KMT2A promotes melanoma cell growth by targeting hTERT signaling pathway.

Melanoma is an aggressive cutaneous malignancy, illuminating the exact mechanisms and finding novel therapeutic targets are urgently needed. In this study, we identified KMT2A as a potential target, which promoted the growth of human melanoma cells. KMT2A knockdown significantly inhibited cell viability and cell migration and induced apoptosis, whereas KMT2A overexpression effectively promoted cell proliferation in various melanoma cell lines. Further study showed that KMT2A regulated melanoma cell growth by targeting the hTERT-dependent signal pathway. Knockdown of KMT2A markedly inhibited the promoter activity and expression of hTERT, and hTERT overexpression rescued the viability inhibition caused by KMT2A knockdown. Moreover, KMT2A knockdown suppressed tumorsphere formation and the expression of cancer stem cell markers, which was also reversed by hTERT overexpression. In addition, the results from a xenograft mouse model confirmed that KMT2A promoted melanoma growth via hTERT signaling. Finally, analyses of clinical samples demonstrated that the expression of KMT2A and hTERT were positively correlated in melanoma tumor tissues, and KMT2A high expression predicted poor prognosis in melanoma patients. Collectively, our results indicate that KMT2A promotes melanoma growth by activating the hTERT signaling, suggesting that the KMT2A/hTERT signaling pathway may be a potential therapeutic target for melanoma

Hypothalamic mitochondria in energy homeostasis and obesity

Citation: Guo X, Wu L, Wang W, Medeiros DM, Clarke S, et al.(2016) Hypothalamic mitochondria in energy homeostasis and obesity. Integr Mol Med 3: DOI: 10.15761/IMM.1000209.Obesity, which is largely due to energy imbalance, has emerged as one of the most serious health issues in the world. The hypothalamus is the most important organ to regulate feeding behavior and energy expenditure through nutrient sensing and signal integration from central and peripheral pathways. As the main organelle to produce energy, mitochondria play a critical role in energy homeostasis from the organelle level. Besides providing a platform for the oxidation of fuel substrates, mitochondria are also involved in a variety of cell signaling pathways and modulate energy homeostasis through mitochondrial dynamics. Mitochondrial dysfunction may lead to obesity due to inadequate ATP production, oxidative stress, endoplasmic reticulum stress, and inflammation. ?, ?-carotene-9’,10’-oxygenase2 (BCO2) is a mitochondrial enzyme that catalyzes the asymmetric cleavage of both provitamin A and non-provitamin A carotenoids. This enzyme is localized to the inner mitochondrial membrane, where the electron transport chain is located. Besides the enzymatic function, BCO2 is important for mitochondrial function and is genetically associated with interleukin-18. Moreover, BCO2 protein expression is suppressed in obese and diabetic mice. Given that the important role of BCO2 in mitochondrial structure and function, and the key position of the hypothalamus in energy balance, BCO2 may play a new role in maintaining metabolic homeostasis that has been overlooked before. The mutation of BCO2 might lead to the impairment of whole body energy homeostasis through hypothalamic mitochondrial dysfunction. Here we will be presenting the updates on hypothalamic mitochondria in cellular energy homeostasis and discussing the potential of BCO2 in regulation of hypothalamic mitochondria in health and obesity. 

Creation of Hypoallergenic Mustard (Brassica juncea) through Genome Editing and Development of Precise Base Editing Tools for Plants

Eine verbesserte landwirtschaftliche Sicherheit durch neue Züchtungsverfahren ist dringend erforderlich, um den Zugang zu nahrhaften Lebensmitteln weltweit zu verbessern. Das Genom-Engineering mit Hilfe von CRISPR (clustered regularly interspaced short palindromic repeats)-Technologien oder TALE (transcription activator-like effector)-Technologien bietet die einzigartige Möglichkeit, gezielt Gene für eine präzise Züchtung zu verändern. Diese Technologie ist vielversprechend für verschiedene Anwendungen in der Allergieforschung. Das Hauptallergen Bra j I aus braunem Senf (Brassica juncea) ist ein Saatgut-Speicherprotein, das zur 2S-Albuminfamilie gehört. Ein Ziel dieser Arbeit war die Schaffung einer hypoallergenen Senfsorte durch den Einsatz von Genome Editing-Techniken und ein zweites Ziel war die Entwicklung neuer Baseneditoren für Pflanzen. Zunächst wurden zwei CRISPR/Cas9-Konstrukte mit Multiplex-Single-Guide-RNAs eingesetzt, um große Deletionen oder Frameshift-Mutationen in den beiden Homöologen Bra j IA und Bra j IB in zwei Linien des braunen Senf (Terratop und CR2664) zu induzieren. In den transgenen T0-Senfpflanzen wurden hohe Mutationseffizienzen beobachtet. Das Bra j IB-Allel wies in vier Linien große Deletionen zwischen 566 und 790 bp auf. Außerdem wiesen neun von 18 Terratop-T0-Linien kleine Indels in den Zielregionen auf. In ähnlicher Weise wiesen 14 der 16 analysierten CR2664 T0-Linien Indels auf, während drei Linien Mutationen in allen vier Bra j I-Allelen aufwiesen. Die Mutationen wurden stabil an die T1-Nachkommen vererbt. Darüber hinaus zeigten Immunoblotting-Ergebnisse eine Abnahme oder ein vollständiges Fehlen des Bra j I-Proteins in den Samenextrakten ausgewählter T1-Linien. Diese Arbeit unterstreicht den Wert von Genom Editing Technologien für die Schaffung hypoallergener Lebensmittelpflanzen. Zweitens wurden zwei Baseneditoren entwickelt: TALE-abgeleitete DddA-basierte Cytosin-Baseneditoren (TALE-DdCBEs) und TALE-abgeleitete Adenin-Baseneditoren (TALE-ABEs). Sie wurden entwickelt, um eine präzise Bearbeitung von C•G-to-T•A bzw. A•T-to-G•C zu erstellen. TALE-DdCBEs, die DddA-Varianten (DddA6 oder DddA11) enthielten, zeigten eine deutliche Verbesserung der Editierungseffizienz sowohl in Nicotiana benthamiana als auch in Reisprotoplasten. TALE-DdCBEs mit DddA11 wiesen eine bessere Sequenzkompatibilität für die Bearbeitung von Nicht-TC-Zielen auf. Darüber hinaus wurden verschiedene TALE-ABEs mit unterschiedlichen Desaminase-Fusionsarchitekturen in Reis und N. benthamiana getestet. Die Ergebnisse zeigten, dass TALE-ABEs A•T-to-G•C Umwandlungen im Reisprotoplasten ermöglichen. TALE-Base-Editoren können für nukleare Gene eingesetzt werden oder alternativ über N-terminale Targeting-Sequenzen die Genome von Plastiden oder Mitochondrien zum Ziel haben.Improved agricultural safety through novel breeding techniques is urgently required to increase access to nutritious foods worldwide. Genome engineering using clustered regularly interspaced short palindromic repeats (CRISPR)-based or transcription activator-like effector (TALE)-based technologies provides a unique ability to modify targeted genes for precise breeding. This technology shows promise in various applications of allergy research. The major allergen Bra j I from brown mustard (Brassica juncea) is a seed storage protein that belongs to the 2S albumin family. One aim of this thesis was to create a hypoallergenic variety of mustard by utilizing genome editing techniques and a second aim was to develop novel base editing tools for plants. Firstly, two CRISPR/Cas9 constructs with multiplex single guide RNAs were employed to induce large deletions or frameshift mutations in both Bra j IA and Bra j IB homoeologs in two brown mustard lines (Terratop and CR2664). High mutation efficiencies were observed in the T0 transgenic mustard plants. The Bra j IB allele exhibited large deletions ranging from 566 to 790 bp in four lines. Additionally, nine out of 18 Terratop T0 lines exhibited small indels in the targeted regions. Similarly, 14 out of 16 CR2664 T0 lines analyzed had indels, while three lines exhibited mutations in all four Bra j I alleles. The mutations were stably inherited to the T1 progeny. Moreover, immunoblotting results demonstrated a decrease or complete absence of the Bra j I protein in the seed extracts of selected T1 lines. This work highlights the value of genome editing technologies in creating hypoallergenic food plants. Secondly, two base editing tools: TALE-derived DddA-based cytosine base editors (TALEDdCBEs) and TALE-derived adenine base editors (TALE-ABEs) were developed for precise C•G-to-T•A and A•T-to-G•C editing, respectively. TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice protoplasts. TALE-DdCBEs containing DddA11 exhibited broader sequence compatibility for editing non-TC targets. Furthermore, a series of TALE-ABEs with different deaminase fusion architectures were tested in N. benthamiana and rice. The results showed that TALE-ABEs enable the conversion of A•T-to-G•C in rice protoplast. The application of TALE-base editors can result in a dramatic change because they can be deployed for nuclear genes or, alternatively, target the genomes of plastids or mitochondria by N-terminal targeting sequences

Development of TALE-adenine base editors in plants

Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes

Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants

BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a β-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement