Combination Effect of Rotator Cuff Repair with Secretome-hypoxia MSCs Ameliorates TNMD, RUNX2, and Healing Histology Score in Rotator Cuff Tear Rats.

Objectives: In order to treat a rat model of rotator cuff rupture, this work concentrated on the expression of TNMD and RUNX2, followed by rotator cuff repair and secretome-hMSCs. Methods: A total of thirty 10-weeks-old male Sprague-Dawley rats were separated into five groups randomly, RC on week 0, lesion treated with a rotator cuff repair and saline (RC + NaCl group, n = 6) for 2 and 8 weeks, and lesion treated with a rotator cuff repair and secretome-hMSCs (RC + secretome-hMSC group, n = 6) for 2 and 8 weeks. The supraspinatus and infraspinatus muscle-tendon units were obtained for histological and biomechanical investigation at 0, 2 and 8 weeks following injury. Results: The findings showed that, in comparison with the RC + NaCl group, secretome-hMSCs significantly improved tendon repair by upregulating TNMD and RUNX2 expression and histology score. Conclusion: Combining Secretome-hypoxia MSCs with RC healing may help rats with rotator cuff tears.

The predictive value of infrared thermal imaging (IRT) for peripheral artery disease: A systematic review.

BACKGROUND: Medical infrared thermal imaging (IRT) has been applied to research blood flow, breast cancer detection, and human body muscle performance. The benefits of IRT include the fact that it is noninvasive, quick, dependable, non-contact, capable of creating several recordings in a short period of time, and secure for both patients and medical professionals. We aimed to determine the predictive value of IRT for identifying and evaluating any interventional procedure in patients affected by peripheral artery disease (PAD) of any severity. METHODS: We searched the Cochrane Library, EMBASE, and PubMed on the topic of IRT and PAD until January 20,2023. We excluded gray literature as it is lacking credibility for not undergoing a peer-reviewed process. The search strategy includes the medical topic headings for "infrared thermal imaging" and "peripheral vascular disorders." The primary outcome of this systematic review was the variation in tissue perfusion in PAD patients. Each technique's technical characteristics and therapeutic use within PAD must be described in each included study. RESULTS: This systematic review included 2 case reports and 3 observational studies. By comparing the temperatures of PAD patients hands, legs, and feet, IRT might prove to be an unduly valuable tool for treating vascular illnesses, especially in light of the knowledge gained from the temperature distribution maps. CONCLUSION: This noninvasive method demonstrated encouraging results in the detection of various areas of foot perfusion and the screening of PAD, and it gave good findings in gauging the effects of any type of intervention.

The pathology of oxidative stress-induced autophagy in a chronic rotator cuff enthesis tear.

Partial-thickness rotator cuff tears (PTRCTs) are often found in daily orthopedic practice, with most of the tears occurring in middle-aged patients. An anaerobic process and imbalanced oxygenation have been observed in PTRCTs, resulting in oxidative stress. Studies have shown the roles of oxidative stress in autophagy and the potential of unregulated mechanisms causing disturbance in soft tissue healing. This article aims to review literature works and summarize the potential pathology of oxidative stress and unregulated autophagy in the rotator cuff enthesis correlated with chronicity. We collected and reviewed the literature using appropriate keywords, in addition to the manually retrieved literature. Autophagy is a normal mechanism of tissue repair or conversion to energy needed for the repair of rotator cuff tears. However, excessive mechanisms will degenerate the tendon, resulting in an abnormal state. Chronic overloading of the enthesis in PTRCTs and the hypovascular nature of the proximal tendon insertion will lead to hypoxia. The hypoxia state results in oxidative stress. An autophagy mechanism is induced in hypoxia via hypoxia-inducible factors (HIFs) 1/Bcl-2 adenovirus E1B 19-kDa interacting protein (BNIP) 3, releasing beclin-1, which results in autophagy induction. Reactive oxygen species (ROS) accumulation would induce autophagy as the regulator of cell oxidation. Oxidative stress will also remove the mammalian target of rapamycin (mTOR) from the induction complex, causing phosphorylation and initiating autophagy. Hypoxia and endoplasmic reticulum (ER) stress would initiate unfolded protein response (UPR) through protein kinase RNA-like ER kinase (PERK) and activate transcription factor 4, which induces autophagy. Oxidative stress occurring in the hypovascularized chronic rotator cuff tear due to hypoxia and ROS accumulation would result in unregulated autophagy directly or autophagy mediated by HIF-1, mTOR, and UPR. These mechanisms would disrupt enthesis healing.

Secretome of hypoxia-preconditioned mesenchymal stem cells enhance the expression of HIF-1a and bFGF in a rotator cuff tear model.

Aim To determine the effect of secretome hypoxia mesenchymal stem cells (SH-MSCs) on the relative gene expression of hypoxia inducible factor-1a (HIF-1a) and basic fibroblast growth factor (bFGF) in accelerating histomorphometric repair of tendon to bone interface healing in rats acute rotator cuff tear (RCT) model. Methods This is experimental research with posttest control group design. Thirty-male Wistar rats were divided into five treatment groups: healthy group and rotator cuff reconstruction group included four groups: SH-MSCs W2 (the treatment group was given a SH-MSCs 0.5 mL and terminated at weeks 2), NaCl W2 (the control vehicle group was given NaCl 0.5 mL and terminated at weeks 2), SH-MSCs W8 (the treatment group was given a SH-MSCs 0.5 mL and terminated at weeks 8), and NaCl W8 (the control vehicle group was given NaCl 0.5 mL and terminated at weeks 8). On the termination day, all the rats were terminated and HIF-1a and bFGF gene expression were analysed using qRT-PCR. Results SH-MSCs significantly increased the HIF-1a and bFGF gene expression than NaCl group even in week 2 and week 8. The highest increased gene expression of HIF-1a and bFGF was on week 8. Conclusion SH-MSCs are important in the healing repair process of tendon-to-bone interface in acute RCT model rats through increasing gene expression of HIF-1α and bFGF.

Physiological Roles of Hippo Signaling Pathway and Autophagy in Dementia.

BACKGROUND: Dementia is a neurocognitive disorder associated with the aging brain and mainly affects the hippocampus and cerebral cortex. The Hippo signaling pathway and autophagy proteins have been found to be perturbed in the brain affected by dementia processes. OBJECTIVE: This systematic review aims to elaborate on the involvement of the Hippo signaling pathway and autophagy in modulating the progression and severity of dementia in aging. METHODS: Searches were conducted on MEDLINE, Google Scholar, Scopus, and Web of Science databases. RESULTS: The Hippo signaling pathway is dependent upon the transcriptional co-activator YAP/TAZ, which forms complexes with TEAD in the nucleus in order to maintain cell homeostasis. When the expression YAP/TAZ is reduced, transcriptional repression-induced atypical cell death, ballooning cell death, and necrosis will consequently occur in the neurons. Moreover, the autophagic proteins, such as LC3, ATG proteins, and Beclin, are reduced, resulting in the disruption of autophagosome formation and accumulation and the spread of misfolded proteins in the brain suffering from dementia. CONCLUSION: The impairment of the Hippo signaling pathway and autophagy in the dementia process in aging should be considered since it might predict the severity, treatment, and prevention of dementia.

Ultrasound-Guided Suprascapular Nerve Block at Spinoglenoid Notch and Glenohumeral Joint Hydrodilation.

Hydrodilation of the glenohumeral joint is commonly employed as a nonsurgical intervention for the frozen shoulder. Accuracy and pain during the procedure can be regarded as difficulties in performing this procedure. Ultrasonography (USG) guided injection and suprascapular nerve block can improve the accuracy and can decrease pain during the hydrodilation procedure. We present the step-by-step method for performing USG-guided injections for suprascapular nerve block and hydrodilation.

Combine Approach of Proximal Fibula Osteotomy (PFO) Followed by Intra-Articular Dextrose Prolotherapy in Severe Medial Knee Osteoarthritis.

Background: Knee osteoarthritis (OA) is a chronic and progressive degenerative disease. It resulted from mechanical and chemical disorders that damage the joint and the underlying bone. The management of knee OA is challenging due to poor self-regeneration of connective tissues. Surgical treatment with prolotherapy approaches was conducted to treat medial compartment knee OA. Aim: To know the injection frequency to reach a 50% improvement in VAS score and WOMAC index. Methods: Six patients who suffered from late-stage medial compartment knee OA underwent PFO followed by twelve sessions of intra-articular dextrose prolotherapy. The subjective pain score, visual analog scale (VAS), was assessed based on the patient subjectiveness before and after treatment. Patients marked the score from 0 to 10 cm to describe the current pain state. The functional index, the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) index used to evaluate the Patient's clinical symptoms. It ranges from 0 to 96 points consisting of three main sections: pain (total 20 points), stiffness (total: eight points), and physical function disability (total 68 points). Higher scores indicate severe symptoms and function. Results: Four patients showed pain relief and functional improvement with more than 50% scores of VAS and WOMAC after the treatment. Two patients received more than twelve doses of intra-articular dextrose prolotherapy due to a lack of progress. Conclusion: This study provides clinical evidence for a new treatment strategy for advanced knee OA. This combined therapy improves the patient's daily activity function and postpones the need for total knee arthroplasty (TKA).

The Potential Use of Propolis as an Adjunctive Therapy in Breast Cancers.

Propolis is a resinous beehive product that has a wide range of biological activities, namely antimicrobial, antioxidant, and anti-inflammatory properties. Propolis is collected by the bees from plant resin and exudates to protect hives and maintain hive homeostasis. The aim of the present systematic scoping review is to explore the potential and suitability of propolis as an adjunctive treatment in breast cancers, based on the latest available experimental evidence (2012-2021). After applying the exclusion criteria, a total of 83 research publications were identified and retrieved from Scopus, Web of Science, and Pubmed. Several relevant key themes identified from the included studies were cytotoxicity, synergistic/combination treatment, improvement in bioavailability, human clinical trials, and others. A majority of the studies identified were still in the in vitro and in vivo stages. Nonetheless, we managed to identify 4 human clinical trials that demonstrated the successful use of propolis in alleviating side effects of chemotherapy and radiotherapy while increasing the quality of life of breast cancer patients, with minimal adverse effects. In conclusion, propolis, as an adjunctive treatment, may have therapeutic benefits in alleviating symptoms related to breast cancers. However, further clinical trials, preferably with higher number of participants/subjects/patients, are urgently needed.

Lumbar interlaminar epidural steroid injections for chronic low back- and lower extremity-pain in Sjogren's syndrome: A case report.

INTRODUCTION AND IMPORTANCE: Peripheral nervous system involvement is very common in Sjogren's syndrome (SS); however, polyradiculopathy has been reported rarely in association with SS, and predominantly chronic forms have been described. Here, we reported a case from our Neurosurgery Department in Intan Medika KIM Hospital, Bangka Island, Pangkalpinang, Indonesia; as Academic Health System of Universitas Padjadjaran. CASE PRESENTATION: A 32-year-old woman, diagnosed with Sjogren's syndrome that was characterized by anti-nuclear, anti-Ro, anti-La and anti dsDNA-antibodies positives since 3 years ago; consulted to our department for a chronic low back with a radicular pain in both lower limbs from the gluteal area to both feet together with numbness, hyperstesis and allodynia. The pain was evaluated by the visual analogue scale (VAS) score of 8; we then performed cervico-lumbal computed tomography (CT) scan that demonstrated multiple protruded discs of the cervical- and lumbar-spine. CLINICAL DISCUSSION: Pain was treated with lumbar interlaminar epidural steroid injections as a safe technique that allows relieving patient symptoms; after 10 min, the patient experienced an improvement in her pain with reduced scores to 0-1 in VAS, as well as a significant improvement on her quality of life later on. CONCLUSION: The use of lumbar interlaminar epidural steroid injections for an alternative therapeutic for neuropathic pain in SS gives a satisfactory result in terms of improvement of pain as well as a significant improvement on patients' quality of life.

The Potential Use of Propolis as a Primary or an Adjunctive Therapy in Respiratory Tract-Related Diseases and Disorders: A Systematic Scoping Review.

Propolis is a resinous beehive product that is collected by the bees from plant resin and exudates, to protect and maintain hive homeostasis. Propolis has been used by humans therapeutically to treat many ailments including respiratory tract-related diseases and disorders. The aim of the present systematic scoping review is to evaluate the experimental evidence to support the use of propolis as a primary or an adjunctive therapy in respiratory tract-related diseases and disorders. After applying the exclusion criteria, 158 research publications were retrieved and identified from Scopus, Web of Science, Pubmed, and Google Scholar. The key themes of the included studies were pathogenic infection-related diseases and disorders, inflammation-related disorders, lung cancers, and adverse effects. Furthermore, the potential molecular and biochemical mechanisms of action of propolis in alleviating respiratory tract-related diseases and disorders are discussed. In conclusion, the therapeutic benefits of propolis have been demonstrated by various in vitro studies, in silico studies, animal models, and human clinical trials. Based on the weight and robustness of the available experimental and clinical evidence, propolis is effective, either as a primary or an adjunctive therapy, in treating respiratory tract-related diseases.

High-Grade Bursal Side Rotator-Cuff Repair: A Surgical Outcome Review.

PURPOSE: We aimed to evaluate surgical outcomes of high-grade bursal rotator cuff-tear repairs. METHODS: This systematic review was performed in May 2020 in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines using PubMed. Inclusion criteria were English-language studies reporting the results of pain improvement, functional outcome scores, and radiographic examinations after repair of bursal side partial rotator-cuff tears at any time point in patients of any age and with all levels of evidence. Exclusion criteria were articles not in English, in vitro or animal studies, epidemiological studies, and such article types as technical notes or narrative reviews. RESULTS: Of 58 articles, five were included in this study, of which three and two had level III and IV evidence, respectively, four were comparative studies, and one was a case series. Visual analogue scales were used in four of the five studies, all showing improvement in pain assessment from 5.87 preoperatively to 1.02 postoperatively. All five studies showed significant improvement on each functional outcome score at the final follow-up. The retear rate for all studies was 10.97% (27 of 246). CONCLUSION: High-grade bursal side partial-thickness rotator cuff-tear repair gave satisfactory results in terms of pain scores, range of motion, and functional outcomes. The retear rate was still considerably high (10.9%), necessitating better understanding of the basic science, such as molecular mechanisms during adaptation, to improve the surgical technique.

Diabetic foot: Which one comes first, the ulcer or the contracture?

Diabetic foot is among the most common complications of patients with diabetes. One of the known causes of foot ulceration is ankle equinus, which increases the pressure on the plantar surface during ambulation. Conversely, equinus contracture can be caused by a complicated wound, and it may be due to prolonged immobilization. In this paper, we reviewed the pathogenesis of both conditions and their clinical considerations. Poor glycemic control in patients with diabetes may result in angiopathy and neuropathy as an underlying condition. An ulcer can be precipitated by an injury, improper foot care, or increased biomechanical loading as seen in elevated plantar pressure following equinus contracture. Equinus contracture may be a direct effect of hyperglycemia or can arise in combination with another pathway, for example, involving the activation of transforming growth factor ß. Static positioning resulting from any prior foot wound may develop fibrotic changes leading to contracture. Wound healing promoting factors can also result in overhealing outcomes such as hypertrophic scarring and fibrosis. The body's repair mechanism during the healing cascade activates repair cells and myofibroblasts, which also serve as the main producers and organizers of the extracellular matrix. Considering this intricate pathogenesis, appropriate interventions are essential for breaking the vicious cycle that may disturb wound healing.

Traumatic Inferior Glenohumeral Dislocation Associated With Rotator Cuff Avulsion Fracture: Arthroscopic-Assisted Fixation: A Technical Note.

Traumatic inferior glenohumeral dislocation with rotator cuff avulsion fracture rarely occurs and may cause chronic pain and diminished shoulder function. Several treatment options are available for this injury, such as open reduction internal fixation and arthroscopic-assisted reduction internal fixation. This technique describes a step-by-step technique to manage traumatic inferior glenohumeral dislocation with rotator cuff avulsion fracture using the simultaneous closed reduction procedure for traumatic inferior glenohumeral dislocation and the arthroscopic procedure with suture bridge technique for the treatment of rotator cuff avulsion fracture.

NAD- and NADPH-Contributing Enzymes as Therapeutic Targets in Cancer: An Overview.

Actively proliferating cancer cells require sufficient amount of NADH and NADPH for biogenesis and to protect cells from the detrimental effect of reactive oxygen species. As both normal and cancer cells share the same NAD biosynthetic and metabolic pathways, selectively lowering levels of NAD(H) and NADPH would be a promising strategy for cancer treatment. Targeting nicotinamide phosphoribosyltransferase (NAMPT), a rate limiting enzyme of the NAD salvage pathway, affects the NAD and NADPH pool. Similarly, lowering NADPH by mutant isocitrate dehydrogenase 1/2 (IDH1/2) which produces D-2-hydroxyglutarate (D-2HG), an oncometabolite that downregulates nicotinate phosphoribosyltransferase (NAPRT) via hypermethylation on the promoter region, results in epigenetic regulation. NADPH is used to generate D-2HG, and is also needed to protect dihydrofolate reductase, the target for methotrexate, from degradation. NAD and NADPH pools in various cancer types are regulated by several metabolic enzymes, including methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, and aldehyde dehydrogenase. Thus, targeting NAD and NADPH synthesis under special circumstances is a novel approach to treat some cancers. This article provides the rationale for targeting the key enzymes that maintain the NAD/NADPH pool, and reviews preclinical studies of targeting these enzymes in cancers.

Description of mutation spectrum and polymorphism of Wilms' tumor 1 (WT1) gene in hypospadias patients in the Indonesian population.

INTRODUCTION: Hypospadias is one of the most common congenital anomalies of the penis. Previous studies reported mutation of the Wilms' tumor 1 (WT1) gene as a cause of hypospadias. The aim of this study is to describe the WT1 mutation spectrum and polymorphism in hypospadias patients in Indonesia. MATERIAL AND METHODS: DNA was isolated from 74 hypospadias patients at the Division of Pediatric Surgery, Department of Surgery Hasan Sadikin Hospital. All exons in the WT1 gene were amplified by a PCR method, followed by Sanger sequencing. Mutation analysis was performed using BioEdit software and in silico analysis using Mutation Taster, Polymorphism Phenotyping-2 (PolyPhen-2), and Sorting Intolerant from Tolerant (SIFT). RESULT: DNA analysis results showed two types of heterozygous mutations in five subjects (Table), hence the frequency of WT1 mutations was 6.7% (10/148 allele). The first mutation was a missense mutation identified in twin boys. The second was a novel heterozygous alteration in the non-coding region nine bp upstream of exon 6 (c.366-9T>C), which was identified in three patients. One heterozygous polymorphism in the coding region of exon 7 (c.471A>G/rs16754) was identified in 10 subjects. This variant did not cause any change in amino acid products (silence polymorphism). Allele frequency for the G allele (mutant allele) and A allele (wild type) was 13.5% and 86.5%, respectively. DISCUSSION: WT1 is one of the best known hypospadias genes. The WT1 gene is involved in male genital development in the early and late periods of sex determination, and hence is known as a long-term expression gene in genitalia development. Mutation analysis of WT1 in a Chinese population identified that the WT1 mutation frequency was 4.4%. The WT1 mutation frequency identified in the present study was higher, at 6.7%. Coincidentally, research subjects with p.R158H variants were monozygotic twin siblings with midshaft hypospadias accompanied by undescended testis in one and penoscrotal hypospadia with micropenis in the other. The incidence of familial hypospadias in male siblings suffering from hypospadias was reported to be 9.6% in a study conducted by Sorensen et al. Moreover, in the present study polymorphism c.471A>G(rs16754) at exon 7 was identified heterozygously in 10 research subjects (minor allele frequency 13.5%). CONCLUSION: WT1 mutations were identified in only a few cases of hypospadias and most of these were syndromic. This result implies that mutation of WT1 is not a common cause of hypospadias in the Indonesian population.

Intergenerational effects of maternal birth weight, BMI, and body composition during pregnancy on infant birth weight: Tanjungsari Cohort Study, Indonesia.

BACKGROUND AND OBJECTIVES: Infant birth weight might be partly contributed to by maternal nutritional status at birth and maternal nutrition during pregnancy. The objective of this study was to analyze intergenerational maternal birth weight, maternal BMI, weight change during pregnancy, and maternal body composition (FM, FFM, and TBW) changes during pregnancy. METHODS AND STUDY DESIGN: We analyzed the associations between the maternal birth weight and body composition of 94 women and infant birth weight by using multiple regression adjusted for socioeconomic and reproductive history. RESULTS: All associations with infant birth weight were positive. The association between infant birth weight and maternal birth weight was 0.28 (95% CI: 0.02-0.54); that for the association between infant birth weight and maternal body weight in the first, second, and third trimesters was 15.1 (95% CI: 4.92-25.3), 13.7 (95% CI: 2.78-24.6), and 16.1 (95% CI: 5.22-27.0), respectively. The association between infant birth weight and fat mass in the second and third trimesters were 18.4 (95% CI: 3.38-33.5) and 16.1 (95% CI: 5.23-27.0), respectively, and those for the association between infant birth weight and fat-free mass in the first and third trimesters were 33.6 (6.38, 60.9) and 34.8 (95% CI: 3.47-66.1), respectively. CONCLUSIONS: This study confirms previous findings that maternal birth weight and body composition during pregnancy are associated with infant birth weight.

The NRAMP1 polymorphism as a risk factor for tuberculous spondylitis.

UNLABELLED: In the present study, we analysed the association between the incidence of tuberculous spondylitis with the Natural Resistance Associated Macrophage Protein 1 (NRAMP1, also known as Solute Carrier Family 11a member1) polymorphism by studying the genetic segregation of this polymorphism and the incidence of the disease among members of the West Javanese population undergoing surgery for tuberculous spondylitis at our institution. We compared the distribution of NRAMP1 polymorphism at two specific sites, namely D543N, and 3'UTR, among subjects with pulmonary tuberculosis and tuberculous spondylitis. We found no significant differences in distribution of polymorphism between the two groups, or between pulmonary tuberculosis and tuberculous spondylitis compared to healthy subjects. However, a pattern emerged in that polymorphisms at the two sites seemed to be protective against development of tuberculous spondylitis in our study population. We concluded that in the West Javanese population, there is no association between NRAMP1 polymorphism with the propensity for development of pulmonary tuberculosis or tuberculous spondylitis. In fact, NRAMP1 may provide protection against the development of tuberculous spondylitis. KEY WORDS: tuberculous spondylitis, NRAMP1, polymorphism.

Sequences sufficient for programming imprinted germline DNA methylation defined.

Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD) requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC); the second carries only the DMD and repeats (DR) from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.

Antagonism between DNA and H3K27 methylation at the imprinted Rasgrf1 locus.

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.

Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker.

Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5' of the promoter. A repeated sequence 3' of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.