Siah2 integrates mitogenic and extracellular matrix signals linking neuronal progenitor ciliogenesis with germinal zone occupancy.

Evidence is lacking as to how developing neurons integrate mitogenic signals with microenvironment cues to control proliferation and differentiation. We determine that the Siah2 E3 ubiquitin ligase functions in a coincidence detection circuit linking responses to the Shh mitogen and the extracellular matrix to control cerebellar granule neurons (CGN) GZ occupancy. We show that Shh signaling maintains Siah2 expression in CGN progenitors (GNPs) in a Ras/Mapk-dependent manner. Siah2 supports ciliogenesis in a feed-forward fashion by restraining cilium disassembly. Efforts to identify sources of the Ras/Mapk signaling led us to discover that GNPs respond to laminin, but not vitronectin, in the GZ microenvironment via integrin ß1 receptors, which engages the Ras/Mapk cascade with Shh, and that this niche interaction is essential for promoting GNP ciliogenesis. As GNPs leave the GZ, differentiation is driven by changing extracellular cues that diminish Siah2-activity leading to primary cilia shortening and attenuation of the mitogenic response.

The vitamin B12 processing enzyme, mmachc, is essential for zebrafish survival, growth and retinal morphology.

Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.

Oxygen Tension and the VHL-Hif1α Pathway Determine Onset of Neuronal Polarization and Cerebellar Germinal Zone Exit.

Postnatal brain circuit assembly is driven by temporally regulated intrinsic and cell-extrinsic cues that organize neurogenesis, migration, and axo-dendritic specification in post-mitotic neurons. While cell polarity is an intrinsic organizer of morphogenic events, environmental cues in the germinal zone (GZ) instructing neuron polarization and their coupling during postnatal development are unclear. We report that oxygen tension, which rises at birth, and the von Hippel-Lindau (VHL)-hypoxia-inducible factor 1α (Hif1α) pathway regulate polarization and maturation of post-mitotic cerebellar granule neurons (CGNs). At early postnatal stages with low GZ vascularization, Hif1α restrains CGN-progenitor cell-cycle exit. Unexpectedly, cell-intrinsic VHL-Hif1α pathway activation also delays the timing of CGN differentiation, germinal zone exit, and migration initiation through transcriptional repression of the partitioning-defective (Pard) complex. As vascularization proceeds, these inhibitory mechanisms are downregulated, implicating increasing oxygen tension as a critical switch for neuronal polarization and cerebellar GZ exit.

Path Tortuosity in Virtual Reality: A Novel Approach for Quantifying Behavioral Process in a Food Choice Context.

There is a pressing need to better understand how parents make feeding decisions for their children, but extant measures focus primarily on outcomes rather than examining the process of food choice as it unfolds. This exploratory study examined parents' translational movement as they moved throughout a virtual reality-based buffet restaurant to select a lunch for their child. Our aim was to explore whether translational movement would be related to cognitive and affective variables that underlie motivation, effort, and ultimate choices within food decision-making contexts (e.g., guilt, self-efficacy). Movement data were quantified in terms of path tortuosity: the degree of straightness of one's path while traveling through a space. Greater path tortuosity predicted a reduction in parents' guilt about their child feeding, above and beyond actual food chosen. Results suggest path tortuosity serves as an implicit measure of effort put forth by parents throughout the food decision-making process. Future work should continue to explore the utility of novel metrics that can be obtained from unique data sources, such as location tracking, for elucidating complicated behavioral processes such as food choice.

Macrophage derived TNFα promotes hepatic reprogramming to Warburg-like metabolism.

During infection, hepatocytes must undergo a reprioritization of metabolism, termed metabolic reprogramming. Hepatic metabolic reprogramming in response to infection begins within hours of infection, suggesting a mechanism closely linked to pathogen recognition. Following injection with polyinosinic:polycytidylic acid, a mimic of viral infection, a robust hepatic innate immune response could be seen involving the TNFα pathway at 2 h. Repeated doses led to the adoption of Warburg-like metabolism in the liver as determined by in vivo metabolic imaging, expression analyses, and metabolomics. Hepatic macrophages, Kupffer cells, were able to induce Warburg-like metabolism in hepatocytes in vitro via TNFα. Eliminating macrophages in vivo or blocking TNFα in vitro or in vivo resulted in abrogation of the metabolic phenotype, establishing an immune-metabolic axis in hepatic metabolic reprogramming. Overall, we suggest that macrophages, as early sensors of pathogens, instruct hepatocytes via TNFα to undergo metabolic reprogramming to cope with challenges to homeostasis initiated by infection. This work not only addresses a key component of end-organ physiology, but also raises questions about the side effects of biologics in the treatment of inflammatory diseases. KEY MESSAGES: • Hepatocytes develop Warburg-like metabolism in vivo during viral infection. • Macrophage TNFα promotes expression of glycolytic enzymes in hepatocytes. • Blocking this immune-metabolic axis abrogates Warburg-like metabolism in the liver. • Implications for patients being treated for inflammatory diseases with biologics.

Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

FGF21 underlies a hormetic response to metabolic stress in methylmalonic acidemia.

Methylmalonic acidemia (MMA), an organic acidemia characterized by metabolic instability and multiorgan complications, is most frequently caused by mutations in methylmalonyl-CoA mutase (MUT). To define the metabolic adaptations in MMA in acute and chronic settings, we studied a mouse model generated by transgenic expression of Mut in the muscle. Mut-/-;TgINS-MCK-Mut mice accurately replicate the hepatorenal mitochondriopathy and growth failure seen in severely affected patients and were used to characterize the response to fasting. The hepatic transcriptome in MMA mice was characterized by the chronic activation of stress-related pathways and an aberrant fasting response when compared with controls. A key metabolic regulator, Fgf21, emerged as a significantly dysregulated transcript in mice and was subsequently studied in a large patient cohort. The concentration of plasma FGF21 in MMA patients correlated with disease subtype, growth indices, and markers of mitochondrial dysfunction but was not affected by renal disease. Restoration of liver Mut activity, by transgenesis and liver-directed gene therapy in mice or liver transplantation in patients, drastically reduced plasma FGF21 and was associated with improved outcomes. Our studies identify mitocellular hormesis as a hepatic adaptation to metabolic stress in MMA and define FGF21 as a highly predictive disease biomarker.

A direct link between MITF, innate immunity, and hair graying.

Melanocyte stem cells (McSCs) and mouse models of hair graying serve as useful systems to uncover mechanisms involved in stem cell self-renewal and the maintenance of regenerating tissues. Interested in assessing genetic variants that influence McSC maintenance, we found previously that heterozygosity for the melanogenesis associated transcription factor, Mitf, exacerbates McSC differentiation and hair graying in mice that are predisposed for this phenotype. Based on transcriptome and molecular analyses of Mitfmi-vga9/+ mice, we report a novel role for MITF in the regulation of systemic innate immune gene expression. We also demonstrate that the viral mimic poly(I:C) is sufficient to expose genetic susceptibility to hair graying. These observations point to a critical suppressor of innate immunity, the consequences of innate immune dysregulation on pigmentation, both of which may have implications in the autoimmune, depigmenting disease, vitiligo.

Drebrin-mediated microtubule-actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection.

Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule-actomyosin crosstalk is required for a neuron's 'two-stroke' nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule-actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbations of drebrin demonstrate that proximal leading process microtubule-actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule-actomyosin interfaces during neuronal differentiation.

Identifying eating behavior phenotypes and their correlates: A novel direction toward improving weight management interventions.

Common reports of over-response to food cues, difficulties with calorie restriction, and difficulty adhering to dietary guidelines suggest that eating behaviors could be interrelated in ways that influence weight management efforts. The feasibility of identifying robust eating phenotypes (showing face, content, and criterion validity) was explored based on well-validated individual eating behavior assessments. Adults (n = 260; mean age 34 years) completed online questionnaires with measurements of nine eating behaviors including: appetite for palatable foods, binge eating, bitter taste sensitivity, disinhibition, food neophobia, pickiness and satiety responsiveness. Discovery-based visualization procedures that have the combined strengths of heatmaps and hierarchical clustering were used to investigate: 1) how eating behaviors cluster, 2) how participants can be grouped within eating behavior clusters, and 3) whether group clustering is associated with body mass index (BMI) and dietary self-efficacy levels. Two distinct eating behavior clusters and participant groups that aligned within these clusters were identified: one with higher drive to eat and another with food avoidance behaviors. Participants' BMI (p = 0.0002) and dietary self-efficacy (p < 0.0001) were associated with cluster membership. Eating behavior clusters showed content and criterion validity based on their association with BMI (associated, but not entirely overlapping) and dietary self-efficacy. Identifying eating behavior phenotypes appears viable. These efforts could be expanded and ultimately inform tailored weight management interventions.

Zeb1 controls neuron differentiation and germinal zone exit by a mesenchymal-epithelial-like transition.

In the developing mammalian brain, differentiating neurons mature morphologically via neuronal polarity programs. Despite discovery of polarity pathways acting concurrently with differentiation, it's unclear how neurons traverse complex polarity transitions or how neuronal progenitors delay polarization during development. We report that zinc finger and homeobox transcription factor-1 (Zeb1), a master regulator of epithelial polarity, controls neuronal differentiation by transcriptionally repressing polarity genes in neuronal progenitors. Necessity-sufficiency testing and functional target screening in cerebellar granule neuron progenitors (GNPs) reveal that Zeb1 inhibits polarization and retains progenitors in their germinal zone (GZ). Zeb1 expression is elevated in the Sonic Hedgehog (SHH) medulloblastoma subgroup originating from GNPs with persistent SHH activation. Restored polarity signaling promotes differentiation and rescues GZ exit, suggesting a model for future differentiative therapies. These results reveal unexpected parallels between neuronal differentiation and mesenchymal-to-epithelial transition and suggest that active polarity inhibition contributes to altered GZ exit in pediatric brain cancers.

Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/ß-catenin defects in neuronal cholesterol synthesis phenotypes.

Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7, which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment, behavioral abnormalities and nervous system defects, though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS, we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation, not cholesterol deficiency, is critical for SLOS-associated defects. We further identified downregulation of Wnt/ß-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs, suggesting that Wnt signaling may be a promising therapeutic target for SLOS.

Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.

Global Gene Expression Profiling in Omental Adipose Tissue of Morbidly Obese Diabetic African Americans.

BACKGROUND: Adipose tissues play important role in the pathophysiology of obesity-related diseases including type 2 diabetes (T2D). To describe gene expression patterns and functional pathways in obesity-related T2D, we performed global transcript profiling of omental adipose tissue (OAT) in morbidly obese individuals with or without T2D. METHODS: Twenty morbidly obese (mean BMI: about 54 kg/m2) subjects were studied, including 14 morbidly obese individuals with T2D (cases) and 6 morbidly obese individuals without T2D (reference group). Gene expression profiling was performed using the Affymetrix U133 Plus 2.0 human genome expression array. Analysis of covariance was performed to identify differentially expressed genes (DEGs). Bioinformatics tools including PANTHER and Ingenuity Pathway Analysis (IPA) were applied to the DEGs to determine biological functions, networks and canonical pathways that were overrepresented in these individuals. RESULTS: At an absolute fold-change threshold of 2 and false discovery rate (FDR) < 0.05, 68 DEGs were identified in cases compared to the reference group. Myosin X (MYO10) and transforming growth factor beta regulator 1 (TBRG1) were upregulated. MYO10 encodes for an actin-based motor protein that has been associated with T2D. Telomere extension by telomerase (HNRNPA1, TNKS2), D-myo-inositol (1, 4, 5)-trisphosphate biosynthesis (PIP5K1A, PIP4K2A), and regulation of actin-based motility by Rho (ARPC3) were the most significant canonical pathways and overlay with T2D signaling pathway. Upstream regulator analysis predicted 5 miRNAs (miR-320b, miR-381-3p, miR-3679-3p, miR-494-3p, and miR-141-3p,) as regulators of the expression changes identified. CONCLUSION: This study identified a number of transcripts and miRNAs in OAT as candidate novel players in the pathophysiology of T2D in African Americans.

Necdin is a breast cancer metastasis suppressor that regulates the transcription of c-Myc.

Metastasis is the primary cause of death in breast cancer. Earlier studies using a mammary tumorigenesis mouse model identified Necdin (Ndn)as a germline modifier of metastasis. Differential expression of Ndn induces a gene-expression signature that predicts prognosis in human breast cancer. Additionally, a non-synonymous germline single nucleotide polymorphism (T50C; V17A) in Ndn distinguishes mouse strains with differing metastatic capacities. To better understand how hereditary factors influence metastasis in breast cancer, we characterized NDN-mediated transcription. Haplotype analysis in a well-characterized breast cancer cohort revealed that NDN germline variation is associated with both NDN expression levels and patient outcome. To examine the role of NDN in mammary tumor metastasis and transcriptional regulation, mouse mammary tumor cell lines stably over-expressing either the wildtype 50T or variant 50C Ndn allele were generated. Cells over-expressing Ndn 50T, but not Ndn 50C, exhibited significant decrease in cell invasiveness and pulmonary metastases compared to control cells. Transcriptome analyses identified a 71-gene expression signature that distinguishes cells over-expressing the two Ndn allelic variants. Furthermore, ChIP assays revealed c-Myc, a target gene of NDN, to be differentially regulated by the allelic variants. These data demonstrate that NDN and the T50C allele regulate gene expression and metastasis efficiency.

Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy.

The use of adeno-associated virus (AAV) as a gene therapy vector has been approved recently for clinical use and has demonstrated efficacy in a growing number of clinical trials. However, the safety of AAV as a vector has been challenged by a single study that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. Most studies have not noted genotoxicity following AAV-mediated gene delivery; therefore, the possibility that there is an association between AAV and HCC is controversial. Here, we performed a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the RNA imprinted and accumulated in nucleus (Rian) locus, and the resulting overexpression of proximal microRNAs and retrotransposon-like 1 (Rtl1) were associated with HCC. In addition, we demonstrated that the AAV vector dose, enhancer/promoter selection, and the timing of gene delivery are all critical factors for determining HCC incidence after AAV gene delivery. Together, our results define aspects of AAV-mediated gene therapy that influence genotoxicity and suggest that these features should be considered for design of both safer AAV vectors and gene therapy studies.

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons.

BACKGROUND: During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. RESULTS: We show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. CONCLUSIONS: We propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.

Metastasis-associated protein ribosomal RNA processing 1 homolog B (RRP1B) modulates metastasis through regulation of histone methylation.

UNLABELLED: Overexpression of ribosomal RNA processing 1 homolog B (RRP1B) induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. Here, the chromatin-binding properties of RRP1B were examined to define how it regulates metastasis-associated transcription. To identify genome-wide RRP1B-binding sites, high-throughput ChIP-seq was performed in the human breast cancer cell line MDA-MB-231 and HeLa cells using antibodies against endogenous RRP1B. Global changes in repressive marks such as histone H3 lysine 9 trimethylation (H3K9me3) were also examined by ChIP-seq. Analysis of these samples identified 339 binding regions in MDA-MB-231 cells and 689 RRP1B-binding regions in HeLa cells. Among these, 136 regions were common to both cell lines. Gene expression analyses of these RRP1B-binding regions revealed that transcriptional repression is the primary result of RRP1B binding to chromatin. ChIP-reChIP assays demonstrated that RRP1B co-occupies loci with decreased gene expression with the heterochromatin-associated proteins, tripartite motif-containing protein 28 (TRIM28/KAP1), and heterochromatin protein 1-α (CBX5/HP1α). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1α complex. In addition, RRP1B upregulation, which is associated with metastasis suppression, induced global changes in histone methylation. IMPLICATIONS: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochromatinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer.

Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects.

Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease of the hematopoietic system that is caused by heterozygous mutations in RUNX1. FPD/AML patients have a bleeding disorder characterized by thrombocytopenia with reduced platelet numbers and functions, and a tendency to develop AML. No suitable animal models exist for FPD/AML, as Runx11/2 mice and zebra fish do not develop bleeding disorders or leukemia. Here we derived induced pluripotent stem cells (iPSCs) from 2 patients in a family with FPD/AML, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in 1 FPD iPSC line through gene targeting, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients.

Distinct microRNA expression signatures are associated with melanoma subtypes and are regulated by HIF1A.

The complex genetic changes underlying metastatic melanoma need to be deciphered to develop new and effective therapeutics. Previously, genome-wide microarray analyses of human melanoma identified two reciprocal gene expression programs, including transcripts regulated by either transforming growth factor, beta 1 (TGFß1) pathways, or microphthalmia-associated transcription factor (MITF)/SRY-box containing gene 10 (SOX10) pathways. We extended this knowledge by discovering that melanoma cell lines with these two expression programs exhibit distinctive microRNA (miRNA) expression patterns. We also demonstrated that hypoxia-inducible factor 1 alpha (HIF1A) is increased in TGFß1 pathway-expressing melanoma cells and that HIF1A upregulates miR-210, miR-218, miR-224, and miR-452. Reduced expression of these four miRNAs in TGFß1 pathway-expressing melanoma cells arrests the cell cycle, while their overexpression in mouse melanoma cells increases the expression of the hypoxic response gene Bnip3. Taken together, these data suggest that HIF1A may regulate some of the gene expression and biological behavior of TGFß1 pathway-expressing melanoma cells, in part via alterations in these four miRNAs.