Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons

Integrative and perturbation-based analysis of the transcriptional dynamics of TGFβ/BMP system components in transition from embryonic stem cells to neural progenitors

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type β (TGFβ) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFβ system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions

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

The Cooperation between hMena Overexpression and HER2 Signalling in Breast Cancer

hMena and the epithelial specific isoform hMena11a are actin cytoskeleton regulatory proteins belonging to the Ena/VASP family. EGF treatment of breast cancer cell lines upregulates hMena/hMena11a expression and phosphorylates hMena11a, suggesting cross-talk between the ErbB receptor family and hMena/hMena11a in breast cancer. The aim of this study was to determine whether the hMena/hMena11a overexpression cooperates with HER-2 signalling, thereby affecting the HER2 mitogenic activity in breast cancer. In a cohort of breast cancer tissue samples a significant correlation among hMena, HER2 overexpression, the proliferation index (high Ki67), and phosphorylated MAPK and AKT was found and among the molecular subtypes the highest frequency of hMena overexpressing tumors was found in the HER2 subtype. From a clinical viewpoint, concomitant overexpression of HER2 and hMena identifies a subgroup of breast cancer patients showing the worst prognosis, indicating that hMena overexpression adds prognostic information to HER2 overexpressing tumors. To identify a functional link between HER2 and hMena, we show here that HER2 transfection in MCF7 cells increased hMena/hMena11a expression and hMena11a phosphorylation. On the other hand, hMena/hMena11a knock-down reduced HER3, AKT and p44/42 MAPK phosphorylation and inhibited the EGF and NRG1-dependent HER2 phosphorylation and cell proliferation. Of functional significance, hMena/hMena11a knock-down reduced the mitogenic activity of EGF and NRG1. Collectively these data provide new insights into the relevance of hMena and hMena11a as downstream effectors of the ErbB receptor family which may represent a novel prognostic indicator in breast cancer progression, helping to stratify patients

Measurement of the total cross section from elastic scattering in pp collisions at s√ = 7 TeV with the ATLAS detector

A measurement of the total pp cross section at the LHC at √s = 7 TeV is presented. In a special run with high-β beam optics, an integrated luminosity of 80 µb−1 was accumulated in order to measure the differential elastic cross section as a function of the Mandelstam momentum transfer variable t. The measurement is performed with the ALFA sub-detector of ATLAS. Using a fit to the differential elastic cross section in the |t| range from 0.01 GeV2 to 0.1 GeV2 to extrapolate to |t| → 0, the total cross section, σtot(pp → X), is measured via the optical theorem to be: σtot(pp → X) = 95.35 ± 0.38 (stat.) ± 1.25 (exp.) ± 0.37 (extr.) mb, where the first error is statistical, the second accounts for all experimental systematic uncertainties and the\ud last is related to uncertainties in the extrapolation to |t| → 0. In addition, the slope of the elastic cross section at small |t| is determined to be B = 19.73 ± 0.14 (stat.) ± 0.26 (syst.) GeV−2

Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care

Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS.MethodsIn a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations.ResultsAll anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations.ConclusionKnowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.GENETICS in MEDICINE advance online publication, 4 January 2018; doi:10.1038/gim.2017.221

Phenotype and genotype of 87 patients with Mowat–Wilson syndrome and recommendations for care

Purpose: Mowat–Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype–phenotype correlations of MWS. Methods: In a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations. Results: All anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluati

Measurement of inclusive jet charged-particle fragmentation functions in Pb plus Pb collisions at root S-NN=2.76 TeV with the ATLAS detector

© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/license/by/3.0/). Funded by SCOAP3

ZEB2, the Mowat-Wilson syndrome transcription factor

After its publication in 1999 as a DNA-binding and SMAD-binding transcription factor (TF) that co-determines cell fate in amphibian embryos, ZEB2 was from 2003 studied by embryologists mainly by documenting the consequences of conditional, cell-type specific Zeb2 knockout (cKO) in mice. In between, it was further identified as causal gene causing Mowat-Wilson Syndrome (MOWS) and novel regulator of epithelial–mesenchymal transition (EMT). ZEB2’s functions and action mechanisms in mouse embryos were first addressed in its main sites of expression, with focus on those that helped to explain neurodevelopmental and neural crest defects seen in MOWS patients. By doing so, ZEB2 was identified in the forebrain as the first TF that determined timing of neuro-/gliogenesis, and thereby also the extent of different layers of the cortex, in a cell non-autonomous fashion, i.e., by its cell-intrinsic control within neurons of neuron-to-progenitor paracrine signaling. Transcriptomics-based phenotyping of Zeb2 mutant mouse cells have identified large sets of intact-ZEB2 dependent genes, and the cKO approaches also m

Different E-box binding transcription factors, similar neuro-developmental defects: ZEB2 (Mowat-Wilson syndrome) and TCF4 (Pitt-Hopkins syndrome)

ZEB2 and TCF4 are transcription factors (TFs) whose locations in embryos overlap in many sites and developmental phases, including in the forebrain and its cortical neurons. De novo mutations cause the phenotypically overlapping, haploinsufficient Mowat-Wilson (MOWS, in the ZEB2 gene) and Pitt-Hopkins (PTHS, in TCF4) syndromes, which currently cannot be cured. Mutant alleles have been mapped and defects documented (also in brain function) in MOWS and PTHS patients. Appropriately designed mouse models and cells derived from these, as well as cellular models including cultured pluripotent cells, enable investigating the genetic and molecular mechanisms underlying the developmental deficiencies that manifest after birth in the nervous systems and their multiple cell types, as well as those of organs other than the brain, in MOWS and PTHS. Biochemical analyses of cell type-specific transcriptomic changes in these perturbation models as compared to control cells, the identification of the intact-factor dependent and direct target genes, and of partner proteins including chromatin modulators, are revealing complex and multiple modes of action that eventually will explain target gene selectivity for these TFs. Both TFs have also been found to operate in acute and chronic diseases and cell-based repair processes after tissue or organ injury. In addition, the defective function also arises from their aberrant gene expression, which will require a deeper investigation of how the transcription of these TF genes is regulated. Furthermore, these two factors genetically and biochemically interact. This review combines the essentials and recent progress for both TFs for the first time, with a focus on MOWS and PTHS