Acute myeloid leukemia (AML) is a heterogeneous disease caused by a variety of mutations in transcription factors, epigenetic regulators and signaling molecules. To determine how different mutant regulators establish AML subtype-specific transcriptional networks we performed a comprehensive global analysis of cis-regulatory element activity and interaction, transcription factor occupancy and gene expression patterns in purified leukemic blast cells. Here, we focussed on specific sub-groups of patients carrying mutations in genes encoding transcription factors ( RUNX1, CEBPA) and signaling molecules ( FTL3-ITD, RAS, NPM1). Integrated analyses of these data demonstrates that each mutant regulator establishes a specific transcriptional and signaling network unrelated to that seen in normal cells, sustaining the expression of unique sets of genes required for AML growth and maintenance.
Disorders of pain neural systems are frequently chronic and, when recalcitrant to treatment, can severely degrade the quality of life. The pain pathway begins with sensory neurons in dorsal root or trigeminal ganglia and the neuronal subpopulations that express the TRPV1 ion channel transduce sensations of painful heat and inflammation, and play a fundamental role in clinical pain arising from cancer and arthritis. In the present study we elucidate the complete transcriptomes of neurons from the TRPV1 lineage and a non-TRPV1 neuro-glial population in sensory ganglia through the combined application of next-gen deep RNA-Seq, genetic neuronal labeling with fluorescence-activated cell sorting, or neuron-selective chemoablation. RNA-Seq accurately quantitates gene expression, a difficult parameter to determine with most other methods especially for very low and very high expressed genes. Differentially expressed genes are present at every level of cellular function from the nucleus to the plasma membrane. We identified many ligand receptor pairs in the TRPV1 population suggesting that autonomous presynaptic regulation maybe a major regulatory mechanism in nociceptive neurons. The data define, in a quantitative, cell population specific fashion, the molecular signature of a distinct and clinically important group of pain-sensing neurons and provide an overall framework for understanding the transcriptome of TRPV1 nociceptive neurons.
Proteins that interact with 14-3-3 isoforms are involved in regulation of the cell cycle, intracellular trafficking/targeting, signal transduction, cytoskeletal structure and transcription. Recent novel roles for 14-3-3 isoforms include nuclear trafficking the direct interaction with cruciform DNA and with a number of receptors, small G-proteins and their regulators. Recent findings also show that the mechanism of interaction is also more complex than the initial finding of the novel phosphoserine/threonine motif. Non-phosphorylated binding motifs that can also be of high affinity may show a more isoform-dependent interaction and binding of a protein through two distinct binding motifs to a dimeric 14-3-3 may also be essential for full interaction. Phosphorylation of specific 14-3-3 isoforms can also regulate interactions. In many cases, they show a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of dimer formation may influence which of the 14-3-3-interacting proteins could be brought together. Mammalian and yeast 14-3-3 isoforms show a preference for dimerization with specific partners in vivo.
SummaryAcute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.
Although technically possible, few clinical laboratories across the world have implemented non-invasive prenatal diagnosis (NIPD) for selected single-gene disorders, mostly owing to the elevated costs incurred. Having previously proven that NIPD for X-linked disorders can be feasibly implemented in clinical practice, we have now developed a test for the NIPD of an autosomal-recessive disorder, spinal muscular atrophy (SMA). Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single-nucleotide polymorphisms across a 6 Mb genomic window on chromosome 5 containing the SMN1 gene. Maternal, paternal and proband DNA samples were also tested for haplotyping purposes. Sequencing data was analysed by relative haplotype dosage (RHDO). Six pregnant SMA carriers and 10 healthy pregnant donors were recruited through the NIPSIGEN study. Inheritance of the maternally and paternally derived alleles of the affected SMN1 gene was determined in the foetus by RHDO analysis for autosomal-recessive disorders. DNA from the proband (for SMA carriers) or an invasively obtained foetal sample (for healthy pregnant donors) was used to identify the maternal and paternal reference haplotypes associated with the affected SMN1 gene. Results for all patients correlated with known outcomes and showed a testing specificity and sensitivity of 100%. On top of showing high accuracy and reliability throughout the stages of validation, our novel test for NIPD of SMA is also affordable and viable for implementation into clinical service.
ObjectiveDevelopment of an accurate and affordable test for the non‐invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies (DMD/BMD) to implement in clinical practice.MethodCell‐free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single nucleotide polymorphisms (SNPs) across the dystrophin gene on chromosome X. Sequencing data were analysed by relative haplotype dosage.ResultsSeven healthy pregnant donors and two pregnant DMD carriers all bearing a male fetus were recruited through the non‐invasive prenatal diagnosis for single gene disorders study. Non‐invasive prenatal diagnosis testing was conducted by relative haplotype dosage analysis for X‐linked disorders where the genomic DNA from the chorionic villus sampling (for healthy pregnant donors) or from the proband (for pregnant DMD carriers) was used to identify the reference haplotype. Results for all patients showed a test accuracy of 100%, when the calculated fetal fraction was >4% and correlated with known outcomes. A recombination event was also detected in a DMD patient.ConclusionOur new test for NIPD of DMD/BMD has been shown to be accurate and reliable during initial stages of validation. It is also feasible for implementation into clinical service. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.
Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2-to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system.RNA sequencing | neuroendocrine regulation | differential expression | chronobiology
Lhx9 is a member of the LIM homeobox gene family. It is expressed during mammalian embryogenesis in the brain including the pineal gland. Deletion of Lhx9 results in sterility due to failure of gonadal development. The current study was initiated to investigate Lhx9 biology in the pineal gland. Lhx9 is highly expressed in the developing pineal gland of the rat with transcript abundance peaking early in development; transcript levels decrease postnatally to nearly undetectable levels in the adult, a temporal pattern that is generally similar to that reported for Lhx9 expression in other brain regions. Studies with C57BL/6J Lhx9−/− mutant mice revealed marked alterations in brain and pineal development. Specifically, the superficial pineal gland is hypoplastic, being reduced to a small cluster of pinealocytes surrounded by meningeal and vascular tissue. The deep pineal gland and the pineal stalk are also reduced in size. Although the brains of neonatal Lhx9−/− mutant mice appear normal, severe hydrocephalus develops in about 70 % of the Lhx9−/− mice at 5–8 weeks of age; these observations are the first to document that deletion of Lhx9 results in hydrocephalus and as such indicate that Lhx9 contributes to the maintenance of normal brain structure. Whereas hydrocephalus is absent in neonatal Lhx9−/−mutant mice, the neonatal pineal gland in these animals is hypoplastic. Accordingly, it appears that Lhx9 is essential for early development of the mammalian pineal gland and that this effect is not secondary to hydrocephalus.
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