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Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol III alpha) and the related POLR3GL in the other form (Pol III beta). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

Background Skin biopsies promote our understanding of atopic dermatitis/AD pathomechanisms in infants/toddlers with early-onset AD, but are not feasible in pediatric populations. Tape strips are an emerging, minimally invasive alternative, but global transcriptomic profiling in early pediatric AD is lacking. We aimed to provide global lesional and nonlesional skin profiles of infants/toddlers with recent-onset, moderate-to-severe AD using tape strips. Methods Sixteen tape strips were collected for RNA-seq profiling from 19 infants/toddlers (<5 years old; lesional and nonlesional) with early-onset moderate-to-severe AD (<= 6 months) and 17 healthy controls. Results We identified 1829 differentially expressed genes/DEGs in lesional AD and 662 DEGs in nonlesional AD, vs healthy skin (fold-change >= 2, FDR <0.05), with 100% sample recovery. Both lesional and nonlesional skin showed significant dysregulations of Th2 (CCL17 and IL4R) and Th22/Th17 (IL36G, CCL20, and S100As)-related genes, largely lacking significant Th1-skewing. Significant down-regulation of terminal differentiation (FLG and FLG2), lipid synthesis/metabolism (ELOVL3 and FA2H), and tight junction (CLDN8) genes were primarily seen in lesional AD. Significant negative correlations were identified between Th2 measures and epidermal barrier gene-subsets and individual genes (FLG with IL-4R and CCL17;r < -0.4,P < .05). Significant correlations were also identified between clinical measures (body surface area/BSA, pruritus ADQ, and transepidermal water loss/TEWL) with immune and barrier mRNAs in lesional and/or nonlesional AD (FLG/FLG2 with TEWL;r < -0.4,P < .05). Conclusion RNA-seq profiling using tape strips in early-onset pediatric AD captures immune and barrier alterations in both lesional and nonlesional skin. Tape strips provide insight into disease pathomechanisms and cutaneous disease activity.

Growth factor deficiency in adulthood constitutes a distinct clinical syndrome with significant morbidities including abnormal body composition, reduced energy, affective disturbances, dyslipidemia, and increased cardiovascular risk. Protein replacement therapies using recombinant proteins or enzymes represent the only approved treatment. Combinatorial antibodies have shown great promise as a new class of therapeutic molecules because they act as "mechanism-based antibodies" with both agonist and antagonist activities. Using leptin, a key hormone in energy metabolism, as an example, a function-guided approach is developed to select combinatorial antibodies with high potency and full agonist activity that substitute natural growth factors in vivo. The identified antibody shows identical biochemical properties and cellular profiles as leptin, and rescues leptin-deficiency in ob/ob mice. Remarkably, the antibody activates leptin receptors that are otherwise nonfunctional because of mutations (L372A and A409E). Combinatorial antibodies have significant advantages over recombinant proteins for chronical usage in terms of immunological tolerance and biological stability.

E2A, a basic helix-loop-helix transcription factor, plays a crucial role in determining tissue - specific cell fate, including differentiation of B-cell lineages. In 5% of childhood acute lymphoblastic leukemia (ALL), the t(1,19) chromosomal translocation specifically targets the E2A gene and produces an oncogenic E2A-PBX1 fusion protein. Although previous studies have shown the oncogenic functions of E2A-PBX1 in cell and animal models, the E2A-PBX1-enforced cistrome, the E2A-PBX1 interactome, and related mechanisms un-derlying leukemogenesis remain unclear. Here, by unbiased genomic profiling approaches, we identify the direct target sites of E2A-PBX1 in t(1,19)positive pre-B ALL cells and show that, compared with normal E2A, E2A-PBX1 preferentially binds to a subset of gene loci cobound by RUNX1 and gene-activating machineries (p300, MED1, and H3K27 acetyla- tion). Using biochemical analyses, we further document a direct interaction of E2A-PBX1, through a region spanning the PBX1 homeodomain, with RUNX1. Our results also show that E2A-PBX1 binding to gene enhancers is dependent on the RUNX1 interaction but not the DNA-binding activity harbored within the PBX1 homeodomain of E2A-PBX1. Tran-scriptome analyses and cell transformation assays further establish a significant RUNX1 requirement for E2A-PBX1-mediated target gene activation and leukemogenesis. Notably, the RUNX1 locus itself is also directly activated by E2A-PBX1, indicating a multilayered interplay between E2A-PBX1 and RUNX1. Collectively, our study provides the first unbiased profiling of the E2A-PBX1 cistrome in pre-B ALL cells and reveals a previously unappreciated pathway in which E2A-PBX1 acts in concert with RUNX1 to enforce transcriptome alterations for the development of pre-B ALL.

During development, neurons adjust their energy balance to meet the high demands of robust axonal growth and branching. The mechanisms that regulate this tuning are largely unknown. Here, we show that sensory neurons lacking liver kinase B1 (Lkb1), a master regulator of energy homeostasis, exhibit impaired axonal growth and branching. Biochemical analysis of these neurons revealed reduction in axonal ATP levels, whereas transcriptome analysis uncovered down-regulation of Efhdl (EF-hand domain family member D1), a mitochondrial Ca2+-binding protein. Genetic ablation of Efhd1 in mice resulted in reduced axonal morphogenesis as well as enhanced neuronal death. Strikingly, this ablation causes mitochondrial dysfunction and a decrease in axonal ATP levels. Moreover, Efhd1 KO sensory neurons display shortened mitochondria at the axonal growth cones, activation of the AMP-activated protein kinase (AMPK)-Ulk (Unc-51-like autophagy-activating kinase 1) pathway and an increase in autophagic flux. Overall, this work uncovers a new mitochondrial regulator that is required for axonal morphogenesis.

Cell death is an important facet of animal development. In some developing tissues, death is the ultimate fate of over 80% of generated cells. Although recent studies have delineated a bewildering number of cell death mechanisms, most have only been observed in pathological contexts, and only a small number drive normal development. This Primer outlines the important roles, different types and molecular players regulating developmental cell death, and discusses recent findings with which the field currently grapples. We also clarify terminology, to distinguish between developmental cell death mechanisms, for which there is evidence for evolutionary selection, and cell death that follows genetic, chemical or physical injury. Finally, we suggest how advances in understanding developmental cell death may provide insights into the molecular basis of developmental abnormalities and pathological cell death in disease.

Centromeres are the complex structures responsible for the proper segregation of chromosomes during cell division. Structural or functional alterations of the centromere cause aneuploidies and other chromosomal aberrations that can induce cell death with consequences on health and survival of the organism as a whole. Because of their essential function in the cell, centromeres have evolved high flexibility and mechanisms of tolerance to preserve their function following stress, whether it is originating from within or outside the cell. Here, we review the main epigenetic mechanisms of centromeres' adaptability to preserve their functional stability, with particular reference to neocentromeres and holocentromeres. The centromere position can shift in response to altered chromosome structures, but how and why neocentromeres appear in a given chromosome region are still open questions. Models of neocentromere formation developed during the last few years will be hereby discussed. Moreover, we will discuss the evolutionary significance of diffuse centromeres (holocentromeres) in organisms such as nematodes. Despite the differences in DNA sequences, protein composition and centromere size, all of these diverse centromere structures promote efficient chromosome segregation, balancing genome stability and adaptability, and ensuring faithful genome inheritance at each cellular generation.

Insulin signaling is critical for neuroplasticity, cerebral metabolism as well as for systemic energy metabolism. In rodent studies, impaired brain insulin signaling with resultant insulin resistance (IR) modulates synaptic plasticity and the corresponding behavioral functions. Despite discoveries of central actions of insulin, in vivo molecular mechanisms of brain IR until recently has proven difficult to study in the human brain. In the current study, we leveraged recent technological advances in molecular biology and herein report an increased number of exosomes enriched for L1CAM, a marker predominantly expressed in the brain, in subjects with major depressive disorder (MDD) as compared with age- and sex-matched healthy controls (HC). We also report increased concentration of the insulin receptor substrate-1 (IRS-1) in L1CAM(+)exosomes in subjects with MDD as compared with age- and sex-matched HC. We found a relationship between expression of IRS-1 in L1CAM(+)exosomes and systemic IR as assessed by homeostatic model assessment of IR in HC, but not in subjects with MDD. The increased IRS-1 levels in L1CAM(+)exosomes were greater in subjects with MDD and were associated with suicidality and anhedonia. Finally, our data suggested sex differences in serine-312 phosphorylation of IRS-1 in L1CAM(+)exosomes in subjects with MDD. These findings provide a starting point for creating mechanistic framework of brain IR in further development of personalized medicine strategies to effectively treat MDD.

Cerebral amyloid angiopathy (CAA), where beta-amyloid (A beta)deposits around cerebral blood vessels, is a major contributor of vascular dysfunction in Alzheimer's disease (AD) patients. However, the molecular mechanism underlying CAA formation and CAA-induced cerebrovascular pathology is unclear. Hereditary cerebral amyloid angiopathy (HCAA) is a rare familial form of CAA in which mutations within the (A beta) peptide cause an increase in vascular deposits. Since the interaction between A beta and fibrinogen increases CAA and plays an important role in cerebrovascular damage in AD, we investigated the role of the A beta-fibrinogen interaction in HCAA pathology. Our work revealed the most common forms of HCAA-linked mutations, Dutch (E22Q) and Iowa (D23N), resulted in up to a 50-fold stronger binding affinity of A beta for fibrinogen. In addition, the stronger interaction between fibrinogen and mutant A beta s led to a dramatic perturbation of clot structure and delayed fibrinolysis. Immunofluorescence analysis of the occipital cortex showed an increase of fibrin(ogen)/A beta codeposition, as well as fibrin deposits in HCAA patients, compared to early-onset AD patients and nondemented individuals. Our results suggest the HCAA-type Dutch and Iowa mutations increase the interaction between fibrinogen and A beta, which might be central to cerebrovascular pathologies observed in HCAA.

Multicellular eukaryotes emerged late in evolution from an ocean of viruses, bacteria, archaea, and unicellular eukaryotes. These macroorganisms are exposed to and infected by a tremendous diversity of microorganisms. Those that are large enough can even be infected by multicellular fungi and parasites. Each interaction is unique, if only because it operates between two unique living organisms, in an infinite diversity of circumstances. This is neatly illustrated by the extraordinarily high level of interindividual clinical variability in human infections, even for a given pathogen, ranging from a total absence of clinical manifestations to death. We discuss here the idea that the determinism of human life-threatening infectious diseases can be governed by single-gene inborn errors of immunity, which are rarely Mendelian and frequently display incomplete penetrance. We briefly review the evidence in support of this notion obtained over the last two decades, referring to a number of focused and thorough reviews published by eminent colleagues in this issue of Human Genetics. It seems that almost any life-threatening infectious disease can be driven by at least one, and, perhaps, a great many diverse monogenic inborn errors, which may nonetheless be immunologically related. While the proportions of monogenic cases remain unknown, a picture in which genetic heterogeneity is combined with physiological homogeneity is emerging from these studies. A preliminary sketch of the human genetic architecture of severe infectious diseases is perhaps in sight.