A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a 'super-enhancer' is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a 'blood enhancer cluster' (BENC). We show that BENC is also essential for the maintenance of MLL-AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.

Author Correction: A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

Elucidating the Morphology of the Endoplasmic Reticulum: Puzzles and Perspectives.

Artificial or synthetic organelles are a key challenge for bottom-up synthetic biology. So far, synthetic organelles have typically been based on spherical membrane compartments, used to spatially confine selected chemical reactions. In vivo, these compartments are often far from being spherical and can exhibit rather complex architectures. A particularly fascinating example is provided by the endoplasmic reticulum (ER), which extends throughout the whole cell by forming a continuous network of membrane nanotubes connected by three-way junctions. The nanotubes have a typical diameter of between 50 and 100 nm. In spite of much experimental progress, several fundamental aspects of the ER morphology remain elusive. A long-standing puzzle is the straight appearance of the tubules in the light microscope, which form irregular polygons with contact angles close to 120°. Another puzzling aspect is the nanoscopic shapes of the tubules and junctions, for which very different images have been obtained by electron microcopy and structured illumination microscopy. Furthermore, both the formation and maintenance of the reticular networks require GTP and GTP-hydrolyzing membrane proteins. In fact, the networks are destroyed by the fragmentation of nanotubes when the supply of GTP is interrupted. Here, it is argued that all of these puzzling observations are intimately related to each other and to the dimerization of two membrane proteins anchored to the same membrane. So far, the functional significance of this dimerization process remained elusive and, thus, seemed to waste a lot of GTP. However, this process can generate an effective membrane tension that stabilizes the irregular polygonal geometry of the reticular networks and prevents the fragmentation of their tubules, thereby maintaining the integrity of the ER. By incorporating the GTP-hydrolyzing membrane proteins into giant unilamellar vesicles, the effective membrane tension will become accessible to systematic experimental studies.

The dependence of Ig class-switching on the nuclear export sequence of AID likely reflects interaction with factors additional to Crm1 exportin.

Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that - beyond a requirement for threshold Crm1 binding - there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1.

Label-free quantification using MALDI mass spectrometry: considerations and perspectives.

Profound knowledge of protein abundances in healthy tissues and their changes in disease is crucial for understanding biological processes in basic science and for the development of novel diagnostics and therapeutics. Mass spectrometrybased label-free protein quantification is used increasingly often to gain insights into physiological changes observed in perturbed systems. Although the soft ionization techniques electrospray ionization and matrix-assisted laser desorption/ionization have both been used for protein quantification, this article focuses on instrumental setups with a MALDI ion source. Beside reviewing current bioinformatic data-processing tools for label-free quantification and elaborating on the technical benefits of combining UHPLC and MALDI-MS, we outline the potential of state-of-the-art instruments by reporting unpublished results obtained from twenty-four complex biological samples. This review points out that the capabilities of LC-MALDI MS systems have not yet been fully utilized because of a lack of suitable software tools.

Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.

Impaired integrin α5 /ß1 -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5 /ß1 is an important mechanosensor in HSC involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and ß1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α5 and ß1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin ß1 remains unaffected. In the aged liver, laminin ß2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5 /ß1 in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration.

BMP-2 Signaling and Mechanotransduction Synergize to Drive Osteogenic Differentiation via YAP/TAZ.

Growth factors and mechanical cues synergistically affect cellular functions, triggering a variety of signaling pathways. The molecular levels of such cooperative interactions are not fully understood. Due to its role in osteogenesis, the growth factor bone morphogenetic protein 2 (BMP-2) is of tremendous interest for bone regenerative medicine, osteoporosis therapeutics, and beyond. Here, contribution of BMP-2 signaling and extracellular mechanical cues to the osteogenic commitment of C2C12 cells is investigated. It is revealed that these two distinct pathways are integrated at the transcriptional level to provide multifactorial control of cell differentiation. The activation of osteogenic genes requires the cooperation of BMP-2 pathway-associated Smad1/5/8 heteromeric complexes and mechanosensitive YAP/TAZ translocation. It is further demonstrated that the Smad complexes remain bound onto and active on target genes, even after BMP-2 removal, suggesting that they act as a "molecular memory unit." Thus, synergistic stimulation with BMP-2 and mechanical cues drives osteogenic differentiation in a programmable fashion.

NTA-Co3+-His6 versus NTA-Ni2+-His6 mediated E-Cadherin surface immobilization enhances cellular traction.

Understanding the biological impact of strategies for protein immobilization onto bioactive surfaces is crucial for the design of biomimetic materials. A common strategy used to immobilize or label recombinant proteins is to exploit the Ni2+-mediated interaction of nitrilotriacetic acid (NTA) with the hexahistidine tag (His6-tag) present on recombinant proteins. While this method ensures a controlled orientation and functionality of the protein, the kinetically labile nature of the bond ensures only its weak immobilization onto the surface. Recently, it has been shown that the oxidation of Co2+ to Co3+ greatly stabilizes the bond between NTA and the His6-tagged proteins, making it inert to ligand exchange and resistant to chelators. This approach not only has the potential to improve the quality of biomimetic material functionalization and molecule labeling but could also affect cellular mechanical responses for which the mechanical strength of the protein-surface bond is crucial. Here, we compared gold (Au) nanopatterned polyacrylamide (PAA) hydrogels functionalized with E-cadherin via Co3+ with those functionalized via Ni2+ for studying adhesion-mediated responses in keratinocytes. We show that keratinocytes develop higher and a broader range of adhesion forces, leading to extended cell spreading and colony organization on Co3+ vs. Ni2+. This work uniquely shows that stabilizing the NTA/His6-tag bond via Co3+ for protein immobilization significantly impacts cellular phenotype on biomimetic materials by impacting cell signaling.

Laminin-521 promotes quiescence in isolated stellate cells from rat liver.

The laminin α5 protein chain is an element of basement membranes and important to maintain stem cells. Hepatic stellate cells (HSC) are liver-resident mesenchymal stem cells, which reside in a quiescent state on a basement membrane-like structure in the space of Dissé. In the present study, laminin α5 chain was detected in the space of Dissé of normal rat liver. Since HSC are critical for liver regeneration and can contribute to fibrosis in chronic liver diseases, the effect of laminins on HSC maintenance was investigated. Therefore, isolated rat HSC were seeded on uncoated polystyrene (PS) or PS coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC on PS/LN-211 or PS developed into myofibroblasts-like cells. To improve the homogeneity as well as the presentation of laminin molecules on the culture surface to HSC, laminin-functionalized, gold-nanostructured glass surfaces were generated. This approach further enhanced the expression of quiescence-associated genes in HSC. In conclusion, the results indicate that LN-521 supports the quiescent state of HSC and laminin α5 can be regarded as an important element of their niche in the space of Dissé.