Small hydrophobic hormones like steroids control many tissue-specific physiological responses in higher organisms. Hormone response is characterized by changes in gene expression, but the molecular details connecting target-gene transcription to the physiology of responding cells remain elusive. The salivary glands of Drosophila provide an ideal model system to investigate gaps in our knowledge, because exposure to the steroid 20-hydroxyecdysone (20E) leads to a robust regulated secretion of glue granules after a stereotypical pattern of puffs (activated 20E-regulated genes) forms on the polytene chromosomes. Here, we describe a convenient bioassay for glue secretion and use it to analyze mutants in components of the puffing hierarchy. We show that 20E mediates secretion through the EcR/USP receptor, and two early-gene products, the rbp(+) function of BR-C and the Ca2+ binding protein E63-1, are involved. Furthermore, we demonstrate that 20E treatment of salivary glands leads to Ca2+ elevations by a genomic mechanism and that elevated Ca2+ levels are required for ectopically produced E63-1 to drive secretion. The results presented establish a connection between 20E exposure and changes in Ca2+ levels that are mediated by Ca2+ effector proteins, and thus establish a mechanistic framework for future studies.
Novel biological subtypes and clinically important genetic aberrations (druggable lesions, prognostic factors) have been described in B-other acute lymphoblastic leukemia (ALL) during the last decade; however, due to a lack of studies on unselected cohorts, their population frequency and mutual associations still have to be established. We studied 110 consecutively diagnosed and uniformly treated childhood B-other patients using single nucleotide polymorphism arrays and whole exome/transcriptome sequencing. The frequency of DUX4 -rearranged, BCR-ABL1 -like, ZNF384 -rearranged, ETV6-RUNX1 -like, iAMP21 and MEF2D -rearranged subtypes was 27%, 15%, 5%, 5%, 4%, and 2%, respectively; 43% of cases were not classified into any of these subtypes (B-rest). We found worse early response to treatment in DUX4 -rearranged leukemia and a strong association of ZNF384 -rearranged leukemia with B-myeloid immunophenotype. Of the druggable lesions, JAK/STAT-class and RAS/RAF/MAPK-class aberrations were found in 21% and 43% of patients, respectively; an ABL-class aberration was found in one patient. A recently described negative prognostic factor, IKZF1 plus , was found in 14% of patients and was enriched in (but not exclusive for) BCR-ABL1 -like subtype. PAX5 fusions (including 4 novel), intragenic amplifications and P80R mutations were mutually exclusive and only occurred in the B-rest subset, altogether accounting for 20% of the B-other group. PAX5 P80R was associated with a specific gene expression signature, potentially defining a novel leukemia subtype. Our study shows unbiased European population-based frequencies of novel ALL subtypes, recurrent (cyto)genetic aberrations and their mutual associations. This study also strengthens and widens the current knowledge of B-other ALL and provides an objective basis for optimization of current genetic diagnostics.
We used the genomic breakpoint between and genes for the DNA-based monitoring of minimal residual disease (MRD) in 48 patients with childhood acute lymphoblastic leukemia (ALL). Comparing the results with standard MRD monitoring based on immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and with quantification of deletion, we observed very good correlation for the methods in a majority of patients; however,>20% of children (25% [8/32] with minor and 12.5% [1/8] with major- variants in the consecutive cohorts) had significantly (>1 log) higher levels of fusion than Ig/TCR rearrangements and/or deletion. We performed cell sorting of the diagnostic material and assessed the frequency of -positive cells in various hematopoietic subpopulations; 12% to 83% of non-ALL B lymphocytes, T cells, and/or myeloid cells harbored the fusion in patients with discrepant MRD results. The multilineage involvement of the -positive clone demonstrates that in some patients diagnosed with-positive ALL, a multipotent hematopoietic progenitor is affected by the fusion. These patients have-positive clonal hematopoiesis resembling a chronic myeloid leukemia (CML)-like disease manifesting in "lymphoid blast crisis." The biological heterogeneity of -positive ALL may impact the patient outcomes and optimal treatment (early stem cell transplantation vs long-term administration of tyrosine-kinase inhibitors) as well as on MRD testing. Therefore, we recommend further investigations on CML-like-positive ALL.
The transmembrane proteins Delta and Notch act as ligand and receptor in a conserved signaling pathway required for a variety of cell fate specification events in many organisms. Binding of Delta to Notch results in a proteolytic cascade that releases the Notch intracellular domain, allowing it to participate in transcriptional activation in the nucleus. Recent research has implicated the endocytic and ubiquitylation machinery as essential components of Delta-Notch signaling. Our analysis of chimeric and missense Delta variants has delineated a number of structural requirements for Delta trafficking, receptor binding, and signaling. We find that while the Delta N-terminal domain is necessary and sufficient for binding to Notch, the integrity of the epidermal-growth-factor-l ike repeat (ELR) 2 is also required for Notch binding. Screening of 117 Delta mutant lines for proteins that exhibit aberrant subcellular trafficking has led to the identification of 18 Delta alleles (Dl TD alleles) that encode ''trafficking-defective'' Delta proteins. We find, unexpectedly, that many Dl TD alleles contain missense mutations in ELRs within the Delta extracellular domain. Finally, we find that two Dl TD alleles contain lysine missense mutations within the Delta intracellular domain (DeltaICD) that may identify residues important for DeltaICD mono-ubiquitylation and subsequent Delta endocytosis and signaling.
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