scholarly journals Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization

2017 ◽  
Author(s):  
Leslie Y. Beh ◽  
Galia T. Debelouchina ◽  
Kelsi A. Lindblad ◽  
Katarzyna Kulej ◽  
Elizabeth R. Hutton ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
De Novo ◽  
Nucleosome Occupancy ◽  
List Type ◽  
Eukaryotic Chromosome ◽  
Nucleosome Organization ◽  
Eukaryotic Chromatin ◽  
Dna Substrates ◽  
The Impact

SummaryBiochemical studies of chromatin have typically used either artificial DNA templates with unnaturally high affinity for histones, or small genomic DNA fragments deprived of their cognate physical environment. It has thus been difficult to dissect chromatin structure and function within fully native DNA substrates. Here, we circumvent these limitations by exploiting the minimalist genome of the eukaryoteOxytricha trifallax, whose notably small ~3kb chromosomes mainly encode single genes. Guided by high-resolution epigenomic maps of nucleosome organization, transcription, and DNA N6-methyladenine (m6dA) locations, we reconstruct full-lengthOxytrichachromosomesin vitroand use these synthetic facsimiles to dissect the influence of m6dA and histone post-translational modifications on nucleosome organization. We show that m6dA directly disfavors nucleosomes in a quantitative manner, leading to local decreases in nucleosome occupancy that are synergistic with histone acetylation. The effect of m6dA can be partially reversed by the action of an ATP-dependent chromatin remodeler. Furthermore, erasing m6dA marks fromOxytrichachromosomes leads to proportional increases in nucleosome occupancy across the genome. This work showcasesOxytrichachromosomes as powerful yet practical models for studying eukaryotic chromatin and transcription in the context of biologically relevant DNA substrates.HighlightsDe novosynthesis of complete, epigenetically definedOxytrichachromosomesEpigenomic profiles of chromatin organization inOxytricha’sminiature chromosomesm6dA directly disfavors nucleosome occupancy in natural and synthetic chromosomesHistone acetylation and chromatin remodelers temper the impact of m6dA on chromatin

scholarly journals Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

2018 ◽  
Author(s):  
Leslie Y. Beh ◽  
Galia T. Debelouchina ◽  
Derek M. Clay ◽  
Robert E. Thompson ◽  
Kelsi A. Lindblad ◽  
...  
Keyword(s):  
De Novo ◽  
Nucleosome Occupancy ◽  
Dna Methyltransferases ◽  
Chromatin Organization ◽  
Sexual Cycle ◽  
List Type ◽  
Adenine Methylation ◽  
The Impact

SummaryDNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1in vivoleads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes in chromatin organization or RNA expression upon loss of 6mA, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We determine the impact of 6mA on chromatin organizationin vitroby reconstructing complete, full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomesin vitroin a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined synthetic chromosomes.HighlightsThe MT-A70 protein MTA1 mediates DNA N6-adenine methylation inOxytrichaMTA1 mutants exhibit subtle changes in nucleosome organization and transcriptionin vivo6mA directly disfavors nucleosome occupancy in natural and synthetic chromosomesin vitroDe novosynthesis of complete, epigenetically definedOxytrichachromosomes

The Rho GTPase Rac1 Is Not Required for Definitive Hematopoietic Specification in ES-Derived Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1494-1494
Author(s):  
Michael D. Milsom ◽  
Akiko Yabuuchi ◽  
George Q. Daley ◽  
David A. Williams
Keyword(s):  
Rho Gtpase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Embryoid Body ◽  
Es Cells ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitors ◽  
The Impact

Abstract Abstract 1494 Poster Board I-517 Rac1 is a Rho GTPase involved in integrating signaling pathways that regulate numerous cellular processes including adhesion, migration, proliferation and HSC engraftment. Homozygous deletion of Rac1 is lethal in the murine embryo prior to E9.5 and Rac1−/− embryos demonstrate defective gastrulation associated with reduced epiblast adhesion and motility. We have recently demonstrated using lineage-specific conditional deletion that Rac1 insufficiency results in severely impaired hematopoiesis in the embryonic sites of hematopoiesis (AGM, aortic clusters and fetal liver) in the setting of normal hematopoietic development in the yolk sac (YS) and reduced HSC and progenitors in the fetal circulation. This data appears to support the controversial hypothesis that YS derived HSC seed embryonic sites, but an alternative explanation is that Rac1 is essential for some aspect of the induction of intraembryonic hematopoiesis in situ. Another possibility is that Vav1-Cre-mediated excision of Rac1 occurs prior to the onset of hematopoiesis in the embryo proper but not early enough to affect yolk sac hematopoiesis. To test whether Rac1 insufficiency perturbs the normal early differentiation of hematopoietic cells in vitro, we used a lentivirus expressing a Rac1-specific shRNA to knock down expression in an ES line previously characterized to have good hemogenic potential. We observed that the de novo knockdown of Rac1 expression appeared to have no impact upon derivation of hematopoietic progenitors. To demonstrate that this was not the result of inefficient knockdown of Rac1, we derived Rac1−/− ES lines from blastomeres resulting from the mating of Rac1+/− mice. Rac1−/− ES lines were produced in normal Mendelian ratios (4 Rac1+/+: 9 Rac1+/−: 3 Rac1−/−) and did not demonstrate any evidence of abnormal expansion on murine embryonic fibroblasts. In order to assess the impact of Rac1 deficiency on the hemogenic potential of ES cells, standard in vitro differentiation via embryoid body formation was utilized. Neither Rac1 haploinsufficiency nor complete absence of Rac1 had any impact on the production of CD41+/c-Kit+ hematopoietic progenitors within embryoid bodies (Table 1). Furthermore, colony forming assays demonstrated that Rac1 insufficiency did not alter the relative frequency of hematopoietic progenitor compartments (Table 2). We conclude that in the absence of a requirement for vascular migration of HSC, Rac1 is not required for the specification of definitive hematopoiesis. These data, together with our previously published in vivo data continue to support the hypothesis that HSC migration from the YS to the embryo may be required for development of hematopoiesis in the embryo proper. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distribution and vulnerability of transcriptional outputs across the genome in Myc-amplified medulloblastoma cells

2021 ◽  
Author(s):  
Rui Yang ◽  
Wenzhe Wang ◽  
Meichen Dong ◽  
Kristen Roso ◽  
Paula Greer ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tumor Cells ◽  
Target Genes ◽  
De Novo ◽  
Inhibitory Effect ◽  
Nucleotide Synthesis ◽  
High Level ◽  
The Impact

Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes1-4. While it is well established that Myc functions by binding to its target genes to regulate their transcription5, the distribution of the transcriptional output across the human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals that a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructure, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly only in genes involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on infrastructure genes, which was accompanied by the abrogation of MB cells proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, provide new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in cancer cells empowered by a high level of Myc oncoprotein.

scholarly journals Peptidomes and Structures Illustrate Two Distinguishing Mechanisms of Alternating the Peptide Plasticity Caused by Swine MHC Class I Micropolymorphism

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaohui Wei ◽  
Song Wang ◽  
Zhuolin Li ◽  
Zibin Li ◽  
Zehui Qu ◽  
...  
Keyword(s):  
De Novo ◽  
Peptide Binding ◽  
Class I ◽  
Class I Mhc ◽  
Mhc I ◽  
Anchor Residue ◽  
Binding Characteristics ◽  
Peptide Presentation ◽  
The Impact

The micropolymorphism of major histocompatibility complex class I (MHC-I) can greatly alter the plasticity of peptide presentation, but elucidating the underlying mechanism remains a challenge. Here we investigated the impact of the micropolymorphism on peptide presentation of swine MHC-I (termed swine leukocyte antigen class I, SLA-I) molecules via immunopeptidomes that were determined by our newly developed random peptide library combined with the mass spectrometry (MS) de novo sequencing method (termed RPLD–MS) and the corresponding crystal structures. The immunopeptidomes of SLA-1*04:01, SLA-1*13:01, and their mutants showed that mutations of residues 156 and 99 could expand and narrow the ranges of peptides presented by SLA-I molecules, respectively. R156A mutation of SLA-1*04:01 altered the charge properties and enlarged the volume size of pocket D, which eliminated the harsh restriction to accommodate the third (P3) anchor residue of the peptide and expanded the peptide binding scope. Compared with 99Tyr of SLA-1*0401, 99Phe of SLA-1*13:01 could not form a conservative hydrogen bond with the backbone of the P3 residues, leading to fewer changes in the pocket properties but a significant decrease in quantitative of immunopeptidomes. This absent force could be compensated by the salt bridge formed by P1-E and 170Arg. These data illustrate two distinguishing manners that show how micropolymorphism alters the peptide-binding plasticity of SLA-I alleles, verifying the sensitivity and accuracy of the RPLD-MS method for determining the peptide binding characteristics of MHC-I in vitro and helping to more accurately predict and identify MHC-I restricted epitopes.

scholarly journals Nucleosomes impede Cas9 access to DNA in vivo and in vitro

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Max A Horlbeck ◽  
Lea B Witkowsky ◽  
Benjamin Guglielmi ◽  
Joseph M Replogle ◽  
Luke A Gilbert ◽  
...  
Keyword(s):  
Large Scale ◽  
Nucleosome Occupancy ◽  
Critical Role ◽  
Genetic Screens ◽  
Guide Rnas ◽  
Highly Active ◽  
Bacterial Enzyme ◽  
Eukaryotic Chromatin

The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.

scholarly journals Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels

2017 ◽  
Vol 292 (8) ◽  
pp. 3312-3322 ◽  
Author(s):  
Alessandro Carrer ◽  
Joshua L. D. Parris ◽  
Sophie Trefely ◽  
Ryan A. Henry ◽  
David C. Montgomery ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
High Fat Diet ◽  
Cell Types ◽  
High Fat ◽  
Atp Citrate Lyase ◽  
Acetyl Coa ◽  
Citrate Lyase ◽  
Liver And Pancreas ◽  
The Impact

Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro. This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.

scholarly journals Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

2020 ◽  
Author(s):  
Quentin M. Dudley ◽  
Ashty S. Karim ◽  
Connor J. Nash ◽  
Michael C. Jewett
Keyword(s):  
Protein Synthesis ◽  
Metabolic Engineering ◽  
Biosynthetic Enzymes ◽  
List Type ◽  
Free Protein ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
The Impact ◽  
Cell Free Protein Synthesis

AbstractMetabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.TOC FigureHighlightsApplied the iPROBE framework to build the nine-enzyme pathway to produce limoneneAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performanceIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactionsExtended pathway to biofuel precursors pinene and bisabolene

scholarly journals IL-21 Receptor Blockade Shifts the Follicular T Cell Balance and Reduces De Novo Donor-Specific Antibody Generation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yeqi Nian ◽  
Zhilei Xiong ◽  
Panpan Zhan ◽  
Zhen Wang ◽  
Yang Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Germinal Center ◽  
De Novo ◽  
Antibody Mediated Rejection ◽  
Treg Differentiation ◽  
The Impact

Donor-specific antibodies (DSAs) play a key role in chronic kidney allograft injury. Follicular T helper (Tfh) cells trigger the humoral alloimmune response and promote DSA generation, while T-follicular regulatory (Tfr) cells inhibit antibody production by suppressing Tfh and B cells. Interleukin (IL)-21 exerts a distinct effect on Tfh and Tfr. Here, we studied whether blocking IL-21R with anti-IL-21R monoclonal antibody (αIL-21R) changes the Tfh/Tfr balance and inhibits DSA generation. First, we investigated the impact of αIL-21R on CD4+ T cell proliferation and apoptosis. The results showed that αIL-21R did not have cytotoxic effects on CD4+ T cells. Next, we examined Tfh and regulatory T cells (Tregs) in an in vitro conditioned culture model. Naïve CD4+ T cells were isolated from 3-month-old C57BL/6 mice and cultured in Tfh differentiation inducing conditions in presence of αIL-21R or isotype IgG and differentiation was evaluated by CXCR5 expression, a key Tfh marker. αIL-21R significantly inhibited Tfh differentiation. In contrast, under Treg differentiation conditions, FOXP3 expression was inhibited by IL-21. Notably, αIL-21R rescued IL-21-inhibited Treg differentiation. For in vivo investigation, a fully mismatched skin transplantation model was utilized to trigger the humoral alloimmune response. Consistently, flow cytometry revealed a reduced Tfh/Tfr ratio in recipients treated with αIL-21R. Germinal center response was evaluated by flow cytometry and lectin histochemistry. We observed that αIL-21R significantly inhibited germinal center reaction. Most importantly, DSA levels after transplantation were significantly inhibited by αIL-21R at different time points. In summary, our results demonstrate that αIL-21R shifts the Tfh/Tfr balance toward DSA inhibition. Therefore, αIL-21R may be a useful therapeutic agent to prevent chronic antibody mediated rejection after organ transplantation.

scholarly journals BIOL-10. DISTRIBUTION AND VULNERABILITY OF TRANSCRIPTIONAL OUTPUTS ACROSS THE GENOME IN MYC-AMPLIFIED MEDULLOBLASTOMA CELLS

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i5-i5
Author(s):  
Rui Yang ◽  
Wenzhe Wang ◽  
Meichen Dong ◽  
Kristen Roso ◽  
Xuhui Bao ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Target Genes ◽  
De Novo ◽  
Inhibitory Effect ◽  
Nucleotide Synthesis ◽  
Expression Of Genes ◽  
High Level ◽  
The Impact

Abstract Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes. While it is well established that Myc functions by binding to its target genes to regulate their transcription, the distribution of the transcriptional output across human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructures, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly in genes of involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc’s expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on the aforementioned infrastructure genes, which was accompanied by the abrogation of MB cell’s proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, gain new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in MB cells empowered by a high level of Myc oncoprotein.

scholarly journals Ferredoxin 1b (Fdx1b) Is the Essential Mitochondrial Redox Partner for Cortisol Biosynthesis in Zebrafish

Endocrinology ◽  
2016 ◽  
Vol 157 (3) ◽  
pp. 1122-1134 ◽  
Author(s):  
Aliesha Griffin ◽  
Silvia Parajes ◽  
Meltem Weger ◽  
Andreas Zaucker ◽  
Angela E. Taylor ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Light Adaptation ◽  
De Novo ◽  
Transcription Activator ◽  
Dark Light ◽  
Cyp Enzymes ◽  
Redox Partner ◽  
The Impact

Abstract Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo.

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