Three-dimensional chromatin reorganization regulates B cell development during ageing.

The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age.

Regularly spaced tyrosines in EBF1 mediate BRG1 recruitment and formation of nuclear subdiffractive clusters.

B lineage priming by pioneer transcription factor EBF1 requires the function of an intrinsically disordered region (IDR). Here, we examine the role of regularly spaced tyrosines in the IDR as potential determinants of IDR function and activity of EBF1. We found that four Y > A mutations in EBF1 reduced the formation of condensates in vitro and subdiffractive clusters in vivo. Notably, Y > A mutant EBF1 was inefficient in promoting B cell differentiation and showed impaired chromatin binding, recruitment of BRG1, and activation of specific target genes. Thus, regularly spaced tyrosines in the IDR contribute to the biophysical and functional properties of EBF1.

EBF1 is continuously required for stabilizing local chromatin accessibility in pro-B cells.

The establishment of de novo chromatin accessibility in lymphoid progenitors requires the "pioneering" function of transcription factor (TF) early B cell factor 1 (EBF1), which binds to naïve chromatin and induces accessibility by recruiting the BRG1 chromatin remodeler subunit. However, it remains unclear whether the function of EBF1 is continuously required for stabilizing local chromatin accessibility. To this end, we replaced EBF1 by EBF1-FKBPF36V in pro-B cells, allowing the rapid degradation by adding the degradation TAG13 (dTAG13) dimerizer. EBF1 degradation results in a loss of genome-wide EBF1 occupancy and EBF1-targeted BRG1 binding. Chromatin accessibility was rapidly diminished at EBF1-binding sites with a preference for sites whose occupancy requires the pioneering activity of the C-terminal domain of EBF1. Diminished chromatin accessibility correlated with altered gene expression. Thus, continuous activity of EBF1 is required for the stable maintenance of the transcriptional and epigenetic state of pro-B cells.

Tnpo3 enables EBF1 function in conditions of antagonistic Notch signaling.

Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of Ebf1 -/- progenitors. By RNA-seq analysis of EBF1E271A-expressing progenitors, we found an up-regulation of T lineage determinants and down-regulation of early B genes, although similar chromatin binding of EBF1E271A and EBF1wt was detected in pro-B cells expressing activated Notch1. B lineage-specific inactivation of Tnpo3 in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions.

How to resist Notch-targeted T-leukemia therapy: Lineage- and MYC enhancer switch.

Gain-of-function NOTCH1 mutations drive oncogenic MYC expression in T-ALL cells. Zhou et al. (2022) reveal that Notch-targeted therapy-resistant T-ALL cells activate EBF1, which promotes a T-to-B lineage shift and maintains oncogenic MYC expression in the absence of Notch signaling.

EBF1 and Pax5 safeguard leukemic transformation by limiting IL-7 signaling, Myc expression, and folate metabolism.

EBF1 and PAX5 mutations are associated with the development of B progenitor acute lymphoblastic leukemia (B-ALL) in humans. To understand the molecular networks driving leukemia in the Ebf1+/-Pax5+/- (dHet) mouse model for B-ALL, we interrogated the transcriptional profiles and chromatin status of leukemic cells, preleukemic dHet pro-B, and wild-type pro-B cells with the corresponding EBF1 and Pax5 cistromes. In dHet B-ALL cells, many EBF1 and Pax5 target genes encoding pre-BCR signaling components and transcription factors were down-regulated, whereas Myc and genes downstream from IL-7 signaling or associated with the folate pathway were up-regulated. We show that blockade of IL-7 signaling in vivo and methotrexate treatment of leukemic cells in vitro attenuate the expansion of leukemic cells. Single-cell RNA-sequencing revealed heterogeneity of leukemic cells and identified a subset of wild-type pro-B cells with reduced Ebf1 and enhanced Myc expression that show hallmarks of dHet B-ALL cells. Thus, EBF1 and Pax5 may safeguard early stage B cells from transformation to B-ALL by limiting IL-7 signaling, folate metabolism and Myc expression.

m6A modulates neuronal functions and sex determination in Drosophila.

N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. Here we carry out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. We identify the member of the split ends protein family, Spenito, as a novel bona fide subunit of the methyltransferase complex. We further demonstrate important roles of this complex in neuronal functions and sex determination, and implicate the nuclear YT521-B protein as a main m6A effector in these processes. Altogether, our work substantially extends our knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.

Xylan from corn cobs, a promising polymer for drug delivery: production and characterization.

Although many authors have reported several beneficial effects ascribed to xylan, such as inhibitory action on mutagenicity activity, antiphlogistic effects, and mitogenic and comitogenic activities, few papers have investigated a systematic study on the technological properties of this polymer. The aim of the present work was to evaluate xylan as a promise raw material for the pharmaceutical industry. The water-insoluble xylan samples were extracted from corn cobs following several steps. The obtained powered sample was analyzed by infrared and RMN spectroscopy, and characterized regarding their particle size, bulk and tap densities, compressibility index, compactability, Hausner ratio, and angle of repose. According to the results, infrared and RMN spectroscopy were shown to be able to evaluate the xylan structural conformation and composition, respectively. In addition, rheological data demonstrated that xylan powder obtained from corn cobs may be characterized as a material with low density and very cohesive flow properties.

1H HR/MAS NMR in the suspended state: molecular recognition processes in liquid chromatography between steroids and a silica hydride-based cholesterol phase.

A mixture of eight steroids was separated by HPLC on a silica hydride-based stationary phase modified with a moiety of cholesteryl 10-undecanoate. Within the steroid mixture progesterone showed the longest retention time suggesting the strongest interaction with the stationary phase material. To obtain more detailed information upon the effective separation process, interaction studies were performed by high resolution/magic angle spinning (HR/MAS) NMR spectroscopy recorded in the suspended state. (1)H saturation transfer difference (STD) NMR experiments with T(1rho) suppression showed significant hydrophobic binding affinities of progesterone toward the cholesterol column material. These NMR results can be correlated directly to the chromatographic data obtained by HPLC.

Synthesis, characterization, and high-performance liquid chromatographic evaluation of C14 stationary phases containing branched and unbranched alkyl groups.

Reversed-phase materials with branched and unbranched alkyl groups were prepared by modifying porous, spherical silica gel in a two-step reaction-immobilization of a trifunctional alkoxysilane (3-glycidoxypropyltrimethoxysilane) on the silica surface followed by reaction with a branched and an unbranched octanoic acid. The chromatographic sorbents were characterized by solid-state (29)Si and (13)C NMR spectroscopy. The chromatographic behaviour of the stationary phases was evaluated by use of a test mixture according to the Standard Reference Material 870 set from the US National Institute of Standards and Technology, in order to study the effect of branched and unbranched alkyl chains.

Characterization of bixin by LC-MS and LC-NMR.

An overview upon modern analytical techniques for the isolation, separation, and structural identification of the essential bioactive carotenoid bixin is given. Isolation from biological matrices is performed by matrix solid phase dispersion (MSPD). The extract is separated with shape-selective C(30 )columns. Structural assignment of the separated compounds is done by online LC-MS and capillary HPLC-NMR.