During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome ...activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 103 different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation i.e., (i) 59%, (ii) 42%, and (iii) 58% was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.
Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies
. Mitochondrial malfunction needs to be relayed to the cytosol, where an ...integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells
. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress
. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown
. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.
Psoriasis vulgaris is a common T cell-mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis ...risk gene. Epidermal CD8(+) T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific target cells of an HLA-C*06:02-restricted psoriatic T cell response. We found that a Vα3S1/Vβ13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8(+) T cell clone of an HLA-C*06:02-positive psoriasis patient specifically recognizes HLA-C*06:02-positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02-presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. Consistent with the Vα3S1/Vβ13S1-TCR reactivity, we observed numerous CD8(+) T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8(+) T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8(+) T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway.
Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues ...and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.
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► LBR- and lamin-A/C-dependent tethers maintain peripheral heterochromatin ► In their absence, all heterochromatin clusters in the nuclear interior ► During cellular differentiation, the LBR tether precedes the lamin A/C tether ► Lamin A/C promotes, whereas LBR delays myogenic differentiation
The lamin B receptor and lamin A/C have sequential roles in the tethering of peripheral heterochromatin during cellular differentiation, corresponding to their differential effects on heterochromatin positioning and transcription of tissue-specific genes.
The scope of this paper is the design of an interval observer bundle for the guaranteed state estimation of an uncertain induction machine with linear, time-varying dynamics. These guarantees are of ...particular interest in the case of safety-critical systems. In many cases, interval observers provide large intervals for which the usability becomes impractical. Hence, based on a reduced-order hybrid interval observer structure, the guaranteed enclosure within intervals of the magnetizing current's estimates is improved using a bundle of interval observers. One advantage of such an interval observer bundle is the possibility to reinitialize the interval observers at specified timesteps during runtime with smaller initial intervals, based on previously observed system states, resulting in decreasing interval widths. Thus, unstable observer dynamics are considered so as to take advantage of their transient behavior, whereby the overall stability of the interval estimation is maintained. An algorithm is presented to determine the parametrization of reduced-order interval observers. To this, an adaptive observer gain is introduced with which the system states are observed optimally by considering a minimal interval width at variable operating points. Furthermore, real-time capability and validation of the proposed methods are shown. The results are discussed with simulations as well as experimental data obtained with a test bench.
•We review studies on bovine embryonic genome activation (EGA).•We provide new insights into EGA by RNA sequencing (RNA-Seq).•A gene ontology analysis of genes activated at the 8-cell, 16-cell, and ...blastocyst stage is provided.•An outlook into potential applications of single cell RNA-Seq is given.
Maternal-to-embryonic transition (MET) is the period in early embryonic development when maternal RNAs and proteins stored in the oocyte are gradually degraded and transcription of the embryonic genome is activated. First insights into the timing of embryonic genome activation (EGA) came from autoradiographic analyses of embryos following incorporation of 3Huridine. These studies identified the eight- to 16-cell stage of bovine embryos as the period of major EGA, but detected first transcriptional activity already in one-cell embryos. Subsequent studies compared the transcriptome profiles of untreated embryos and of embryos incubated with the transcription inhibitor α-amanitin to reveal transcripts of embryonic origin. In addition, candidate gene-based and global gene expression studies over several stages of early development were performed and characteristic profiles were revealed. However, the onset of embryonic transcription was obscured by the presence of maternal transcripts and could only be determined for genes which are not expressed in oocytes. Using RNA sequencing of bovine germinal vesicle and metaphase II oocytes, and of four-cell, eight-cell, 16-cell and blastocyst stage embryos, we established the most comprehensive transcriptome data set of bovine oocyte maturation and early development. EGA was analyzed by (i) detection of embryonic transcripts which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. Using these three approaches we were able to map the onset of embryonic transcription for almost 7400 genes. Genes activated at the four-cell stage or before were functionally related to RNA processing, translation, and transport, preparing the embryo for major EGA at the eight-cell stage, when genes from a broad range of functional categories were found to be activated. These included transcriptional and translational functions as well as protein ubiquitination. The functions of the genes activated at the 16-cell stage were consistent with ongoing transcription and translation, while the genes activated in blastocysts included regulators of early lineage specification. Fine mapping of EGA provides a new layer of information for detecting disturbances of early development due to genetic, epigenetic, and environmental factors.
Mechanistic understanding of human SLFN11 Metzner, Felix J; Wenzl, Simon J; Kugler, Michael ...
Nature communications,
09/2022, Letnik:
13, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Abstract
Schlafen 11 (SLFN11) is an interferon-inducible antiviral restriction factor with tRNA endoribonuclease and DNA binding functions. It is recruited to stalled replication forks in response to ...replication stress and inhibits replication of certain viruses such as the human immunodeficiency virus 1 (HIV-1) by modulating the tRNA pool. SLFN11 has been identified as a predictive biomarker in cancer, as its expression correlates with a beneficial response to DNA damage inducing anticancer drugs. However, the mechanism and interdependence of these two functions are largely unknown. Here, we present cryo-electron microscopy (cryo-EM) structures of human SLFN11 in its dimeric apoenzyme state, bound to tRNA and in complex with single-strand DNA. Full-length SLFN11 neither hydrolyses nor binds ATP and the helicase domain appears in an autoinhibited state. Together with biochemical and structure guided mutagenesis studies, our data give detailed insights into the mechanism of endoribonuclease activity as well as suggestions on how SLFN11 may block stressed replication forks.
Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a high-risk subgroup of T-lineage ALL characterized by specific stem cell and myeloid features. In adult ETP-ALL, no comprehensive ...studies on the genetic background have been performed to elucidate molecular lesions of this distinct subgroup. We performed whole-exome sequencing of 5 paired ETP-ALL samples. In addition to mutations in genes known to be involved in leukemogenesis (ETV6, NOTCH1, JAK1, and NF1), we identified novel recurrent mutations in FAT1 (25%), FAT3 (20%), DNM2 (35%), and genes associated with epigenetic regulation (MLL2, BMI1, and DNMT3A). Importantly, we verified the high rate of DNMT3A mutations (16%) in a larger cohort of adult patients with ETP-ALL (10/68). Mutations in epigenetic regulators support clinical trials, including epigenetic-orientated therapies, for this high-risk subgroup. Interestingly, more than 60% of adult patients with ETP-ALL harbor at least a single genetic lesion in DNMT3A, FLT3, or NOTCH1 that may allow use of targeted therapies.
•Exome sequencing of adult ETP-ALL reveals new recurrent mutations; in particular, DNMT3A is frequently mutated in adult ETP-ALL.•More than 60% of all adult patients with ETP-ALL harbor a mutation that could potentially be targeted by a specific therapy.
The fusion genes CBFB/MYH11 and RUNX1/RUNX1T1 block differentiation through disruption of the core binding factor (CBF) complex and are found in 10-15% of adult de novo acute myeloid leukemia (AML) ...cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum ('mutatome') in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11- and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2, and ZBTB7A being strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A, and DHX15 and identified novel, recurrent mutations in NFE2 (3%), MN1 (4%), HERC1 (3%), and ZFHX4 (5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia.
Nucleosome-to-protamine exchange during mammalian spermiogenesis is essential for compaction and protection of paternal DNA. It is interesting that, depending on the species, 1% to 15% of nucleosomes ...are retained, but the generalizability and biological function of this retention are unknown. Here, we show concordantly in human and bovine that nucleosomes remained in sperm chromatin predominantly within distal intergenic regions and introns and associated with centromere repeats and retrotransposons (LINE1 and SINEs). In contrast, nucleosome depletion concerned particularly exons, 5′-UTR, 3′-UTR, TSS, and TTS and was associated with simple and low-complexity repeats. Overlap of human and bovine genes exhibiting nucleosome preservation in the promoter and gene body revealed a significant enrichment of signal transduction and RNA- and protein-processing factors. Our study demonstrates the genome-wide uniformity of the nucleosome preservation pattern in mammalian sperm and its connection to repetitive DNA elements and suggests a function in preimplantation processes for paternally derived nucleosomes.
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•Nucleosome preservation in mammalian sperm underlies a universal pattern•Centromeres LINE1 and SINE preserve nucleosomes•Nucleosomes occur largely intergenically, while exons and most promoters are depleted•Nucleosome-rich genes are hypomethylated in promoter and code RNA-processing factors
The study demonstrates concordantly in human and bovine the uniformity of the nucleosome-preservation pattern in sperm in a genome-wide manner. A potential role in preimplantation development is suggested for sperm-derived nucleosomes frequently detected in centromere repeats, LINE1 and SINE, and predominantly in genes relevant for RNA and protein processing.
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