Transcription Factors, Chromatin Loops & Blood Cells

__Abstract__ A key aspect of genome biology is the proper regulation of gene expression, which is found perturbed in many diseases and disorders. Here we conducted studies on differentiating hematopoietic cells, with the aim to generate new knowledge of the regulatory mechanisms operated by transcription factors and the impact of these processes on cellular differentiation. Our results provide a collection of novel insights into the biology of hematopoietic transcription factor complexes and how they govern complex cellular processes such as the regulation of gene transcription and V(D)J recombination. Additionally, we have also developed and implemented new tools to study transcription factor actions

3D genome organization during lymphocyte development and activation

Chromosomes have a complex three-dimensional (3D) architecture comprising A/B compartments, topologically associating domains and promoter-enhancer interactions. At all these levels, the 3D genome has functional consequences for gene transcription and therefore for cellular identity. The development and activation of lymphocytes involves strict control of gene expression by transcription factors (TFs) operating in a three-dimensionally organized chromatin landscape. As lymphocytes are indispensable for tissue homeostasis and pathogen defense, and aberrant lymphocyte activity is involved in a wide range of human morbidities, acquiring an in-depth understanding of the molecular mechanisms that control lymphocyte identity is highly relevant. Here we review current knowledge of the interplay between 3D genome organization and transcriptional control during B and T lymphocyte development and antigen-dependent activation, placing special emphasis on the role of TFs

Integrating transposable elements in the 3D genome

Chromosome organisation is increasingly recognised as an essential component of genome regulation, cell fate and cell health. Within the realm of transposable elements (TEs) however, the spatial information of how genomes are folded is still only rarely integrated in experimental studies or accounted for in modelling. Whilst polymer physics is recognised as an important tool to understand the mechanisms of genome folding, in this commentary we discuss its potential applicability to aspects of TE biology. Based on recent works on the relationship between genome organisation and TE integration, we argue that existing polymer models may be extended to create a predictive framework for the study of TE integration patterns. We suggest that these models may offer orthogonal and generic insights into the integration profiles (or "topography") of TEs across organisms. In addition, we provide simple polymer physics arguments and preliminary molecular dynamics simulations of TEs inserting into heterogeneously flexible polymers. By considering this simple model, we show how polymer folding and local flexibility may generically affect TE integration patterns. The preliminary discussion reported in this commentary is aimed to lay the foundations for a large-scale analysis of TE integration dynamics and topography as a function of the three-dimensional host genome

Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles

Control of developmentally primed erythroid genes by combinatorial co-repressor actions

How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2-IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation

Changes in the ceIl membrane of Lactobacillus bulgaricus during storage following freeze-drying

The mechanism of inactivation of freeze-dried Lactobacillus bulgaricus during storage in maltodextrin under controlled humidity was investigated. Evidence is presented supporting the hypothesis that membrane damage occurs during storage. A study on the lipid composition of the cells by gas chromatography showed a decrease in the unsaturated and saturated fatty acid content of the cell. Further evidence indicating membrane damage includes a decrease in membrane bound proton-translocating ATPase activity

Group 2 innate lymphoid cells exhibit a dynamic phenotype in allergic airway inflammation

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33-and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought, whereby their surface marker and gene expression profile are highly dynamic

Increased group 2 innate lymphoid cells in peripheral blood of adults with mastocytosis

Background: Systemic mastocytosis is a hematological disease in which aberrant mast cells accumulate because of gain-of-function mutations in the KIT receptor. Group 2 innate lymphoid cells (ILC2s) are effector cells of type 2 immune responses that also express KIT and colocalize with mast cells at barrier tissue sites. In mouse models, mast cell-ILC2 crosstalk can drive local inflammation. However, a possible role for ILC2s in the pathophysiology of mastocytosis remains unexplored. Objective: We sought to characterize circulating ILC2s in a clinically diverse cohort of patients with mastocytosis. Methods: We included 21 adults with systemic mastocytosis and 18 healthy controls. Peripheral blood ILC2 abundance and phenotype were analyzed by flow cytometry and correlated to clinical characteristics, including the presence of the D816V KIT mutation. Results: ILC2 levels were significantly higher in D816V+ patients with mastocytosis compared with D816V− patients or healthy controls. We observed increased proportions of KIT+ ILC2s among patients with mastocytosis, regardless of D816V status. Patients with skin involvement and itch showed the highest levels of ILC2s, which was independent from atopy or serum tryptase levels. Allele-specific quantitative PCR showed that the vast majority of ILC2s did not carry the D816V mutation. Conclusions: Our findings suggest a role for ILC2s and pathogenic ILC2-mast cell crosstalk in mastocytosis. We hypothesize that a high cutaneous D816V+

Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling

Background: Short-chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell–mediated diseases. However, the effects of SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell–mediated pathology and human mast cell activation, including the molecular mechanisms involved. Method: Precision-cut lung slices (PCLS) of allergen-exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co-cultured with SCFAs and assessed for degranulation after IgE- or non–IgE-mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays. Results: Butyrate treatment inhibited allergen-induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE- and non–IgE-mediated human or mouse mast cell degranulation in a concentration-dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate-induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI-mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions. Conclusion: Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation

NLS-tagging: an alternative strategy to tag nuclear proteins

The characterization of transcription factor com-plexes and their binding sites in the genome by affin-ity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity bind-ing of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biolog-ical function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the ‘tag ’ close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the con-tribution of Fli-1 to hematopoiesis