DUX is a non-essential synchronizer of zygotic genome activation.

Some of the earliest transcripts produced in fertilized human and mouse oocytes code for DUX, a double homeodomain protein that promotes embryonic genome activation (EGA). Deleting Dux by genome editing at the one- to two-cell stage in the mouse impairs EGA and blastocyst maturation. Here, we demonstrate that mice carrying homozygous Dux deletions display markedly reduced expression of DUX target genes and defects in both pre- and post-implantation development, with, notably, a disruption of the pace of the first few cell divisions and significant rates of late embryonic mortality. However, some Dux-/- embryos give rise to viable pups, indicating that DUX is important but not strictly essential for embryogenesis.

DPPA2 and DPPA4 are necessary to establish a 2C-like state in mouse embryonic stem cells.

After fertilization of the transcriptionally silent oocyte, expression from both parental chromosomes is launched through zygotic genome activation (ZGA), occurring in the mouse at the 2-cell (2C) stage. Among the first elements to be transcribed are the Dux gene, the product of which induces a wide array of ZGA genes, and a subset of evolutionary recent LINE-1 retrotransposons that regulate chromatin accessibility in the early embryo. The maternally inherited factors that activate Dux and LINE-1 transcription have so far remained unknown. Mouse embryonic stem cells (mESCs) recapitulate some aspects of ZGA in culture, owing to their ability to cycle through a 2C-like stage when Dux, its target genes, and LINE-1 integrants are expressed. Here, we identify the paralog proteins DPPA2 and DPPA4 as necessary for the activation of Dux and LINE-1 expression in mESCs. Since their encoding RNAs are maternally transmitted to the zygote, it is likely that these factors are important upstream mediators of murine ZGA.

Prospective study on microangiopathy in type 2 diabetic foot ulcer.

AIMS/HYPOTHESIS: We investigated the significance of microangiopathy in the development of foot ulcer, which is still disputed. METHODS: We assessed microangiopathy by histological analysis of the capillary ultrastructure using transmission electron microscopy and capillary density and arteriolar morphology in paraffin-embedded sections from the skin of type 2 diabetic patients: 30 neuroischaemic patients (Isc) revascularised with peripheral angioplasty and 30 neuropathic patients (Neu) with foot ulcer, compared with ten non-diabetic volunteers. RESULTS: In the diabetic patients, capillaries in the dermal papillary layer were fewer (-22.2%, 159 ± 43 vs 205 ± 52 mm(2) in non-diabetic volunteers, p < 0.01). They also showed detrimental remodelling, with a 2.2-fold increase in capillary basement membrane thickness (3.44 ± 1.19 vs 1.53 ± 0.34 µm in non-diabetic volunteers, p < 0.001) and a 57.7% decrease in lumen area (14.6 ± 11.1 vs 34.7 ± 27.5 µm(2), p < 0.001). No differences were observed between the diabetic Isc or Neu patients. Isc were more prone to develop arteriolar occlusion than Neu (16.8 ± 6.9% vs 6.7 ± 3.7%, respectively, p < 0.001). No patient had been amputated at 30 days and healing time was significantly longer in Isc (180 ± 120 vs 64 ± 50 days in Neu, p < 0.001). CONCLUSIONS/INTERPRETATION: Capillary microangiopathy is present in equal measure in neuroischaemic and neuropathic diabetic foot skin. The predominance of arteriolar occlusions with neuroischaemia indicated the existence of an additional 'small vessel disease' that did not affect an effective revascularisation and did not worsen the prognosis of major amputations but slowed the healing process of the neuroischaemic foot ulcer. TRIAL REGISTRATION: ClinicalTrials.gov NCT02610036.

Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA.

BACKGROUND: The human peptidyl-prolyl isomerase Cyclophilin A (CypA) binds HIV-1 capsid (CA) and influences early steps in the HIV-1 replication cycle. The mechanism by which CypA regulates HIV-1 transduction efficiency is unknown. Disruption of CypA binding to CA, either by genetic means or by the competitive inhibitor cyclosporine A (CsA), reduces the efficiency of HIV-1 transduction in some cells but not in others. Transduction of certain cell types increases significantly when CypA binding to particular HIV-1 CA mutants, i.e., A92E, is prevented. Previous studies have suggested that this cell type-specific effect is due to a dominant-acting, CypA-dependent restriction factor. RESULTS: Here we investigated the mechanism by which CypA regulates HIV-1 transduction efficiency using 27 different human cell lines, 32 HeLa subclones, and several previously characterized HIV-1 CA mutants. Disruption of CypA binding to wild-type CA, or to any of the mutant CAs, caused a decrease in HIV-1 reverse transcription in all the cell lines analyzed here. This block to reverse transcription, though, did not correlate with cell type-specific effects on transduction efficiency. The level of 2-LTR circles, a marker for nuclear transport of the viral cDNA that results from reverse transcription, correlated closely with effects on infectivity. No correlation was observed between the cell type-specific effects on infectivity and the steady-state CypA protein levels in these cells. Instead, as indicated by a fate-of-capsid assay, CsA released the HIV-1 CA core from an apparent state of hyperstabilization, in a cell type-specific manner. CONCLUSION: These data demonstrate that, while CypA promotes reverse transcription under all conditions tested here, its effect on HIV-1 infectivity correlates more closely with effects on nuclear entry of the viral cDNA. The data also support the hypothesis that a cell-type specific CypA-dependent restriction factor blocks HIV-1 replication by delaying CA core uncoating and hindering nuclear entry.

TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm.

BACKGROUND: Despite intensive investigation the mechanism by which HIV-1 reaches the host cell nucleus is unknown. TNPO3, a karyopherin mediating nuclear entry of SR-proteins, was shown to be required for HIV-1 infectivity. Some investigators have reported that TNPO3 promotes HIV-1 nuclear import, as would be expected for a karyopherin. Yet, an equal number of investigators have failed to obtain evidence that supports this model. Here, a series of experiments were performed to better elucidate the mechanism by which TNPO3 promotes HIV-1 infectivity. RESULTS: To examine the role of TNPO3 in HIV-1 replication, the 2-LTR circles that are commonly used as a marker for HIV-1 nuclear entry were cloned after infection of TNPO3 knockdown cells. Potential explanation for the discrepancy in the literature concerning the effect of TNPO3 was provided by sequencing hundreds of these clones: a significant fraction resulted from autointegration into sites near the LTRs and therefore were not bona fide 2-LTR circles. In response to this finding, new techniques were developed to monitor HIV-1 cDNA, including qPCR reactions that distinguish 2-LTR circles from autointegrants, as well as massive parallel sequencing of HIV-1 cDNA. With these assays, TNPO3 knockdown was found to reduce the levels of 2-LTR circles. This finding was puzzling, though, since previous work has shown that the HIV-1 determinant for TNPO3-dependence is capsid (CA), an HIV-1 protein that forms a mega-dalton protein lattice in the cytoplasm. TNPO3 imports cellular splicing factors via their SR-domain. Attention was therefore directed towards CPSF6, an SR-protein that binds HIV-1 CA and inhibits HIV-1 nuclear import when the C-terminal SR-domain is deleted. The effect of 27 HIV-1 capsid mutants on sensitivity to TNPO3 knockdown was then found to correlate strongly with sensitivity to inhibition by a C-terminal deletion mutant of CPSF6 (R2 = 0.883, p < 0.0001). TNPO3 knockdown was then shown to cause CPSF6 to accumulate in the cytoplasm. Mislocalization of CPSF6 to the cytoplasm, whether by TNPO3 knockdown, deletion of the CPSF6 nuclear localization signal, or by fusion of CPSF6 to a nuclear export signal, resulted in inhibition of HIV-1 replication. Additionally, targeting CPSF6 to the nucleus by fusion to a heterologous nuclear localization signal rescued HIV-1 from the inhibitory effects of TNPO3 knockdown. Finally, mislocalization of CPSF6 to the cytoplasm was associated with abnormal stabilization of the HIV-1 CA core. CONCLUSION: TNPO3 promotes HIV-1 infectivity indirectly, by shifting the CA-binding protein CPSF6 to the nucleus, thus preventing the excessive HIV-1 CA stability that would otherwise result from cytoplasmic accumulation of CPSF6.

Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus.

BACKGROUND: HIV-1 infects non-dividing cells. This implies that the virus traverses the nuclear pore before it integrates into chromosomal DNA. Recent studies demonstrated that TNPO3 is required for full infectivity of HIV-1. The fact that TNPO3 is a karyopherin suggests that it acts by directly promoting nuclear entry of HIV-1. Some studies support this hypothesis, while others have failed to do so. Additionally, some studies suggest that TNPO3 acts via HIV-1 Integrase (IN), and others indicate that it acts via capsid (CA). RESULTS: To shed light on the mechanism by which TNPO3 contributes to HIV-1 infection we engineered a panel of twenty-seven single-cycle HIV-1 vectors each bearing a different CA mutation and characterized them for the ability to transduce cells in which TNPO3 had been knocked down (KD). Fourteen CA mutants were relatively TNPO3-independent, as compared to wild-type (WT) HIV-1. Two mutants were more TNPO3-dependent than the WT, and eleven mutants were actually inhibited by TNPO3. The efficiency of the synthesis of viral cDNA, 2-LTR circles, and proviral DNA was then assessed for WT HIV-1 and three select CA mutants. Controls included rescue of TNPO3 KD with non-targetable coding sequence, RT- and IN- mutant viruses, and pharmacologic inhibitors of RT and IN. TNPO3 KD blocked transduction and establishment of proviral DNA by wild-type HIV-1 with no significant effect on the level of 2-LTR circles. PCR results were confirmed by achieving TNPO3 KD using two different methodologies (lentiviral vector and siRNA oligonucleotide transfection); by challenging three different cell types; by using two different challenge viruses, each necessitating different sets of PCR primers; and by pseudotyping virus with VSV G or using HIV-1 Env. CONCLUSION: TNPO3 promotes HIV-1 infectivity at a step in the virus life cycle that is detectable after the preintegration complex arrives in the nucleus and CA is the viral determinant for TNPO3 dependence.

KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage.

The Krüppel-associated box zinc finger protein (KRAB-ZFP) family diversified in mammals. The majority of human KRAB-ZFPs bind transposable elements (TEs), however, since most TEs are inactive in humans it is unclear whether KRAB-ZFPs emerged to suppress TEs. We demonstrate that many recently emerged murine KRAB-ZFPs also bind to TEs, including the active ETn, IAP, and L1 families. Using a CRISPR/Cas9-based engineering approach, we genetically deleted five large clusters of KRAB-ZFPs and demonstrate that target TEs are de-repressed, unleashing TE-encoded enhancers. Homozygous knockout mice lacking one of two KRAB-ZFP gene clusters on chromosome 2 and chromosome 4 were nonetheless viable. In pedigrees of chromosome 4 cluster KRAB-ZFP mutants, we identified numerous novel ETn insertions with a modest increase in mutants. Our data strongly support the current model that recent waves of retrotransposon activity drove the expansion of KRAB-ZFP genes in mice and that many KRAB-ZFPs play a redundant role restricting TE activity.

DUX-family transcription factors regulate zygotic genome activation in placental mammals.

In animal embryos, transcription is mostly silent for several cell divisions, until the release of the first major wave of embryonic transcripts through so-called zygotic genome activation (ZGA). Maternally provided ZGA-triggering factors have been identified in Drosophila melanogaster and Danio rerio, but their mammalian homologs are still undefined. Here, we provide evidence that the DUX family of transcription factors is essential to this process in mice and potentially in humans. First, human DUX4 and mouse Dux are both expressed before ZGA in their respective species. Second, both orthologous proteins bind the promoters of ZGA-associated genes and activate their transcription. Third, Dux knockout in mouse embryonic stem cells (mESCs) prevents the cells from cycling through a 2-cell-like state. Finally, zygotic depletion of Dux leads to impaired early embryonic development and defective ZGA. We conclude that DUX-family proteins are key inducers of zygotic genome activation in placental mammals.