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Summary Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general., Graphical Abstract, Highlights • TINC allows the isolation of a specific locus for molecular analyses • TINC identified hundreds of proteins at the Nanog promoter • RCOR2 is a component of the pluripotency network in embryonic stem cells • RCOR2 is required for efficient differentiation, Here, Knaupp and colleagues describe TINC, an epigenetic method that allows interrogation of mammalian regulatory complexes at a single-locus resolution. TINC was applied to dissect the transcriptional complex at the Nanog promoter in embryonic stem cells, revealing hundreds of interactors, including RCOR2, hence redefining how this gene is regulated in pluripotency.

Hematopoietic stem cell (HSC) maintenance throughout life and engraftment after transplantation during anticancer treatment, depend on support from various bone marrow (BM) niche compartments. Here, by studying physiological homing of fetal HSCs, we show the critical requirement of balanced crosstalk within the multi-component BM niche for enabling HSC-lodgment. Transgene-induced over-production of VEGF by skeletal progenitor cells elicited stromal and endothelial hyper-activation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSC seeding, settlement and survival were drastically impaired. Transcriptome-profiling, flow-cytometry and high-resolution imaging indicated alterations in peri-/endothelial characteristics, vascular permeability, and cellular metabolism, associated with a higher oxidative status and increased oxidative stress within the VEGF-enriched BM-environment. Thus, developmental HSC-homing to bone is controlled by local inter-niche communication regulating stromal-vascular integrity and theoxidative and metabolic milieu.Since irradiation of adult mice also induced stromal VEGF-expression and similar osteo-angiogenic niche changes, our findings can contribute to improving stem cell therapy.

Aberrant expression of the SNAIL & ZEB families of Epithelial to Mesenchymal Transition (EMT) modulators is an emerging theme in acute leukemia biology. EMT regulation has long been implicated in epithelial tumor pathogenesis, however a role in hematopoietic malignancy has only recently been explored. In this study, we describe a novel mechanism of AML pathogenesis involving SNAI1 induced epigenetic dysregulation via corruption of the histone demethylase LSD1. SNAI1 is expressed ∼10-fold higher in AML blasts compared to normal hematopoietic cells, suggesting a functional role for SNAI1 in AML. Indeed, SNAI1 knockdown induces differentiation in human AML cells, while Snai1 deletion delays tumour onset in mouse AML models. Furthermore, transgenic Snai1 expression perturbs myeloid development, resulting in accumulation of immature myeloid cells with enhanced self-renewal capacity - ultimately predisposing mice to AML. Intriguingly, impaired myeloid development by Snai1 absolutely requires its interaction with Lsd1, a H3K4 histone demethylase that has emerged as a novel therapeutic target in AML. Mechanistically, this interaction results in corruption of Lsd1 function via aberrant recruitment to Snai1 gene targets, and sequestration away from its own targets. Ultimately, this leads to changes in H3K4me1/2 methylation at specific promoters and enhancers, and subsequently altered gene expression. This model provides insight into the mechanisms by which LSD1 can be perturbed in AML, and offers a possible explanation for why LSD1 inhibition/loss has somewhat dichotomous effects on normal and malignant hematopoietic stem cells. Furthermore, as other members of the SNAIL and ZEB families are known to interact with LSD1, it is likely that this model will be more broadly applicable to other EMT modulators in AML, representing a possible new focus for future therapeutic studies.

GABAergic interneurons mainly originate in the medial ganglionic eminence (MGE) of the embryonic ventral telencephalon (VT) and migrate tangentially to the cortex, guided by membrane-bound and secreted factors. We found that Sip1 (Zfhx1b, Zeb2), a transcription factor enriched in migrating cortical interneurons, is required for their proper differentiation and correct guidance. The majority of Sip1 knockout interneurons fail to migrate to the neocortex and stall in the VT. RNA sequencing reveals that Sip1 knockout interneurons do not acquire a fully mature cortical interneuron identity and contain increased levels of the repulsive receptor Unc5b. Focal electroporation of Unc5b-encoding vectors in the MGE of wild-type brain slices disturbs migration to the neocortex, whereas reducing Unc5b levels in Sip1 knockout slices and brains rescues the migration defect. Our results reveal that Sip1, through tuning of Unc5b levels, is essential for cortical interneuron guidance. ispartof: Neuron vol:77 issue:1 pages:70-82 ispartof: location:United States status: published

SummaryGrowing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.

We previously demonstrated that Fibroblast Growth Factor 10 (FGF10) and its receptor FGFR2b play a key role in controlling the very early stages of mammary gland development during embryogenesis [Mailleux, A.A., Spencer-Dene, B., Dillon, C., Ndiaye, D., Savona-Baron, C., Itoh, N., Kato, S., Dickson, C., Thiery, J.P., and Bellusci, S. (2002). Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development 129, 53–60. Veltmaat, J. M., Relaix, F., Le, L.T., Kratochwil, K., Sala, F.G., van Veelen, W., Rice, R., Spencer-Dene, B., Mailleux, A.A., Rice, D.P., Thiery, J.P., and Bellusci, S. (2006). Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes. Development 133, 2325–35.]. However, the role of FGFR2b signaling in postnatal mammary gland development is still elusive. We show that FGF10 is expressed at high level throughout the adipose tissue in the mammary gland of young virgin female mice whereas its main receptor FGFR2 is found mostly in the epithelium. Using a rtTA transactivator/tetracycline promoter approach allowing inducible and reversible attenuation of the FGFR2b signaling throughout the adult mouse, we are now reporting that FGFR2b signaling is also critical during postnatal mammary gland development. Ubiquitous attenuation of FGFR2b signaling in the postnatal mouse for 6 weeks starting immediately after birth is not lethal and leads to minor defects in the animal. Upon dissection of the mammary glands, a 40% reduction in size compared to the WT control is observed. Further examination shows a rudimentary mammary epithelial tree with completely absent terminal end buds (TEBs), compared to a well-branched structure observed in wild type. Transplantation of mammary gland explants into cleared fat pad of wild type mouse recipients indicates that the observed abnormal branching results from defective FGFR2b signaling in the epithelium. We also demonstrate that this rudimentary tree reforms TEBs and resumes branching upon removal of doxycycline suggesting that the regenerative capacities of the mammary epithelial progenitor cells were still functional despite long-term inactivation of the FGFR2b pathway. At the cellular level, upon FGFR2b attenuation, we show an increase in apoptosis associated with a decrease in the proliferation of the mammary luminal epithelium. We conclude that during puberty, there is a differential requirement for FGFR2b signaling in ductal vs. TEBs epithelium. FGFR2b signaling is crucial for the survival and proliferation of the mammary luminal epithelial cells, but does not affect the regenerative potential of the mammary epithelial progenitor cells.