The parkin-coregulated gene product PACRG promotes TNF signaling by stabilizing LUBAC

The Parkin-coregulated gene (PACRG), which encodes a protein of unknown function, shares a bidirectional promoter with Parkin (PRKN), which encodes an E3 ubiquitin ligase. Because PRKN is important in mitochondrial quality control and protection against stress, we tested whether PACRG also affected these pathways in various cultured human cell lines and in mouse embryonic fibroblasts. PACRG did not play a role in mitophagy but did play a role in tumor necrosis factor (TNF) signaling. Similarly to Parkin, PACRG promoted nuclear factor κB (NF-κB) activation in response to TNF. TNF-induced nuclear translocation of the NF-κB subunit p65 and NF-κB–dependent transcription were decreased in PACRG-deficient cells. Defective canonical NF-κB activation in the absence of PACRG was accompanied by a decrease in linear ubiquitylation mediated by the linear ubiquitin chain assembly complex (LUBAC), which is composed of the two E3 ubiquitin ligases HOIP and HOIL-1L and the adaptor protein SHARPIN. Upon TNF stimulation, PACRG was recruited to the activated TNF receptor complex and interacted with LUBAC components. PACRG functionally replaced SHARPIN in this context. In SHARPIN-deficient cells, PACRG prevented LUBAC destabilization, restored HOIP-dependent linear ubiquitylation, and protected cells from TNF-induced apoptosis. This function of PACRG in positively regulating TNF signaling may help to explain the association of PACRG and PRKN polymorphisms with an increased susceptibility to intracellular pathogens.

Functionally Related Genes Cluster into Genomic Regions That Coordinate Transcription at a Distance in Saccharomyces cerevisiae

ABSTRACT Balancing gene expression is a fundamental challenge of all cell types. To properly regulate transcription on a genome-wide level, there are myriad mechanisms employed by the cell. One layer to this regulation is through spatial positioning, with particular chromosomal loci exerting an influence on transcription throughout a region. Many coregulated gene families utilize spatial positioning to coordinate transcription, with functionally related genes clustering together which can allow coordinated expression via adjacent gene coregulation. The mechanisms underlying this process have not been elucidated, though there are many coregulated gene families that exhibit this genomic distribution. In the present study, we tested for a role for the enhancer-promoter (EP) hypothesis, which demonstrates that regulatory elements can exert transcriptional effects over a broad distance, in coordinating transcriptional coregulation using budding yeast, Saccharomyces cerevisiae. We empirically validated the EP model, finding that the genomic distance a promoter can affect varies by locus, which can profoundly affect levels of transcription, phenotype, and the extent of transcriptional disruption throughout a genomic region. Using the nitrogen metabolism, ribosomal protein, toxin response, and heat shock gene families as our test case, we report functionally clustered genes localize to genomic loci that are more conducive to transcriptional regulation at a distance compared to the unpaired members of the same families. Furthermore, we report that the coregulation of functional clusters is dependent, in part, on chromatin maintenance and remodeling, providing one mechanism underlying adjacent gene coregulation. IMPORTANCE The two-dimensional, physical positioning of genes along a chromosome can impact proper transcriptional regulation throughout a genomic region. The transcription of neighboring genes is correlated in a genome-wide manner, which is a characteristic of eukaryotes. Many coregulated gene families can be found clustered with another member of the same set—which can result in adjacent gene coregulation of the pair. Due to the myriad gene families that exhibit a nonrandom genomic distribution, there are likely multiple mechanisms working in concert to properly regulate transcriptional coordination of functionally clustered genes. In this study, we utilized budding yeast in an attempt to elucidate mechanisms that underlie this coregulation: testing and empirically validating the enhancer-promoter hypothesis in this species and reporting that functionally related genes cluster to genomic regions that are more conducive to transcriptional regulation at a distance. These clusters rely, in part, on chromatin maintenance and remodelers to maintain proper transcriptional coordination. Our work provides insight into the mechanisms underlying adjacent gene coregulation.

ALL2, a Homologue ofALL1, Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans

Cryptococcus neoformansis a facultative intracellular fungal pathogen that has a polysaccharide capsule and causes life-threatening meningoencephalitis. Its capsule, as well as its ability to survive in the acidic environment of the phagolysosome, contributes to the pathogen's resilience in the host environment. Previously, we reported that downregulation of allergen 1 (ALL1) results in the secretion of a shorter, more viscous exopolysaccharide with less branching and structural complexity, as well as altered iron homeostasis. Now, we report on a homologous coregulated gene, allergen 2 (ALL2).ALL2's function was characterized by generating null mutants inC. neoformans. In contrast toALL1, loss ofALL2attenuated virulence in the pulmonary infection model. Theall2Δ mutant shed a less viscous exopolysaccharide and exhibited higher sensitivity to hydrogen peroxide than the wild type, and as a result, theall2Δ mutant was more resistant to macrophage-mediated killing. Transcriptome analysis further supported the distinct function of these two genes. UnlikeALL1's involvement in iron homeostasis, we now present data onALL2's unique function in maintaining intracellular pH in low-pH conditions. Thus, our data highlight thatC. neoformans, a human-pathogenic basidiomycete, has evolved a unique set of virulence-associated genes that contributes to its resilience in the human niche.

219. Parkin co-regulated gene (Pacrg) is an axonemal protein involved in sperm tail and ependymal cell function and is a candidate primary ciliary dyskinesia gene

A leading cause of male infertility is genetic variation in genes required for sperm formation or function. Considerable evidence suggests PACRG is involved in spermiogeneis. The loss of Pacrg function causes infertility in mice (Lorenzetti et al. 2004) and we have shown an association between variability in the 5′ untranslated region of PACRG and human male infertility (Wilson et al. in preparation). Evidence from studies in C.reinhardtii and T.brucei indicate Pacrg is crucial for axonome formation and microtubule stability. To assess this possibility in mammals, we generated and characterised Pacrg knockout (Quaking viable, Qkv), wildtype and Pacrg transgenic mice (Qkv-Tg). Using confocal and immunoelectron microscopy we showed that Pacrg was localised to the axonemal microtubule doublets of sperm, tracheal and ependymal cilia. The absence of Pacrg was associated with compromised sperm flagella formation and MRI analyses revealed the occurrence of hydrocephalus. Specifically, Qkv mice showed an inward expansion of the lateral ventricles, resulting in a significant reduction in distance between ventricles (1.0 ± 0.6 mm, mean ± s.d., n = 5) and a ~250% increase in ventricle area (70 ± 13 arbitrary units, mean ± s.d., n = 5) compared with wildtype littermates (1.38 ± 0.09 mm; area 26 ± 12, n = 3). Transgenic expression of Pacrg was necessary and sufficient to correct the hydrocephalus (1.45 ± 0.05 mm; area 26 ± 9, n = 2) and infertility phenotypes (evidenced by daily sperm counts and litter sizes). In conclusion, we have shown Pacrg is a novel axoneme associated protein in a subset of motile cilia/flagella and loss of Pacrg function results in spermiogenic defects and hydrocephalus in mice. Further, we have shown that variations in the human PACRG promoter are a risk factor in human male infertility. Collectively these data suggest PACRG is a candidate gene in the human syndrome of primary ciliary dyskinesia. (1) Lorenzetti D, Bishop CE, Justice MJ. 2004. Deletion of the Parkin coregulated gene causes male sterility in the quaking (viable) mouse mutant. Proc Natl Acad Sci U S A 101(22):8402–8407