Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core oncogenic mechanisms are not well-defined. To identify major effectors, we used CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma cells back to wildtype. ChIP-seq analysis broadly linked K27M to altered H3K27me3 activity including within super-enhancers, which exhibited perturbed transcriptional function. This was largely independent of H3.3 DNA binding. The K27M and G34R mutations induced several of the same pathways suggesting key shared oncogenic mechanisms including activation of neurogenesis and NOTCH pathway genes. H3.3 mutant gliomas are also particularly sensitive to NOTCH pathway gene knockdown and drug inhibition, reducing their viability in culture. Reciprocal editing of cells generally produced reciprocal effects on tumorgenicity in xenograft assays. Overall, our findings define common and distinct K27M and G34R oncogenic mechanisms, including potentially targetable pathways.

Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and β, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and β, increased myoblast differentiation whereas GC1, a selective TRβ agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/β-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRβ PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.

Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into β-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous β-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into β-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo β-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous β-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining β-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of β-cell recovery after injury mediated by hBMSC therapy.

It remains a challenge to achieve the stable and long‐term expression (in human cell lines) of a previously engineered hybrid enzyme [triple‐catalytic (Trip‐cat) enzyme‐2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003–14011], which links cyclo‐oxygenase isoform‐2 (COX‐2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s Trip‐cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)‐mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip‐cat enzyme, in which human cyclo‐oxygenase isoform‐1, which has a different C‐terminal sequence from COX‐2, was linked to PGI2 synthase and called Trip‐cat enzyme‐1. Transient expression of recombinant Trip‐cat enzyme‐1 in HEK293 cells led to 3–5‐fold higher expression capacity and better PGI2‐synthesizing activity as compared to that of the previously engineered Trip‐cat enzyme‐2. Furthermore, an HEK293 cell line that can stably express the active new Trip‐cat enzyme‐1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase‐rich plasma. This study has optimized engineering of the active Trip‐cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2‐producing therapeutic cell line for use against vascular diseases.

Cyclooxygenase isoform-2 (COX-2) and microsomal prostaglandin E(2) synthase-1 (mPGES-1) are inducible enzymes that become up-regulated in inflammation and some cancers. It has been demonstrated that their coupling reaction of converting arachidonic acid (AA) into prostaglandin (PG) E(2) (PGE(2)) is responsible for inflammation and cancers. Understanding their coupling reactions at the molecular and cellular levels is a key step toward uncovering the pathological processes in inflammation. In this paper, we describe a structure-based enzyme engineering which produced a novel hybrid enzyme that mimics the coupling reactions of the inducible COX-2 and mPGES-1 in the native ER membrane. Based on the hypothesized membrane topologies and structures, the C-terminus of COX-2 was linked to the N-terminus of mPGES-1 through a transmembrane linker to form a hybrid enzyme, COX-2-10aa-mPGES-1. The engineered hybrid enzyme expressed in HEK293 cells exhibited strong triple-catalytic functions in the continuous conversion of AA into PGG(2) (catalytic-step 1), PGH(2) (catalytic-step 2) and PGE(2) (catalytic-step 3), a pro-inflammatory mediator. In addition, the hybrid enzyme was also able to directly convert dihomo-gamma-linolenic acid (DGLA) into PGG(1), PGH(1) and then PGE(1) (an anti-inflammatory mediator). The hybrid enzyme retained similar K(d) and V(max) values to that of the parent enzymes, suggesting that the configuration between COX-2 and mPGES-1 (through the transmembrane domain) could mimic the native conformation and membrane topologies of COX-2 and mPGES-1 in the cells. The results indicated that the quick coupling reaction between the native COX-2 and mPGES-1 (in converting AA into PGE(2)) occurred in a way so that both enzymes are localized near each other in a face-to-face orientation, where the COX-2 C-terminus faces the mPGES-1 N-terminus in the ER membrane. The COX-2-10aa-mPGES-1 hybrid enzyme engineering may be a novel approach in creating inflammation cell and animal models, which are particularly valuable targets for the next generation of NSAID screening.