Biology subjects

Duckett, K.

Publications and source records attributed to Duckett, K..

3 recordsLinked to original sources

bioRxiv2026

The HIV-1 restriction factor RPRD2 does not inhibit transcription of HIV-1 or endogenous retroelements

A vital question in transcription regulation is how and to what extent transcription of different DNA species, including viral DNA, episomal DNA and endogenous retroelements, is differentially regulated to transcription of host genes. RPRD2 has previously been characterized as an HIV restriction factor that blocks reverse transcription. However, RPRD2 has also been characterized as a regulator of global transcription of host genes. Therefore, we hypothesized that RPRD2 may also regulate nascent transcription from HIV provirus and endogenous retroelements. First, we used a combination of experimental and computational methods to characterize the binding of RPRD2 to RNA and DNA:RNA hybrids. Using immunoprecipitation, we identified that RPRD2 interacts with both the transcription regulator PAF1 and the HUSH complex member TASOR, independently. Immunofluorescence revealed that GFP-RPRD2 localizes to foci in the nucleus, and these foci overlap with nuclear speckles. To measure the effect of RPRD2 on transcription, we used plasmid-borne HIV LTR-driven reporter constructs and observed that RPRD2 depletion increased transcription of constructs both with and without an intron. We next investigated transcription from integrated proviruses and found no effect of RPRD2 depletion using several different systems. Lastly, we measured transcription of endogenous retroelements and found that RPRD2 depletion did not affect transcription of LINE-1 or HERV-K. Finally, we investigated whether RPRD2 regulates production of IFN in response to nucleic acid species or affects transcription of IFN-stimulated genes. We found that depletion of RPRD2 had no effect on IFN production or ISG expression. Together, our findings demonstrate how regulation of transcription is not universal for host genes, integrated provirus, unintegrated plasmid and endogenous retroviruses, and confirmed that although RPRD2 governs cellular transcription, it does not regulate transcription of HIV-1 provirus or of endogenous retroelements.

microbiology↗

bioRxiv2024

Damaging mutations in LXRα uncouple lipogenesis from hepatotoxicity and implicate hepatic cholesterol sensing in human liver health

The nuclear receptor Liver X Receptor- (LXR) activates lipogenic gene expression in hepatocytes. Its inhibition has therefore been proposed as a strategy to treat metabolic-dysfunction-associated steatotic liver disease (MASLD). In order to understand the impact of reducing LXR activity on human health we first examined the association between the carriage of rare loss of function mutations in NR1H3 (encoding LXR) and metabolic and hepatic phenotypes. We identified 63 rare predicted damaging variants in the ligand binding domain of LXR in 454,787 participants in UK Biobank. On functional characterisation, 42 of these were found to be severely impaired. Consistent with loss of the lipogenic actions of LXR, carriers of damaging mutations in LXR had reduced serum triglycerides ({beta}=-0.13 s.d. {+/-}0.03, P=2.7x10-5, N(carriers)=971). Surprisingly, these carriers also had elevated concentrations of serum liver enzymes (e.g. ALT: {beta}=0.17s.d. {+/-}0.03, P=1.1x10-8, N(carriers)=972) with a 35% increased risk of clinically significant elevations in ALT (OR=1.32, 95%CI:1.15-1.53, P=1.2x10-4, N(carriers)=972), suggestive of hepatotoxicity. We generated a knock-in mouse carrying one of the most severely damaging mutations (Nr1h3 p.W441R) which we demonstrated to have dominant negative properties. Homozygous knock-in mice rapidly developed severe hepatitis and fibrotic liver injury following exposure to western diet despite markedly reduced steatosis, liver triglycerides and lipogenic gene expression. This phenotype was completely rescued by viral over-expression of wildtype LXR specifically in hepatocytes, indicating a cell-autonomous effect of the mutant on hepatocyte health. While homozygous LXR knockout mice showed some evidence of hepatocyte injury under similar dietary conditions, the phenotype of the LXRW441R/W441R mouse was much more severe, suggesting that dominant negative mutations that actively co-repress target genes can result in pathological impacts significantly more severe than those seen with simple absence of the receptor. In summary, our results show that loss of function mutations in LXR occur in at least 1/450 people and are associated with evidence of liver dysfunction. These findings implicate LXR in the maintenance of human liver health, identify a new murine model of rapidly progressive fibrotic liver disease and caution against LXR antagonism as a therapeutic strategy for MASLD.

genetics↗

bioRxiv2023

Identification of a P62-TIF-IA axis that drives nucleolar fusion and the senescence associated secretory phenotype

Two key characteristics of senescent cells are nucleolar fusion and secretion of a plethora of pro-inflammatory cytokines called the senescence-associated secretory phenotype (SASP). The SASP is dependent on NF-{kappa}B but the initial trigger, and links with nucleoli, are unclear. Using multiple in vitro and in vivo models, we show that an early response to oncogene- and therapy-induced senescence (OIS and TIS) is nuclear/nucleolar accumulation of the PolI complex component, TIF-IA. This accumulation is essential for nucleolar fusion, the SASP and senescence, independent of rDNA transcription. We show that in steady state, TIF-IA is targeted for autophagic degradation by the p62 cargo receptor and that accumulation in senescence occurs as a consequence of ATM activation, which disrupts the p62-TIF-IA interaction. In mice, TIF-IA accumulates in colonic mucosa with age, which is further enhanced in the nfkb1-/- model of accelerated ageing. Together, these results reveal a p62-TIF-IA nucleolar stress axis that regulates the SASP and senescence, and that warrants further investigation as an anti-ageing target.

molecular biology↗