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/SummarySpecialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as direct histone variant H2A.Z interactor involved in mitosis and cranial-facial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes including NuRD, and show that it localizes to genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis. Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental defects are pheno-copied in HMG20A-depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CMs). Accordingly, loss of HMG20A caused striking deregulation of transcription programs involved in epithelial- mesenchymal transition (EMT) and cardiac differentiation, thereby providing insights into the regulatory circuits controlled by HMG20A. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of the intricate developmental transcription programs that guide NCC and cardiomyocyte differentiation.

Post-transcriptional gene regulation is complex, dynamic and ensures proper T cell function. The targeted transcripts can simultaneously respond to various factors as evident for Icos, an mRNA regulated by several RNA binding proteins (RBPs), including Roquin. However, fundamental information about the entire RBPome involved in post-transcriptional gene regulation in T cells is lacking. Here, we applied global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS) to human and mouse primary T cells and identified the core T cell RBPome. This defined 798 mouse and 801 human proteins as RBPs, unexpectedly containing signaling proteins like Stat1, Stat4 and Vav1. Based on the vicinity to Roquin-1 in proximity labeling experiments, we selected ∼50 RBPs for testing coregulation of Roquin targets. Induced expression of these candidate RBPs in wildtype and Roquin-deficient T cells unraveled several Roquin-independent contributions, but also revealed Celf1 as a new Roquin-1-dependent and target-specific coregulator of Icos.One sentence statementWe provide an atlas of RNA-binding proteins in human and mouse T helper cells as a resource for studying higher order post-transcriptional gene regulation.

Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene., eLife digest Cells contain a large number of proteins that control the activity of genes in response to various signals and changes in their environment. Often these proteins work together in groups called complexes. In the fruit fly Drosophila melanogaster, one of these complexes is called DOMINO. The DOMINO complex alters gene activity by interacting with other proteins called histones which influence how the genes are packaged and accessed within the cell. DOMINO works in two separate ways. First, it can replace certain histones with other variants that regulate genes differently. Second, it can modify histones by adding a chemical marker to them, which alters how they interact with genes. It was not clear how DOMINO can do both of these things and how that is controlled; but it was known that cells can make two different forms of the central component of the complex, called DOM-A and DOM-B, which are both encoded by the same gene. Scacchetti et al. have now studied fruit flies to understand the activities of these forms. This revealed that they do have different roles and that gene activity in cells changes if either one is lost. The two forms operate as part complexes with different compositions and only DOM-A includes the TIP60 enzyme that is needed to modify histones. As such, it seems that DOM-B primarily replaces histones with variant forms, while DOM-A modifies existing histones. This means that each form has a unique role associated with each of the two known behaviors of this complex. The presence of two different DOMINO complexes is common to flies and, probably, other insects. Yet, in other living things, such as mammals and yeast, their two roles are carried out by protein complexes originating from two distinct genes. This illustrates a concept called convergent evolution, where different organisms find different solutions for the same problem. As such, these findings provide an insight into the challenges encountered through evolution and the diverse solutions that have developed. They will also help us to understand the ways in which protein activities can adapt to different needs over evolutionary time.

Heterogeneous populations of hypothalamic neurons orchestrate energy balance via the release of specific signatures of neuropeptides. However, how specific intracellular machinery controls peptidergic identities and function of individual hypothalamic neuron’s remains largely unknown. The transcription factor T-box 3 (Tbx3) is expressed in hypothalamic neurons sensing and governing energy status, while human TBX3 haploinsufficiency has been linked with obesity. Here we demonstrate that loss-of-Tbx3 function in hypothalamic neurons causes weight gain and other metabolic disturbances by disrupting both peptidergic identity and plasticity of Pomc/Cart and Agrp/Npy neurons. These alterations are observed following loss of Tbx3 in both immature hypothalamic neurons and terminally differentiated murine neurons. We further establish the importance of Tbx3 for body weight regulation in Drosophila melanogaster and show that TBX3 is implicated in the differentiation of human embryonic stem cells (hESC) into hypothalamic Pomc neurons. Our data indicate that Tbx3 directs the terminal specification of neurons as functional components of the melanocortin system and is required for maintaining their peptidergic identity. In summary, we report the discovery of a mechanistic key process underlying the functional heterogeneity of hypothalamic neurons governing body weight and systemic metabolism.

Two-dimensional gas chromatography mass spectrometry (GCxGC-MS) is utilized to an increasing extent in biomedical metabolomics. Here, we established and adapted metabolite extraction and derivatization protocols for cell/tissue biopsy, serum and urine samples according to their individual properties. GCxGC-MS analysis revealed detection of ~600 molecular features from which 165 were characterized representing different classes such as amino acids, fatty acids, lipids, carbohydrates, nucleotides and small polar components of glycolysis and the Krebs cycle using electron impact (EI) spectrum matching and validation using external standard compounds. Advantages of two-dimensional gas chromatography based resolution were demonstrated by optimizing gradient length and separation through modulation between the first and second column, leading to a marked increase in metabolite identification due to improved separation as exemplified for lactate versus pyruvate, talopyranose versus methyl palmitate and inosine versus docosahexaenoic acid. Our results demonstrate that GCxGC-MS represents a robust metabolomics platform for discovery and targeted studies that can be used with samples derived from the clinic., Graphical abstract fx1, Highlights • GCxGC-MS detected ~600 features;165 represented metabolites of different classes. • Optimizing gradient length and separation through modulation improved metabolite ID. • improved separation of lactate/pyruvate, talopyranose/palmitate and inosine/docosahexaenoate.