Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic dysregulation of the chromosome 4q35 D4Z4 macrosatellite. However, this does not account for the tissue specificity of FSHD pathology, which requires stable expression of an alternative full-length mRNA splice form of DUX4 (DUX4-fl) from the D4Z4 array in skeletal muscle. Here, we describe the identification of two enhancers, DUX4 myogenic enhancer 1 (DME1) and DME2 which activate DUX4-fl expression in skeletal myocytes but not fibroblasts. Analysis of the chromatin revealed histone modifications and RNA polymerase II occupancy consistent with DME1 and DME2 being functional enhancers. Chromosome conformation capture analysis confirmed association of DME1 and DME2 with the DUX4 promoter in vivo. The strong interaction between DME2 and the DUX4 promoter in both FSHD and unaffected primary myocytes was greatly reduced in fibroblasts, suggesting a muscle-specific interaction. Nucleosome occupancy and methylome sequencing analysis indicated that in most FSHD myocytes, both enhancers are associated with nucleosomes but have hypomethylated DNA, consistent with a permissive transcriptional state, sporadic occupancy, and the observed DUX4 expression in rare myonuclei. Our data support a model in which these myogenic enhancers associate with the DUX4 promoter in skeletal myocytes and activate transcription when epigenetically derepressed in FSHD, resulting in the pathological misexpression of DUX4-fl.
The early promise of institutional repositories is beginning to bear fruit. Medical libraries with institutional repositories, like other academic libraries, have found that their repositories support new ways of engaging with researchers and meeting the challenges posed by the transformation in scholarly communication over the past decade exemplified by open access, the National Institutes of Health Public Access Policy, campus-based publishing, and the sharing of research data. Institutional repositories can grow and thrive in academic health sciences libraries and be a vital component in the provision of library services to faculty, researchers, staff, and students.
The Nef-like effect of murine leukemia virus glycosylated gag on HIV-1 infectivity is mediated by its cytoplasmic domain and depends on the AP-2 adaptor complex
Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the μ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis.
IMPORTANCE: The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.
The nematode Caenorhabditis elegans (C. elegans) has four Sir2 paralogs, sir-2.1, sir-2.2, sir-2.3, and sir-2.4. Thus far, most of the research tools to study worm sirtuins have been developed for sir-2.1, due to its homology to yeast SIR2 and human SIRT1. Here, we have compiled a listing of the currently available strains (including both loss-of-function alleles and transgenics), antibodies, and RNAi constructs relevant to studies on all C. elegans sirtuin family members. We also describe the methods used in the analysis of C. elegans sirtuin function, including life span analysis, various stress-resistance assays, and fat content analysis and provide an overview of all phenotypic data relevant to C. elegans sir-2.1.
Ceramide transfer protein deficiency compromises organelle function and leads to senescence in primary cells
Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.
Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8
Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5' end of exon 8, a 33nt RNA from 3' end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations in SMN1, a gene that encodes an SMN protein, which regulates assembly/disassembly of U snRNP and is suggested to direct axonal transport of beta-actin mRNA in neurons, are responsible for most cases of SMA disease. However, humans contain a second SMN gene called SMN2. Unlike SMN1, the majority of SMN2 mRNA don't include exon 7. Here we show that increased expression of PSF significantly promotes inclusion of exon 7 in the SMN2 and SMN1 mRNA, whereas reduced expression of PSF promotes exon 7 skipping in various cell lines and in fibroblast cells from SMA patients. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2beta in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA.
To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.
Background: Radiostereometric Analysis (RSA) is a method for performing highly accurate three-dimensional measurements in-vivo using sequential radiographs. RSA has been used extensively for monitoring prosthesis fixation in hip and knee replacements. Recently, there has been increasing interest in applying RSA towards the monitoring of fracture healing.
Objective: The objective of this study was to evaluate the feasibility of using RSA to measure strain, stress, and plate migration in a distal femur fracture model.
Methods: Femoral sawbones with a distal femur fracture were used as models. A distal femur condylar locking compression plate (LCP) was used to reduce the fracture model. Stainless steel screws were used to fasten the plate to the sawbone. In addition, translucent polyester screws were composed, embedded with 1mm steel beads, and fastened to the most proximal and distal portions of the plate. This allowed for recognition by the RSA imaging modality. The femoral sawbones were then placed in a mechanical testing complex and RSA X-rays taken at different forces of compression. The radiographs were analyzed for plate migration using the 1mm steel beads as points of reference.
Results: Preliminary data indicate that it is possible to use a plate model that incorporates a micro-bead system to measure migration. Further analysis will quantify the amount of migration to determine whether significant changes occur at different stages of compression.
Conclusion: The ability to measure plate migration in a Radiostereometric Analysis X-ray model is an important step towards improving the ability of orthopedic surgeons to monitor fracture healing and prevent non-union. The next stage of this research will involve using this model in clinical trials of distal femur fractures and building a database to correlate levels of plate migration with surgical outcome.
Data from: The Effects of Vitamin D Supplementation on Hepatic Dysfunction, Vitamin D Status, and Glycemic Control in Children and Adolescents with Vitamin D Deficiency and Either Type 1 or Type 2 Diabetes Mellitus
Background: The effects of vitamin D supplementation on mild hepatic dysfunction and glycemic control are unclear in children and adolescents with either type 1 (T1D) or type 2 diabetes (T2D).
Hypothesis: Vitamin D supplementation will improve hepatic dysfunction and glycemic control.
Aim: To determine the effect of vitamin D supplementation on alanine transaminase (ALT), hemoglobin A1c (HbA1c), and serum 25-hydroxyvitamin D [25(OH)D] concentration.
Subjects and Methods: A retrospective study of 131 subjects with either T1D (n=88: 46 females, 42 males), or T2D ( n=43: 26 females, 17 males) of ages 3-18 years between 2007-2013. All subjects had (1) a diagnosis of diabetes for >12 mo, (2) received vitamin D supplementation for the management of vitamin D deficiency (3) had baseline and subsequent simultaneous measurements of HbA1c, ALT, and 25(OH)D. Vitamin D deficiency was defined as 25(OH)D concentration of <50nmol/L (20 ng/mL).
Results: At baseline, vitamin D deficiency occurred in 72.1% of patients with T2D and in 37.5% of T1D patients (p
Conclusions: Vitamin D supplementation in subjects with T2D was associated with statistically significant decreases in BMI SDS, ALT, and a clinically-significant decrease in HbA1c.
Background: Osteoporosis is the most common bone disease in the United States, and it is particularly common among women with multiple sclerosis (MS). However, despite this association, the temporal relationship between these two conditions has not been previously studied. Data from the Women’s Health Initiative provides a unique opportunity to examine the risk of developing osteoporosis over time in individuals diagnosed with MS.
Objective: The purpose of this study is to refine the relationship between MS and osteoporosis, clarifying the impact of environmental and pharmacologic factors on each condition, as well as addressing treatment and preventative efforts for a patient population at a greater potential risk for osteoporosis.
Methods: The study sample, derived from the Women’s Health Initiative, included 449 women who reported an MS diagnosis at baseline and 161,359 women without MS who comprised a control group. Baseline measures of self-reported osteoporosis, age, smoking status, steroid and anti-inflammatory use, and supplementary as well as dietary calcium and vitamin D were compared. MS patients reporting osteoporosis at baseline were removed, resulting in 355 women with MS to monitor for time to incident osteoporosis. Survival analyses were performed on follow-up data gathered annually between 1993 and 2005 to factor out significant associations of additional factors. Proportions of participants on osteoporosis-related medications as well as latency to use were compared between the multiple sclerosis and control cohorts.
Results: At baseline, women with MS are nearly three times as likely to report osteoporosis (p
Conclusions: A higher prevalence of osteoporosis at baseline suggests MS may significantly increase the risk of osteoporosis in premenopausal women. In contrast, environmental and pharmacologic variables appear to have a more significant role in the post-menopausal population. While osteoporosis was treated similarly between both groups, the point for intervention or prevention of osteoporosis in MS patients may be earlier in the disease course.
Demographic Characteristics Associated with the Presence of Recalled and Measured Prepregnancy Weights
Background: Gestational weight gain within prepregnancy BMI-specific Institute of Medicine (IOM) recommended ranges are associated with good outcomes for both mother and baby. Availability of measured prepregnancy weight, recalled prepregnancy weight or measured weight at first prenatal visit if the former two weights are not available, influences the accuracy of provider recommendations for gestational weight gain.
Objective: The purpose of this study is to examine demographic characteristics associated with the presence of recalled prepregnancy weight and measured prepregnancy weight in the prenatal care medical record.
Methods: Medical record review of 1,998 randomly selected pregnancies, of which 1,911 met inclusion criteria of delivery between January 2007 and December 2012 and receipt of prenatal care in faculty and resident clinic sites at UMass Memorial Health Care (UMMHC). Subjects' paper prenatal chart and electronic record (AllScripts and QS prenatal EMR) were fully abstracted if available and contained both: (1) a recorded measured weight within one year of conception, and (2) a self-reported prepregnancy weight obtained at first prenatal visit. Additionally, exclusion criteria included those pregnancies with only prenatal weights recorded one year prior to conception for index pregnancy. For women with multiple pregnancies during the study time period, one pregnancy was randomly selected for inclusion in study analyses. Demographic data was abstracted for all available charts regardless of presence or absence of weights of interest. Demographic characteristics considered were age (15-29, 20-24, 25-29, 30-34, 35+ years), prepregnancy BMI calculated based on recalled height and weight (underweight: BMI<18.5 kg/m2, normal weight: 18.5≤ BMI<25 kg/m2, overweight: 25≤BMI<30 kg/m2, and obese: 30 kg/m2≤BMI), race/ethnicity (non-Hispanic white vs. other race/ethnicity), marital status (not married vs. married), primary language (non-English vs. English), gravidity (1, 2, 3+), education (high school diploma or less, some college, 4 year college or more) and prenatal care site (faculty vs. resident obstetric clinic). Logistic regressions were performed to calculate crude and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) and adjusted analyses controlled for demographics.
Results: Of the 1911 pregnancies meeting initial inclusion criteria, 1711 (89.5%) had charts available for abstraction; fifty-three subjects had multiple pregnancies of which only one was included in analyses resulting in an analytic sample of 1656 pregnancies. Of these, 511 (30.9%) were missing a recalled prepregnancy weight at first prenatal visit, 711 (42.9%) had the recalled prepregnancy weight but did not have a measured weight; and only 434 (26.2%) had both weights of interest. Overweight women had decreased odds of having a recalled weight compared to women of normal weight (aOR 0.75; 95% CI 0.56-1.00). Additionally, women with ≥4 years of college compared to those with ≤ high school diploma (aOR 0.54; 95% CI 0.40-0.73), and those receiving care in the faculty compared to the resident clinics (aOR 0.48; 95% CI 0.35-0.65) had decreased odds of having a recalled weight available in the chart. Among women with available recalled prepregnancy weight (n=1101), 390 (35.4%) also had a documented measured weight within one year of conception and 711 (64.6%) did not. Women who were not married (aOR 0.54; 95% 0.39-0.76) had decreased odds of having a measured weight, whereas those receiving care in the faculty compared to resident clinics had greater odds (aOR 1.79; 95% CI 1.26-2.53) of having a measured weight within one year of conception available in their charts.
Conclusions: Our results suggest that approximately 25% of women have both recalled weight at first prenatal visit and at least one weight measured within one year of conception in their medical records. Prepregnancy BMI, education, and prenatal care site were associated with presence or absence of recalled weight. Similarly, amongst those with recalled weight, martial status and prenatal care in faculty practice where associated with decreased and increased odds respectively of having a measured weight within one year of conception. We can use this information to help practitioners target women for which greater efforts are needed to provide accurate IOM-recommended BMI-specific gestational weight gain guidelines. This may be utilized to discern patterns of health care access in this patient population.
Background: Arthritis is the leading cause of disability among adults in the United States affecting twenty-one million adults. In addition, osteoarthritis is the second most costly chronic condition in the U.S. Physical activity is a challenge in all patients and is associated with fewer functional limitations and lower risk for developing illness. Currently, there are no objective measures of physical activity in advanced knee OA.
Objectives: The purpose of this study was to quantify patient-reported changes in pain and function during the natural progression of osteoarthritis at 3, 6, and 9 months, and to correlate these metrics with objective activity monitors.
Methods: 50 patients who were undergoing non-operative management of OA were enrolled. Patients were seen at baseline, 3 months, 6 months, and 9 months. At each visit, basic demographics and patient-reported measures (SF-36, WOMAC, and Charlson Co-morbidity index) were recorded. In addition, patients wore ActiGraph and activPal activity monitors for 7 days following the visit.
Results: The average age of the enrolled participants was 57 with 82% of participants being less than 65 years of age. Most participants were female (64%), and 80% of participants had 1 or fewer medical co-morbidities on the Charlson Co-morbidity Index. Only 4% of patients were using assistive devices. The average WOMAC pain score was 68 and did not change from one time period to the next. The average SF-36 PCS score was 38 and the MCS was 54, and neither changed over time. The average SF-36 PCS score in patients with a WOMAC pain score less than 80 was 36, while in those with a WOMAC pain score greater than 80 it was 42.5. In contrast, analyses of the activPal found a decline in activity over the time period. In the first 19 patients wearing the activPal who were analyzed, 12 of 19 increased sedentary time at 9 months by an average of 18%. In addition, 15 of 19 participants decreased minutes of moderate to vigorous physical activity (MVPA) at 9 months by an average of 26%.
Conclusions: In our study of 50 participants with osteoarthritis, patient-reported function did not change over a 9-month period. However, preliminary activity data suggests a decline. Further work will correlate patient-reported measures to the objective measures recorded by activity monitors to determine if objective monitors are preferable to detect early changes in activity due to OA.
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