Regulation of energy metabolism during early mammalian development: TEAD4 controls mitochondrial transcription

Kumar, Ram P.; Ray, Soma; Home, Pratik; Saha, Biswarup; Bhattacharya, Bhaswati; Wilkins, Heather; Chavan, Hemantkumar; Ganguly, Sudipto; Milano-Foster, Jessica; Paul, Arindam; Krishnamurthy, Partha; Swerdlow, Russell H.; Paul, Soumen

doi:10.1242/dev.162644

Cited by 36 publications

(30 citation statements)

References 83 publications

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“…When tested in vivo by transfer to the uterus, TEAD4-depleted embryos again did not implant when transferred at E3.5, while they succeeded when transferred at E4.5. In order to make sense of this discrepancy we note that the O 2 tension is lower in the uterus, and that the Tead4 −/− phenotype might be contingent on the O 2 tension, having been proposed that TEAD4 operates in mitochondria to protect the cell from reactive oxygen species [55, 63] . Unfortunately a comparison of these studies with our study does not contribute to the discussion, because our TEAD4-depleted embryos were cultured without oil overlay from the 1-cell stage, whereas the rescued ones were cultured with oil overlay in the presence of NAC from the 2- or 8-cell stage [55] .…”

Section: Discussionmentioning

confidence: 99%

A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days

et al. 2019

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BackgroundWhile DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires also protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. When proteins in the oocyte encounter a specific antibody and the TRIpartite Motiv-containing 21 (TRIM21) ubiquitin-protein ligase, they can be committed to degradation in the proteasome, producing a transient functional knock-out that reveals the role of the protein. However, there are doubts about whether this targeted proteolysis could be successfully used to study mammalian development, because duration of the transient effect is unknown, and also because amounts of reagents delivered must be adequate in relation to the amount of target protein, which is unknown, too.ResultsWe show that the mouse egg contains up to 1E-02 picomoles/protein, as estimated by mass spectrometry using the intensity-based absolute quantification (iBAQ) algorithm. However, the egg can only accommodate ≈1E-04 picomoles of antibody or TRIM21 without incurring toxic effects. Within this framework, we demonstrate that TRIM21-mediated protein depletion efficiently disrupts the embryonic process of trophectoderm formation, which critically depends on the TEA domain family member 4 (Tead4) gene. TEAD4 depletion starting at the 1-cell stage lasts for 3 days prior to a return of gene and protein expression to baseline. This time period is long enough to result in a phenotype entirely consistent with that of the published null mutation and RNA interference studies: significant underexpression of trophectodermal genes Cdx2 and Gata3 and strongly impaired ability of embryos to cavitate and implant in the uterus. Omics data are available via ProteomeXchange (PXD012613) and GEO (GSE124844).ConclusionsTRIM21-mediated protein depletion can be an effective means to disrupt gene function in mouse development, provided the target gene is chosen carefully and the method is tuned accurately. The knowledge gathered in this study provides the basic know-how (prerequisites, requirements, limitations) to expedite the protein depletion of other genes besides Tead4.

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Section: Discussionmentioning

confidence: 99%

A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days

et al. 2019

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“…The co-localization of our mt-ASFP sites with regulatory elements, particularly with sites that associate with mtDNA transcription, tempts to assess the connection between our observed footprinting dynamics during development and mtDNA transcription. With this in mind the recent discovery of TEAD4, a critical factor in mouse preimplantation embryogenesis, which also regulates mtDNA transcription in the trophectoderm, is noteworthy (Kumar et al., 2018). Once quantitative techniques that measure nascent RNA formation, such as PRO-seq (Kwak et al., 2013), which was recently adapted to mtDNA analysis (Blumberg et al., 2017), are adapted to single-cell analysis, one will be able to experimentally assess such correlation.…”

Section: Discussionmentioning

confidence: 99%

mtDNA Chromatin-like Organization Is Gradually Established during Mammalian Embryogenesis

et al. 2019

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SummaryUnlike the nuclear genome, the mammalian mitochondrial genome (mtDNA) is thought to be coated solely by mitochondrial transcription factor A (TFAM), whose binding sequence preferences are debated. Therefore, higher-order mtDNA organization is considered much less regulated than both the bacterial nucleoid and the nuclear chromatin. However, our recently identified conserved DNase footprinting pattern in human mtDNA, which co-localizes with regulatory elements and responds to physiological conditions, likely reflects a structured higher-order mtDNA organization. We hypothesized that this pattern emerges during embryogenesis. To test this hypothesis, we analyzed assay for transposase-accessible chromatin sequencing (ATAC-seq) results collected during the course of mouse and human early embryogenesis. Our results reveal, for the first time, a gradual and dynamic emergence of the adult mtDNA footprinting pattern during embryogenesis of both mammals. Taken together, our findings suggest that the structured adult chromatin-like mtDNA organization is gradually formed during mammalian embryogenesis.

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“…This dramatic change heralds an increase in metabolic activity by the differentiating TE, comprising elevated ATP, amino acid turnover and mitochondrial count 6, 7 . The murine embryo is equipped to overcome adverse consequences associated with accumulation of reactive oxygen species during the metabolic transition to oxidative phosphorylation, largely facilitated by the transcriptional enhancer factor TEAD4 8, 9 . TEAD4 becomes intensified in the TE, where it cooperates with nuclear YAP to initiate transcription of TE-specific genes 10, 11 .…”

Section: Introductionmentioning

confidence: 99%

Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

Stirparo

Kurowski

Strawbridge

et al. 2020

Preprint

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44 OCT4 is a fundamental component of the molecular circuitry governing 45 pluripotency in vivo and in vitro. To determine how OCT4 protects the 46 pluripotent lineage from differentiation into trophoblast, we used single cell 47 transcriptomics and quantitative immunofluorescence on blastocysts and 48 established differentially expressed genes and pathways between control and 49 OCT4 null cells. Activation of most pluripotency-associated transcription 50 factors in the early mouse inner cell mass appears independent of OCT4, 51 whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single 52 cell deconvolution, diffusion component and trajectory inference dissected the 53 process of differentiation of OCT4 null cells by activating specific gene-54 network and transcription factors. Downregulation of glycolytic and oxidative 55 metabolism was observed. CHIPseq analysis suggests OCT4 directly targets 56 rate-limiting glycolytic enzymes. Concomitant with significant disruption of the 57 STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null 58 cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos 59 is likely attributable to aberrant metabolism. 60 Results 104 Single cell transcriptional profiling reveals divergence of OCT4 null from wild 105 type and heterozygous ICM cells by the mid blastocyst stage 106 Following observation of NANOG protein in OCT4 null blastocysts24,25, we 107 performed whole transcriptome analysis by scRNAseq of ICM cells isolated 108 from Pou5f1 heterozygous inter se mating at embryonic day (E)3.5 to 109 investigate the entire pluripotency network. Quality control, as previously 110 reported27, eliminated inadequate samples, leaving 29 null mutant (MUT), 42 111wild-type (WT) and 16 heterozygous (HET) cells from 4, 5 and 2 mid 112 blastocysts, respectively (Fig.1A, Suppl.Table1). Pou5f1 RNA expression was 113 not detected in MUT embryos, confirming degradation of maternal transcripts 114 ( Fig.1A, Fig.S1A), consistent with lack of OCT4 protein observed at the 115 morula stage13. To characterize global differences and similarities between 116 genotypes t-SNE analysis was performed (Fig.1B, Fig.S1A) using the most 117 variable genes identified in early blastocysts (n=2232, log2FPKM>0.5, 118 logCV2>0.5). MUT cells cluster separately from HET and WT, suggesting 119 major changes in transcriptome, despite no apparent difference in ICM 120 morphology. Interestingly, HET and WT cells clustered together, indicating no 121 more than a negligible effect of reduced Pou5f1 in HET cells in the developing 122 embryo, contrasting with the elevated and more homogeneous expression of 123 Nanog, Klf4 and Estrogen-related-receptor B (Esrrb) previously reported in 124Oct4 HET ESCs28 (Fig.S1B). 125Weighted gene correlation network analysis (WGCNA) allows 126 extraction of modules defined by co-regulated genes, combined with 127 unsupervised clustering (Fig.1C, Fig.S1C). Two main modules emerged, 128 7 clustering cells according to genotype: module 1 co-clust...

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Regulation of energy metabolism during early mammalian development: TEAD4 controls mitochondrial transcription

Cited by 36 publications

References 83 publications

A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days

A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days

mtDNA Chromatin-like Organization Is Gradually Established during Mammalian Embryogenesis

Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

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