EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

Genes encoding B lineage restricted transcription factors are frequently mutated in B-lymphoid leukemias, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is Early B cell Factor 1 (EBF1), a protein of critical importance for lineage specification and survival of B-lymphoid progenitors. Here, we report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC partially rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC-seq, ChIP-seq and reporter gene assays, we identified six EBF responsive enhancer elements within the Myc locus. CRISPR-Cas9 mediated targeting of EBF1 binding sites identified one element of key importance for Myc expression and pro-B cell expansion. These data provide evidence that Myc is a direct target of EBF1. Furthermore, ChIP-seq analysis revealed that several regulatory elements in the Myc locus are targets of PAX5. However, ectopic expression of PAX5 in EBF1 deficient cells inhibits the cell cycle and reduces Myc expression, suggesting that EBF1 and PAX5 act in an opposing manner to regulate Myc levels. This hypothesis is further substantiated by the finding that Pax5 inactivation reduces requirements for EBF1 in pro-B cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-ALL cell line indicate that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development

Diverse motif ensembles specify non-redundant DNA binding activities of AP-1 family members in macrophages

Mechanisms by which members of the AP-1 family of transcription factors play non-redundant biological roles despite recognizing the same DNA sequence remain poorly understood. To address this question, here we investigate the molecular functions and genome-wide DNA binding patterns of AP-1 family members in primary and immortalized mouse macrophages. ChIP-sequencing shows overlapping and distinct binding profiles for each factor that were remodeled following TLR4 ligation. Development of a machine learning approach that jointly weighs hundreds of DNA recognition elements yields dozens of motifs predicted to drive factor-specific binding profiles. Machine learning-based predictions are confirmed by analysis of the effects of mutations in genetically diverse mice and by loss of function experiments. These findings provide evidence that non-redundant genomic locations of different AP-1 family members in macrophages largely result from collaborative interactions with diverse, locus-specific ensembles of transcription factors and suggest a general mechanism for encoding functional specificities of their common recognition motif

Dissection of progenitor compartments resolves developmental trajectories in B-lymphopoiesis

To understand the developmental trajectories in early lymphocyte differentiation, we identified differentially expressed surface markers on lineage-negative lymphoid progenitors (LPs). Single-cell polymerase chain reaction experiments allowed us to link surface marker expression to that of lineage-associated transcription factors (TFs) and identify GFRA2 and BST1 as markers of early B cells. Functional analyses in vitro and in vivo as well as single-cell gene expression analyses supported that surface expression of these proteins defined distinct subpopulations that include cells from both the classical common LPs (CLPs) and Fraction A compartments. The formation of the GFRA2-expressing stages of development depended on the TF EBF1, critical both for the activation of stage-specific target genes and modulation of the epigenetic landscape. Our data show that consecutive expression of Ly6D, GFRA2, and BST1 defines a developmental trajectory linking the CLP to the CD19(+) progenitor compartment.Peer reviewe

Validation of an open source, remote web‐based eye‐tracking method (WebGazer) for research in early childhood

Measuring eye movements remotely via the participant's webcam promises to be an attractive methodological addition to in-person eye-tracking in the lab. However, there is a lack of systematic research comparing remote web-based eye-tracking with in-lab eye-tracking in young children. We report a multi-lab study that compared these two measures in an anticipatory looking task with toddlers using WebGazer.js and jsPsych. Results of our remotely tested sample of 18-27-month-old toddlers (N = 125) revealed that web-based eye-tracking successfully captured goal-based action predictions, although the proportion of the goal-directed anticipatory looking was lower compared to the in-lab sample (N = 70). As expected, attrition rate was substantially higher in the web-based (42%) than the in-lab sample (10%). Excluding trials based on visual inspection of the match of time-locked gaze coordinates and the participant's webcam video overlayed on the stimuli was an important preprocessing step to reduce noise in the data. We discuss the use of this remote web-based method in comparison with other current methodological innovations. Our study demonstrates that remote web-based eye-tracking can be a useful tool for testing toddlers, facilitating recruitment of larger and more diverse samples; a caveat to consider is the larger drop-out rate

The enzymatic machinery of leukotriene biosynthesis : Studies on ontogenic expression, interactions and function

Leukotrienes (LTs) are biologically active arachidonic acid (AA) derivatives generated by the 5-lipoxygenase (5-LO) pathway. They are produced by myeloid cells. 5-LO converts AA to LTA4 in cooperation with 5-LO activating protein (FLAP). LTA4 is converted to LTB4, by LTA4-hydrolase (LTA4H) or to LTC4 by LTC4-synthase (LTC4S). LTs act on cells through plasma membrane bound G-protein coupled receptors found on leukocytes, smooth muscle and endothelial cells. We report here protein-protein interactions of proteins involved in LTC4 synthesis. 5-LO interacts with cytosolic domains of the integral membrane proteins FLAP and LTC4S at the nuclear envelope, in addition LTC4S interacts with FLAP through its hydrophobic membrane spanning regions. We constructed an LTC4S promoter controlled GFP reporter vector, displaying cell specific expression and sensitivity to agents known to affect LTC4S expression. The vector was used to create transgenic mice expressing GFP as a reporter for LTC4S. Ontogenic mouse expression studies revealed that the complete LT biosynthesis machinery was present at e11.5 primarily in the hematopoietic cells colonizing the liver. Although mature myeloid cells were the main contributors, a substantial amount of FLAP message was also detected in hematopoietic stem and progenitor cells, indicating possible functions for FLAP in hematopoietic regulation. Functional analyses using FLAP knockout mice suggested fine-tuning roles for LTs during differentiation, primarily along the B-lymphocyte differentiation path

Modeling of foundation and soil in FEM-Design : A study of the geo-modules 3D Soil and Pile

Beräkning med finita elementmetoden, FEM, är en vanlig metod vid lastnedräkning för bärande konstruktioner. Vid FEM-modellering inom huskonstruktion måste konstruktören på något sätt även modellera markens egenskaper för att uppnå statisk jämvikt i modellen. Att låta byggnaden vila på oeftergivliga stöd fungerar vid grundläggning på berg, men i övriga fall måste jordens deformation vid belastning simuleras. Ett vanligt tillvägagångssätt är att konstruktören samarbetar med en geotekniker som beräknar sättningar i jorden utifrån de laster konstruktören beräknat. Det görs i ett geotekniskt FEM-program som exempelvis PLAXIS. Utifrån geoteknikerns resultat beräknar konstruktören fjäderstöd som får simulera marken. Därefter kan deformationer i byggnaden studeras.    Denna studie har prövat en alternativ metod. Hus, grundläggning och undergrund har modellerats i en gemensam modell i FEM-Design 3D Structure 17. Syftet med att undersöka detta var möjligheten till effektiviseringar av arbetsflödet. Om konstruktören kan modellera allt i samma modell skulle flera arbetsmoment kunna sparas. Risken för fel då data tolkas och flyttas mellan olika program skulle också elimineras.   Som fallstudie användes ett sexvåningshus med både pål- och plattgrundläggning. En befintlig modell med fjäderstöd beräknade i PLAXIS gjordes om till en komplett modell med byggnad, grundplattor, pålar och jord som finita element. FEM-Design kunde dock inte beräkna något resultat för modellen. Modulen Pile som modellerar pålar och modulen 3D Soil som modellerar jorden som solida element var inte kompatibla. Studien övergick då till att undersöka enskilda grundläggningselement separat. Pålar och plattor lyftes ut från den stora modellen och studerades dels med externt beräknade fjäderstöd och dels med FEM-Designs geotekniska moduler. Resultaten visade större deformationer för de modeller som var modellerade med 3D Soil. För Pile var resultaten att betrakta som likvärdiga.   En tydlig slutsats är att för husprojekt med både pål- och plattgrundläggning kan byggnad och undergrund inte modelleras tillsammans i FEM-Design. Programmets utvecklare StruSoft har planer på att utveckla funktionerna i framtiden så att de kan användas tillsammans, men det finns ingen prognos för när det kan vara klart. För byggnader med endast en grundläggningstyp kan respektive modul däremot användas. Det ska understrykas att en konstruktör som ska modellera undergrunden själv måste ha goda geotekniska kunskaper för att kunna hantera modulerna korrekt. Den optimala arbetsgången skulle enligt författarna vara att konstruktören och geoteknikern arbetade i samma modelleringsprogram i en gemensam modell. The finite element method, FEM, is a common method for load calculation in building construction design. In addition to the structure itself, the structural engineer must also model the soil response to achieve static equilibrium in the model. Using unyielding supports works for structures founded on rock, but in other cases the soil deformation must be simulated somehow. A common approach is that the structural engineer collaborates with a geotechnician who calculates the settlements in the soil due to the loads provided by the structural engineer. This is done in a geotechnical FEM program, e.g. PLAXIS. The structural engineer then uses the PLAXIS results to calculate spring supports simulating the soil response. The settlements in the structure can then be studied.   This study has evaluated a different approach. The structure, foundation slabs, piles and subgrade has been modeled in a common model in the program FEM-Design 3D Structure 17. The study identified several possible benefits if the method proved reliable. If the structural engineer could model everything in one model, several work steps could be excluded. It would also eliminate the risk of errors that may occur when data is to be interpreted and moved between different programs.   The studied case is a six-storey residential building founded on both piles and foundation slabs. An existing model with spring supports calculated in PLAXIS was modified into a complete model with structure, foundation slabs, piles and soil as finite elements. The complete model proved unable to produce any results. The Pile module and the 3D Soil module turned out to be incompatible. Facing this fact, the study decided to evaluate separate foundation elements individually. Piles and foundation slabs were extracted from the full model and studied first with externally calculated spring supports and then with the FEM-Design geotechnical modules. The results displayed larger deformations for the 3D Soil models. For the Pile module, the results should be regarded as equivalent.   The major conclusion is that a building founded on both piles and foundation slabs is not possible to model together with subgrade in FEM-Design. The program developer StruSoft may develop the features in the future so that they can work together, but there is no forecast for when this can be done. However, the features can be used separately for structures with only one type of foundation. It should be emphasized that a structural engineer who is going to model the subgrade must have good geotechnical knowledge in order to handle the modules correctly. According to the authors, the optimal workflow would be that the structural engineer and the geotechnician worked in the same modeling program in a common model