Bromodomain Inhibitors Correct Bioenergetic Deficiency Caused by Mitochondrial Disease Complex I Mutations.

Mitochondrial diseases comprise a heterogeneous group of genetically inherited disorders that cause failures in energetic and metabolic function. Boosting residual oxidative phosphorylation (OXPHOS) activity can partially correct these failures. Herein, using a high-throughput chemical screen, we identified the bromodomain inhibitor I-BET 525762A as one of the top hits that increases COX5a protein levels in complex I (CI) mutant cybrid cells. In parallel, bromodomain-containing protein 4 (BRD4), a target of I-BET 525762A, was identified using a genome-wide CRISPR screen to search for genes whose loss of function rescues death of CI-impaired cybrids grown under conditions requiring OXPHOS activity for survival. We show that I-BET525762A or loss of BRD4 remodeled the mitochondrial proteome to increase the levels and activity of OXPHOS protein complexes, leading to rescue of the bioenergetic defects and cell death caused by mutations or chemical inhibition of CI. These studies show that BRD4 inhibition may have therapeutic implications for the treatment of mitochondrial diseases.

Dissecting the function of the adult ß-globin downstream promoter region using an artificial zinc finger DNA-binding domain.

Developmental stage-specific expression of the ß-type globin genes is regulated by many cis- and trans-acting components. The adult ß-globin gene contains an E-box located 60 bp downstream of the transcription start site that has been shown to bind transcription factor upstream stimulatory factor (USF) and to contribute to efficient in vitro transcription. We expressed an artificial zinc finger DNA-binding domain (ZF-DBD) targeting this site (+60 ZF-DBD) in murine erythroleukemia cells. Expression of the +60 ZF-DBD reduced the recruitment and elongation of RNA polymerase II (Pol II) at the adult ß-globin gene and at the same time increased the binding of Pol II at locus control region (LCR) element HS2, suggesting that Pol II is transferred from the LCR to the globin gene promoters. Expression of the +60 ZF-DBD also reduced the frequency of interactions between the LCR and the adult ß-globin promoter. ChIP-exonuclease-sequencing revealed that the +60ZF-DBD was targeted to the adult ß-globin downstream promoter and that the binding of the ZF-DBD caused alterations in the association of USF2 containing protein complexes. The data demonstrate that targeting a ZF-DBD to the adult ß-globin downstream promoter region interferes with the LCR-mediated recruitment and activity of Pol II.

Artificial zinc finger DNA binding domains: versatile tools for genome engineering and modulation of gene expression.

Genome editing and alteration of gene expression by synthetic DNA binding activities gained a lot of momentum over the last decade. This is due to the development of new DNA binding molecules with enhanced binding specificity. The most commonly used DNA binding modules are zinc fingers (ZFs), TALE-domains, and the RNA component of the CRISPR/Cas9 system. These binding modules are fused or linked to either nucleases that cut the DNA and induce DNA repair processes, or to protein domains that activate or repress transcription of genes close to the targeted site in the genome. This review focuses on the structure, design, and applications of ZF DNA binding domains (ZFDBDs). ZFDBDs are relatively small and have been shown to penetrate the cell membrane without additional tags suggesting that they could be delivered to cells without a DNA or RNA intermediate. Advanced algorithms that are based on extensive knowledge of the mode of ZF/DNA interactions are used to design the amino acid composition of ZFDBDs so that they bind to unique sites in the genome. Off-target binding has been a concern for all synthetic DNA binding molecules. Thus, increasing the specificity and affinity of ZFDBDs will have a significant impact on their use in analytical or therapeutic settings.

Neutralizing the function of a ß-globin-associated cis-regulatory DNA element using an artificial zinc finger DNA-binding domain.

Gene expression is primarily regulated by cis-regulatory DNA elements and trans-interacting proteins. Transcription factors bind in a DNA sequence-specific manner and recruit activities that modulate the association and activity of transcription complexes at specific genes. Often, transcription factors belong to families of related proteins that interact with similar DNA sequences. Furthermore, genes are regulated by multiple, sometimes redundant, cis-regulatory elements. Thus, the analysis of the role of a specific DNA regulatory sequence and the interacting proteins in the context of intact cells is challenging. In this study, we designed and functionally characterized an artificial DNA-binding domain that neutralizes the function of a cis-regulatory DNA element associated with adult ß-globin gene expression. The zinc finger DNA-binding domain (ZF-DBD), comprising six ZFs, interacted specifically with a CACCC site located 90 bp upstream of the transcription start site (-90 ß-ZF-DBD), which is normally occupied by KLF1, a major regulator of adult ß-globin gene expression. Stable expression of the -90 ß-ZF-DBD in mouse erythroleukemia cells reduced the binding of KLF1 with the ß-globin gene, but not with locus control region element HS2, and led to reduced transcription. Transient transgenic embryos expressing the -90 ß-ZF-DBD developed normally but revealed reduced expression of the adult ß-globin gene. These results demonstrate that artificial DNA-binding proteins lacking effector domains are useful tools for studying and modulating the function of cis-regulatory DNA elements.

Nipsnap1-A regulatory factor required for long-term maintenance of non-shivering thermogenesis.

OBJECTIVE: The activation of non-shivering thermogenesis (NST) has strong potential to combat obesity and metabolic disease. The activation of NST however is extremely temporal and the mechanisms surrounding how the benefits of NST are sustained once fully activated, remain unexplored. The objective of this study is to investigate the role of 4-Nitrophenylphosphatase Domain and Non-Neuronal SNAP25-Like 1 (Nipsnap1) in NST maintenance, which is a critical regulator identified in this study. METHODS: The expression of Nipsnap1 was profiled by immunoblotting and RT-qPCR. We generated Nipsnap1 knockout mice (N1-KO) and investigated the function of Nipsnap1 in NST maintenance and whole-body metabolism using whole body respirometry analyses. We evaluate the metabolic regulatory role of Nipsnap1 using cellular and mitochondrial respiration assay. RESULTS: Here, we show Nipsnap1 as a critical regulator of long-term thermogenic maintenance in brown adipose tissue (BAT). Nipsnap1 localizes to the mitochondrial matrix and increases its transcript and protein levels in response to both chronic cold and ß3 adrenergic signaling. We demonstrated that these mice are unable to sustain activated energy expenditure and have significantly lower body temperature in the face of an extended cold challenge. Furthermore, when mice are exposed to the pharmacological ß3 agonist CL 316, 243, the N1-KO mice exhibit significant hyperphagia and altered energy balance. Mechanistically, we demonstrate that Nipsnap1 integrates with lipid metabolism and BAT-specific ablation of Nipsnap1 leads to severe defects in beta-oxidation capacity when exposed to a cold environmental challenge. CONCLUSION: Our findings identify Nipsnap1 as a potent regulator of long-term NST maintenance in BAT.

Ado-Mediated Depletion of Taurine Impairs Mitochondrial Respiratory Capacity and Alters the Chromatin Landscape of Inguinal Adipose Tissue.

Non-shivering thermogenesis (NST) has strong potential to combat obesity; however, a safe molecular approach to activate this process has not yet been identified. The sulfur amino acid taurine has the ability to safely activate NST and confer protection against obesity and metabolic disease in both mice and humans, but the mechanism of this action is unknown. In this study, we discover that a suite of taurine biosynthetic enzymes, especially that of cysteamine dioxygenase (ADO), significantly increases in response to ß3 adrenergic signaling in inguinal adipose tissue (IWAT) in order to increase intracellular concentrations of taurine. We further show that ADO is critical for thermogenic mitochondrial respiratory function as its ablation in adipocytes significantly reduces taurine levels, which leads to declines in mitochondrial oxygen consumption rates. Finally, we demonstrate via assay for transposase-accessible chromatin with sequencing (ATAC-seq) that taurine supplementation in beige adipocytes has the ability to remodel the chromatin landscape to increase the chromatin accessibility and transcription of genes, such as glucose-6-phosphate isomerase 1 (Gpi1), which are critical for NST. Taken together, our studies highlight a potential mechanism for taurine in the activation of NST that can be leveraged toward the treatment of obesity and metabolic disease.

Inhibition of the ER stress IRE1α inflammatory pathway protects against cell death in mitochondrial complex I mutant cells.

Mitochondrial mutations cause bioenergetic defects associated with failures to use the electron transfer chain and oxidize substrates. These defects are exacerbated under energetic stress conditions and ultimately cause cell deterioration and death. However, little is known about cellular strategies that rescue mitochondrial stress failures and maintain cell survival under these conditions. Here, we have designed and performed a high-throughput chemical screen to identify small molecules that rescue human mitochondrial complex I mutations from energetic stress-induced cell death. The top positive hits were a series of sulfonylureas that efficiently maintain prolonged cell survival and growth under energetic stress conditions. The addition of galactose instead of glucose, to experimentally force mitochondrial respiration, triggered an initial ER stress response that was associated with IRE1α-dependent inflammatory signals including JNK and p38 MAP kinases in mutant cells. Sulfonylureas, similar to inhibition of IRE1α and p38 MAP kinase, potently blocked this ER stress inflammatory and cell death pathway and maintained viability and cell growth under severe energetic stress conditions. These studies reveal that sulfonylureas and specific inhibition of the IRE1α inflammatory pathway protect against cell death and can be used to rescue bioenergetic failures in mitochondrial complex I-mutated cells under stress conditions.

Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells.

Zinc finger proteins are the most common among families of DNA-binding transcription factors. Designer transcription factors generated by the fusion of engineered zinc finger DNA-binding domains (ZF-DBDs) to effector domains have been valuable tools for the modulation of gene expression and for targeted genome editing. However, ZF-DBDs without effector domains have also been shown to effectively modulate gene expression by competing with sequence-specific DNA-binding transcription factors. Here, we describe the methodology and provide a detailed workflow for the cloning, expression, purification, and direct cell delivery of engineered ZF-DBDs. Using this protocol, ZF-DBDs can be generated with high efficiency in less than 2 weeks. We also describe a nonradioactive method for measuring DNA binding affinity of the purified ZF-DBD proteins as well as a method for direct delivery of the purified ZF-DBDs to mammalian cells.

Brown Adipose YY1 Deficiency Activates Expression of Secreted Proteins Linked to Energy Expenditure and Prevents Diet-Induced Obesity.

Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight.