TIF1gamma controls erythroid cell fate by regulating transcription elongation.

Recent genome-wide studies have demonstrated that pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1gamma mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1gamma-deficient animals. Biochemical analysis established physical interactions among TIF1gamma, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. Chromatin immunoprecipitation assays in human CD34(+) cells supported a TIF1gamma-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.

Blunt Head Injury in the Elderly: Analysis of the NEXUS II Injury Cohort.

BACKGROUND: Changes with aging make older patients vulnerable to blunt head trauma and alter the potential for injury and the injury patterns seen among this expanding cohort. High-quality care requires a clear understanding of the factors associated with blunt head injuries in the elderly. Our objective was to develop a detailed assessment of the injury mechanisms, presentations, injury patterns, and outcomes among older blunt head trauma patients. METHODS: We conducted a planned secondary analysis of patients aged 65 or greater who were enrolled in the National Emergency X-Radiography Utilization Study (NEXUS) Head Computed Tomography validation study. We performed a detailed assessment of the demographics, mechanisms, presentations, injuries, interventions, and outcomes among older patients. RESULTS: We identified 3,659 patients aged 65 years or greater, among the 11,770 patients enrolled in the NEXUS validation study. Of these older patients, 325 (8.9%) sustained significant injuries, as compared with significant injuries in 442 (5.4%) of the 8,111 younger patients. Older females (1,900; 51.9%) outnumbered older males (1,753; 47.9%), and occult presentations (exhibiting no high-risk clinical criteria beyond age) occurred in 48 (14.8%; 95% confidence interval (CI) 11.1 to 19.1) patients with significant injuries. Subdural hematomas (377 discreet lesions in 299 patients) and subarachnoid hemorrhages (333 discreet instances in 256 patients) were the most frequent types of injuries occurring in our elderly population. A ground-level fall was the most frequent mechanism of injury among all patients (2,211; 69.6%), those sustaining significant injuries (180; 55.7%), and those who died of their injuries (37; 46.3%), but mortality rates were highest among patients experiencing a fall from a ladder (11.8%; 4 deaths among 34 cases [95% CI 3.3% to 27.5%]) and automobile versus pedestrian events (10.7%; 16 deaths among 149 cases [95% CI 6.3% to 16.9%]). Among older patients who required neurosurgical intervention for their injuries, only 16.4% (95% CI 11.1% to 22.9%) were able to return home, 32.1% (95% CI 25.1% to 39.8%) required extended facility care, and 41.8% (95% CI 34.2% to 49.7%) died from their injuries. CONCLUSIONS: Older blunt head injury patients are at high risk of sustaining serious intracranial injuries even with low-risk mechanisms of injury, such as ground-level falls. Clinical evaluation is unreliable and frequently fails to identify patients with significant injuries. Outcomes, particularly after intervention, can be poor, with high rates of long-term disability and mortality.

Characteristics of Intracranial Injuries in Pediatric Patients Following Blunt Head Trauma.

OBJECTIVES: Pediatric head trauma is a frequent reason for presentation to the emergency department. Despite this, there are few reports on specific characteristics and injury patterns in head injured children. The goal of this study was to evaluate head injury patterns in children with blunt head injury and their prevalence by age group. METHODS: This is a planned secondary analysis of the NEXUS II Head CT validation study. Consecutive patients with blunt head trauma were enrolled between 2006 and 2015. Demographics and criteria from 2 clinical decision instruments (NEXUS and Canadian Head CT rules) were gathered at the time of enrollment. We abstracted and cataloged injuries for pediatric patients based on radiologist report. Frequencies of injuries and severity were analyzed by developmental age group. RESULTS: A total of 1018 pediatric patients were enrolled, 128 (12.6%) of whom had an injury on computed tomography scan. Median age was 11.9 (Interquartile range 4.5-15.5) for all patients and 12 (4.8-15.5) for injured patients. Of injured patients, 49 (38.3%) had a significant injury, and 27 (21.1%) received an intervention. Teenagers had the highest rate of significant injury (50%) and intervention (30%). Injuries were most frequently noted in the temporal (46.1%), frontal (45.3%), and parietal (45.3%) regions. Subarachnoid hemorrhage (29.7%) and subdural hematoma (28.9%) were the most common injuries observed.Intraparenchymal hemorrhage and cerebral edema were more prevalent in older age groups. The most common injury mechanism overall was fall from height (24.7%). Motor vehicle accidents and nonmotorized wheeled vehicle accidents were more common in older patients. CONCLUSIONS: Serious injuries requiring intervention were rarely encountered in pediatric patients experiencing blunt head trauma. Mechanisms of injury, type of injury, and rates of intervention varied between developmental age groups.

Physical Examination Sensitivity for Skull Fracture in Pediatric Patients With Blunt Head Trauma: A Secondary Analysis of the National Emergency X-Radiography Utilization Study II Head Computed Tomography Validation Study.

STUDY OBJECTIVE: We evaluated the emergency department (ED) providers' ability to detect skull fractures in pediatric patients presenting with blunt head trauma. METHODS: This was a secondary analysis of the National Emergency X-Radiography Utilization Study (NEXUS) Head computed tomography (CT) validation study. Demographics and clinical characteristics were analyzed for pediatric patients. Radiologist interpretations of head CT imaging were abstracted and cataloged. Detection of skull fractures was evaluated through provider response to specific clinical decision instrument criteria (NEXUS or Canadian head CT rules) at the time of initial patient evaluation. The presence of skull fracture was determined by formal radiologist interpretation of CT imaging. RESULTS: Between April 2006, and December 2015, 1,018 pediatric patients were enrolled. One hundred twenty-eight (12.5%) children had a notable injury reported on CT head. Skull fracture was present in most (66.4%) children with intracranial injuries. The sensitivity and specificity of provider physical examination to detect skull fractures was 18.5% (95% confidence interval 10.5% to 28.7%) and 96.6% (95.3% to 97.7%), respectively. The most common injuries associated with skull fractures were subarachnoid hemorrhage (27%) and subdural hematoma (22.3%). CONCLUSION: Skull fracture is common in children with intracranial injury after blunt head trauma. Despite this, providers were found to have poor sensitivity for skull fractures in this population, and these injuries may be missed on initial emergency department assessment.

Infantile Myelofibrosis and Myeloproliferation with CDC42 Dysfunction.

Studies of genetic blood disorders have advanced our understanding of the intrinsic regulation of hematopoiesis. However, such genetic studies have only yielded limited insights into how interactions between hematopoietic cells and their microenvironment are regulated. Here, we describe two affected siblings with infantile myelofibrosis and myeloproliferation that share a common de novo mutation in the Rho GTPase CDC42 (Chr1:22417990:C>T, p.R186C) due to paternal germline mosaicism. Functional studies using human cells and flies demonstrate that this CDC42 mutant has altered activity and thereby disrupts interactions between hematopoietic progenitors and key tissue microenvironmental factors. These findings suggest that further investigation of this and other related disorders may provide insights into how hematopoietic cell-microenvironment interactions play a role in human health and can be disrupted in disease. In addition, we suggest that deregulation of CDC42 may underlie more common blood disorders, such as primary myelofibrosis.

Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors.

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFß transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFß and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFß deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

ß3-Adrenergic receptor stimulation induces E-selectin-mediated adipose tissue inflammation.

Inflammation induced by wound healing or infection activates local vascular endothelial cells to mediate leukocyte rolling, adhesion, and extravasation by up-regulation of leukocyte adhesion molecules such as E-selectin and P-selectin. Obesity-associated adipose tissue inflammation has been suggested to cause insulin resistance, but weight loss and lipolysis also promote adipose tissue immune responses. While leukocyte-endothelial interactions are required for obesity-induced inflammation of adipose tissue, it is not known whether lipolysis-induced inflammation requires activation of endothelial cells. Here, we show that ß(3)-adrenergic receptor stimulation by CL 316,243 promotes adipose tissue neutrophil infiltration in wild type and P-selectin-null mice but not in E-selectin-null mice. Increased expression of adipose tissue cytokines IL-1ß, CCL2, and TNF-α in response to CL 316,243 administration is also dependent upon E-selectin but not P-selectin. In contrast, fasting increases adipose-resident macrophages but not neutrophils, and does not activate adipose-resident endothelium. Thus, two models of lipolysis-induced inflammation induce distinct immune cell populations within adipose tissue and exhibit distinct dependences on endothelial activation. Importantly, our results indicate that ß(3)-adrenergic stimulation acts through up-regulation of E-selectin in adipose tissue endothelial cells to induce neutrophil infiltration.

Traumatic injury to the posterior fossa: a secondary analysis and description of case series from the NEXUS head injury dataset.

Background: Traumatic brain injuries involving the posterior fossa are rare and case reports indicate they often result in severe outcomes. We seek to describe characteristics and outcomes of traumatic posterior fossa injuries. Methods: We performed a planned secondary analysis of all patients with posterior fossa injuries enrolled in the NEXUS head computed tomography (CT) validation study dataset. The dataset includes prospectively collected data on all patients undergoing non-contrast cranial CT following blunt traumatic head injury from April 2006 to December 2015, at four emergency departments comprising community and university sites, as well as urban, suburban and rural settings in California (Antelope Valley Hospital, San Francisco General Hospital, UCLA Ronald Reagan Medical Center, UCSF Fresno Community Regional Medical Center). We classified each patient into one of three injury patterns: Type I-notable traumatic injuries primarily above the tentorium, with minimal posterior fossa involvement; Type II-notable traumatic injuries both above and within the posterior fossa; and Type III-notable traumatic injuries primarily within the posterior fossa. We extracted demographic data for each patient as well as physician assessments of the NEXUS head CT and Canadian Head CT rule clinical criteria, mechanisms of injury, patient outcomes, and the location and types of intracranial injuries sustained. Findings: Of 11,770 patients in the database, 184 (1.6%) had posterior fossa injuries on CT imaging. Mean age was 55.4 years (standard deviation 22.5 years, range 2-96 years); 131 (71.2%) were males. We identified 63 patients with Type I injuries, 87 with Type II injuries, and 34 Type III injuries. The most common mechanisms of injury were falls (41%), pedestrian vs automobile (15%), and motor vehicle collisions (13%). On presentation most patients had altered mental status (72%), abnormal behavior (53%), or a neurologic deficit (55%). The majority of individuals, 151 (82%), had clinically important injuries and 111 (60%) required neurosurgical intervention. The dispositions for the subjects included 52 deaths (28%), 49 (27%) patients discharged home, and 48 (26%) discharged to rehabilitation facilities. When compared to individuals with Type I and Type II injuries, patients with Type III injuries had lower mortality (6% vs 30% and 35%) and higher percentage of patients discharged home (60% vs 19% and 21%). Interpretation: Patients with Type I and II injury patterns (those that involve both the posterior fossa and supratentorium) experienced high mortality and disability. Patients with Type III injuries (isolated posterior fossa) had a better prognosis. Funding: None.

Mitochondrial retrograde signaling connects respiratory capacity to thermogenic gene expression.

Mitochondrial respiration plays a crucial role in determining the metabolic state of brown adipose tissue (BAT), due to its direct roles in thermogenesis, as well as through additional mechanisms. Here, we show that respiration-dependent retrograde signaling from mitochondria to nucleus contributes to genetic and metabolic reprogramming of BAT. In mouse BAT, ablation of LRPPRC (LRP130), a potent regulator of mitochondrial transcription and respiratory capacity, triggers down-regulation of thermogenic genes, promoting a storage phenotype in BAT. This retrograde regulation functions by inhibiting the recruitment of PPARγ to the regulatory elements of thermogenic genes. Reducing cytosolic Ca2+ reverses the attenuation of thermogenic genes in brown adipocytes with impaired respiratory capacity, while induction of cytosolic Ca2+ is sufficient to attenuate thermogenic gene expression, indicating that cytosolic Ca2+ mediates mitochondria-nucleus crosstalk. Our findings suggest respiratory capacity governs thermogenic gene expression and BAT function via mitochondria-nucleus communication, which in turn leads to either a thermogenic or storage mode.

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via ß-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying ß-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of ß-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease.

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance--ROS and PKCε activity--were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation.

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation.

Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation.

About 10% of Down syndrome (DS) infants are born with a transient myeloproliferative disorder (DS-TMD) that spontaneously resolves within the first few months of life. About 20%-30% of these infants subsequently develop acute megakaryoblastic leukemia (DS-AMKL). Somatic mutations leading to the exclusive production of a short GATA1 isoform (GATA1s) occur in all cases of DS-TMD and DS-AMKL. Mice engineered to exclusively produce GATA1s have marked megakaryocytic progenitor (MkP) hyperproliferation during early fetal liver (FL) hematopoiesis, but not during postnatal BM hematopoiesis, mirroring the spontaneous resolution of DS-TMD. The mechanisms that underlie these developmental stage-specific effects are incompletely understood. Here, we report a striking upregulation of type I IFN-responsive gene expression in prospectively isolated mouse BM- versus FL-derived MkPs. Exogenous IFN-α markedly reduced the hyperproliferation FL-derived MkPs of GATA1s mice in vitro. Conversely, deletion of the α/ß IFN receptor 1 (Ifnar1) gene or injection of neutralizing IFN-α/ß antibodies increased the proliferation of BM-derived MkPs of GATA1s mice beyond the initial postnatal period. We also found that these differences existed in human FL versus BM megakaryocytes and that primary DS-TMD cells expressed type I IFN-responsive genes. We propose that increased type I IFN signaling contributes to the developmental stage-specific effects of GATA1s and possibly the spontaneous resolution of DS-TMD.

Differentiation-dependent interactions between RUNX-1 and FLI-1 during megakaryocyte development.

The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders.