIM: To evaluate four search tools commonly used to answer clinical questions, in terms of accuracy, speed, and user confidence. METHODS: From December 2008 to June 2009, medical students, resident physicians, and attending physicians at the authors' institution were asked to answer a set of four anaesthesia and/or critical care based clinical questions, within 5 min, using Google, Ovid, PubMed, or UpToDate (only one search tool per question). At the end of each search, participants rated their results on a four point confidence scale. One to 3 weeks after answering the initial four questions, users were randomised to one of the four search tools, and asked to answer eight questions, four of which were repeated. The primary outcome was defined as a correct answer with the highest level of confidence. RESULTS: Google was the most popular search tool. Users of Google and UpToDate were more likely than users of PubMed to answer questions correctly. Subjects had the most confidence in UpToDate. Searches with Google and UpToDate were faster than searches with PubMed or Ovid. CONCLUSION: Non-Medline based search tools are not inferior to Medline based search tools for purposes of answering evidence based anaesthesia and critical care questions.

Strict glucose control does not affect mortality after aneurysmal subarachnoid hemorrhage.

BACKGROUND: The effects of both hyperglycemia and hypoglycemia are deleterious to patients with neurologic injury. METHODS: On January 1, 2002, the neurointensive care unit at the University of Virginia Health System initiated a strict glucose control protocol (goal glucose < 120 mg/dl). The authors conducted an impact study to determine the effects of this protocol on patients presenting with aneurysmal subarachnoid hemorrhage. RESULTS: Among the 834 patients admitted between 1995 and 2007, the in-hospital mortality was 11.6%. The median admission glucose for survivors was lower (135 vs. 176 mg/dl); however, on multivariate analysis, increasing admission glucose was not associated with a statistically significant increase in the risk of death (P = 0.064). The median average glucose for survivors was also lower (116 vs. 135 mg/dl). This was significant on multivariate analysis (P < 0.001); however, the effect was small (odds ratio, 1.045). Implementation of the strict glucose protocol decreased median average glucose (121 vs. 116 mg/dl, P < 0.001) and decreased the incidence of hyperglycemia. Implementation of the protocol had no effect on in-hospital mortality (11.7% vs. 12.0%, P = 0.876 [univariate], P = 0.132 [multivariate]). Protocol implementation was associated with an increased incidence of hypoglycemia (P < 0.001). Hypoglycemia was associated with a substantially increased risk of death on multivariate analysis (P = 0.009; odds ratio = 3.818). CONCLUSIONS: The initiation of a tight glucose control regimen lowered average glucose levels but had no effect on overall in-hospital mortality.

Feasibility of Spinal Anesthesia Placement Using Automated Interpretation of Lumbar Ultrasound Images: A Prospective Randomized Controlled Trial.

BACKGROUND: This study evaluated the efficacy of spinal anesthesia administration by resident physicians when using an ultrasound system with automated neuraxial landmark detection capabilities. METHODS: 150 patients were enrolled in this trial. Anesthesiology residents placed spinals in subjects undergoing scheduled cesarean delivery using one of three techniques to identify neuraxial landmarks: palpation, ultrasound, or combined palpation and ultrasound. Ultrasound was performed using a handheld system that automatically identified neuraxial landmarks (e.g. midline, intervertebral spaces). All residents watched a 10-minute video and received 20 minutes of hands-on training prior to participating in the study. First insertion success rate was the primary end point. RESULTS: Among all patients, use of ultrasound resulted in a 11% greater first-insertion success rate (RR: 1.11 [0.85-1.47], p=0.431), a 15% reduction in needle insertions (RR: 0.85, p=0.052), and a 26% decrease in needle passes (RR: 0.74, p=0.070). In obese patients of BMI ≥ 30 kg/m2, use of ultrasound resulted in 26% greater first-insertion success rates (RR: 1.26, p=0.187), a 21% decrease in needle insertions (RR: 0.79, p=0.025), a 38% decrease in needle passes (RR: 0.62, p=0.030), and a 75% decrease in patients reporting neutral or low patient satisfaction with anesthesia administration (RR: 0.25, p=0.004). DISCUSSION: Resident anesthesiologists competently utilized the ultrasound system after receiving minimal training. Technical endpoints and patient satisfaction trended towards improvement when ultrasound was used prior to spinal placement, with stronger trends observed in obese patients. Additional study is required to fully characterize the impact of the ultrasound system on clinical efficacy.

Imaging Performance of a Handheld Ultrasound System With Real-Time Computer-Aided Detection of Lumbar Spine Anatomy: A Feasibility Study.

OBJECTIVES: The aim of this study was to evaluate the imaging performance of a handheld ultrasound system and the accuracy of an automated lumbar spine computer-aided detection (CAD) algorithm in the spines of human subjects. MATERIALS AND METHODS: This study was approved by the institutional review board of the University of Virginia. The authors designed a handheld ultrasound system with enhanced bone image quality and fully automated CAD of lumbar spine anatomy. The imaging performance was evaluated by imaging the lumbar spines of 68 volunteers with body mass index between 18.5 and 48 kg/m. The accuracy, sensitivity, and specificity of the lumbar spine CAD algorithm were assessed by comparing the algorithm's results to ground-truth segmentations of neuraxial anatomy provided by radiologists. RESULTS: The lumbar spine CAD algorithm detected the epidural space with a sensitivity of 94.2% (95% confidence interval [CI], 85.1%-98.1%) and a specificity of 85.5% (95% CI, 81.7%-88.6%) and measured its depth with an error of approximately ±0.5 cm compared with measurements obtained manually from the 2-dimensional ultrasound images. The spine midline was detected with a sensitivity of 93.9% (95% CI, 85.8%-97.7%) and specificity of 91.3% (95% CI, 83.6%-96.9%), and its lateral position within the ultrasound image was measured with an error of approximately ±0.3 cm. The bone enhancement imaging mode produced images with 5.1- to 10-fold enhanced bone contrast when compared with a comparable handheld ultrasound imaging system. CONCLUSIONS: The results of this study demonstrate the feasibility of CAD for assisting with real-time interpretation of ultrasound images of the lumbar spine at the bedside.

Haptoglobin 2-2 Phenotype Is Associated With Increased Acute Kidney Injury After Elective Cardiac Surgery in Patients With Diabetes Mellitus.

BACKGROUND: Recent studies reported an association between the 2-2 phenotype of haptoglobin (Hp 2-2) and increased cardiorenal morbidity in nonsurgical diabetic patients. Our goal was to determine whether the Hp 2-2 phenotype was associated with acute kidney injury (AKI) after elective cardiac surgery in patients with diabetes mellitus. METHODS AND RESULTS: We prospectively enrolled 99 diabetic patients requiring elective cardiac surgery with cardiopulmonary bypass. Haptoglobin phenotypes were determined by gel electrophoresis. Cell-free hemoglobin, haptoglobin, and total serum bilirubin were quantified as hemolysis markers. The primary outcome was postoperative AKI, as defined by the Acute Kidney Injury Network classification. The incidence of AKI was significantly higher in Hp 2-2 patients compared with patients without this phenotype (non-Hp-2-2; 55.6% versus 27%, P<0.01). The need for renal replacement therapy was also significantly higher in the Hp 2-2 group (5 patients versus 1 patient, P=0.02). Thirty-day mortality (3 versus 0 patients, P=0.04) and 1-year mortality (5 versus 0 patients, P<0.01) were also significantly higher in patients with the Hp 2-2 phenotype. In multivariable analysis, Hp 2-2 was an independent predictor of postoperative AKI (P=0.01; odds ratio: 4.17; 95% confidence interval, 1.35-12.48). CONCLUSIONS: Hp 2-2 phenotype is an independent predictor of postoperative AKI and is associated with decreased short and long-term survival after cardiac surgery in patients with diabetes mellitus.

Perioperative statin use is associated with decreased incidence of primary graft dysfunction after lung transplantation.

BACKGROUND: Primary graft dysfunction (PGD) is a major cause of early morbidity and mortality after lung transplantation. Statins reduce the risk of chronic rejection after lung transplantation, but their effects on PGD are unknown. We hypothesized that perioperative statin therapy decreases the risk for PGD after lung transplantation. METHODS: We retrospectively reviewed records of all patients undergoing lung transplantation between January 1999 and December 2014 at the University of Virginia Health System. The primary outcome was PGD (grades 1-3). Secondary outcomes included grade 3 PGD, length of intensive care unit and hospital stay, and mortality. RESULTS: Of 266 patients who met final inclusion criteria, 138 (52%) were diagnosed with PGD. In-hospital mortality among patients with PGD was 6.5%. There were no deaths in patients without PGD (p < 0.001). PGD was diagnosed in 24 patients taking statins (34.8%) and in 114 patients (57.9%) who did not take statins (p = 0.001). After propensity score adjustments, perioperative statin use was independently associated with a reduced risk for PGD (odds ratio [OR] 0.41, 95% confidence interval [CI] 0.20-0.84, p = 0.015) and reduced risk to develop grade 3 PGD (OR 0.42, 95% CI 0.18-0.94, p = 0.036). Other risk factors associated with PGD included intraoperative use of cardiopulmonary bypass (OR 3.74, 95% CI 1.75-8.02, p = 0.001) and positive donor smoking status (OR 2.27, 95% CI 1.18-4.35, p = 0.014). CONCLUSIONS: The results demonstrate that perioperative use of statins is independently associated with reduced risk for PGD after lung transplantation.

The redundancy of the mammalian heterochromatic compartment.

Two chromatin compartments are present in most mammalian cells; the first contains primarily euchromatic, early replicating chromatin and the second, primarily late-replicating heterochromatin, which is the subject of this review. Heterochromatin is concentrated in three intranuclear regions: the nuclear periphery, the perinucleolar space and in pericentromeric bodies. We review recent evidence demonstrating that the heterochromatic compartment is critically involved in global nuclear organization and the maintenance of genome stability, and discuss models regarding how this compartment is formed and maintained. We also evaluate our understanding of how heterochromatic sequences (herein named heterochromatic associated regions (HADs)) might be tethered within these regions and review experiments that reveal the stochastic nature of individual HAD positioning within the compartment. These investigations suggest a substantial level of functional redundancy within the heterochromatic compartment.

Prelamin A processing, accumulation and distribution in normal cells and laminopathy disorders.

Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.

Wash interacts with lamin and affects global nuclear organization.

The cytoplasmic functions of Wiskott-Aldrich syndrome family (WAS) proteins are well established and include roles in cytoskeleton reorganization and membrane-cytoskeletal interactions important for membrane/vesicle trafficking, morphogenesis, immune response, and signal transduction. Misregulation of these proteins is associated with immune deficiency and metastasis [1-4]. Cytoplasmic WAS proteins act as effectors of Rho family GTPases and polymerize branched actin through the Arp2/3 complex [1, 5]. Previously, we identified Drosophila washout (wash) as a new member of the WAS family with essential cytoplasmic roles in early development [6, 7]. Studies in mammalian cells and Dictyostelium suggest that WASH functions primarily in a multiprotein complex that regulates endosome shape and trafficking in an Arp2/3-dependent manner [8-11]. However, roles for classically cytoplasmic proteins in the nucleus are beginning to emerge, in particular, as participants in the regulation of gene expression [12, 13]. Here, we show that Drosophila Wash is present in the nucleus, where it plays a key role in global nuclear organization. wash mutant and knockdown nuclei disrupt subnuclear structures/organelles and exhibit the abnormal wrinkled morphology reminiscent of those observed in diverse laminopathies [14-16]. We find that nuclear Wash interacts with B-type Lamin (Lamin Dm0), and, like Lamin, Wash associates with constitutive heterochromatin. Wash knockdown increases chromatin accessibility of repressive compartments and results in a global redistribution of repressive histone modifications. Thus, our results reveal a novel role for Wash in modulating nucleus morphology and in the organization of both chromatin and non-chromatin nuclear sub-structures.

Functional redundancy in the nuclear compartmentalization of the late-replicating genome.

The eukaryotic nucleus is structurally and functionally organized, as reflected in the distribution of its protein and DNA components. The genome itself is segregated into euchromatin and heterochromatin that replicate in a distinct spatio-temporal manner. We used a combination of fluorescence in situ hybridization (FISH) and DamID to investigate the localization of the early and late replicating components of the genome in a lymphoblastoid cell background. Our analyses revealed that the bulk of late replicating chromatin localizes to the nuclear peripheral heterochromatin (PH) in a chromosome size and gene density dependent manner. Late replicating DNA on small chromosomes exhibits a much lower tendency to localize to PH and tends to associate with alternate repressive subcompartments such as pericentromeric (PCH) and perinucleolar heterochromatin (PNH). Furthermore, multicolor FISH analysis revealed that late replicating loci, particularly on the smaller chromosomes, may associate with any of these 3 repressive subcompartments, including more than one at the same time. These results suggest a functional equivalence or redundancy among the 3 subcompartments. Consistent with this notion, disruption of nucleoli resulted in an increased association of late replicating loci with peripheral heterochromatin. Our analysis reveals that rather than considering the morphologically distinct PH, PCH and PNH as individual subcompartments, they should be considered in aggregate as a functional compartment for late replicating chromatin.

TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends.

Telomeres protect the ends of linear genomes, and the gradual loss of telomeres is associated with cellular ageing. Telomere protection involves the insertion of the 3' overhang facilitated by telomere repeat-binding factor 2 (TRF2) into telomeric DNA, forming t-loops. We present evidence suggesting that t-loops can also form at interstitial telomeric sequences in a TRF2-dependent manner, forming an interstitial t-loop (ITL). We demonstrate that TRF2 association with interstitial telomeric sequences is stabilized by co-localization with A-type lamins (lamin A/C). We also find that lamin A/C interacts with TRF2 and that reduction in levels of lamin A/C or mutations in LMNA that cause an autosomal dominant premature ageing disorder--Hutchinson Gilford Progeria Syndrome (HGPS)-lead to reduced ITL formation and telomere loss. We propose that cellular and organismal ageing are intertwined through the effects of the interaction between TRF2 and lamin A/C on chromosome structure.