NAP1-Related Protein 1 (NRP1) has multiple interaction modes for chaperoning histones H2A-H2B

Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure ofArabidopsisNAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.

Unravelling the disease mechanism for TSPYL1 deficiency

We describe a lethal combined nervous and reproductive systems disease in three affected siblings of a consanguineous family. The phenotype was characterized by visceroautonomic dysfunction (neonatal bradycardia/apnea, feeding problems, hyperactive startle reflex), severe postnatal progressive neurological abnormalities (including abnormal neonatal cry, hypotonia, epilepsy, polyneuropathy, cerebral gray matter atrophy), visual impairment, testicular dysgenesis in males and sudden death at infant age by brainstem-mediated cardiorespiratory arrest. Whole-exome sequencing revealed a novel homozygous frameshift variant p.Val242GlufsTer52 in the TSPY-like 1 gene (TSPYL1). The truncated TSPYL1 protein that lacks the nucleosome assembly protein domain was retained in the Golgi of fibroblasts from the three patients, whereas control fibroblasts express full-length TSPYL1 in the nucleus. Proteomic analysis of nuclear extracts from fibroblasts identified 24 upregulated and 20 downregulated proteins in the patients compared with 5 controls with ‘regulation of cell cycle’ as the highest scored biological pathway affected. TSPYL1-deficient cells had prolonged S and G2 phases with reduced cellular proliferation rates. Tspyl1 depletion in zebrafish mimicked the patients’ phenotype with early lethality, defects in neurogenesis and cardiac dilation. In conclusion, this study reports the third pedigree with recessive TSPYL1 variants, confirming that TSPYL1 deficiency leads to a combined nervous and reproductive systems disease, and provides for the first time insights into the disease mechanism.

Effects of ORAI calcium release-activated calcium modulator 1 (ORAI1) on neutrophil activity in dairy cows with subclinical hypocalcemia1

Hypocalcemia in dairy cows is often associated with inflammation-related disorders such as metritis and mastitis. The protein encoded by the Ca2+ release-activated calcium modulator 1 (ORAI1) gene is a membrane Ca2+ channel subunit that is activated when Ca2+ stores are depleted. Polymorphonuclear neutrophils (PMNL) have a crucial role in the defense against infection through migration, adhesion, chemotaxis, phagocytosis, and reactive oxygen species (ROS) production in response to pathogens. Whether hypocalcemia affects the activity of PMNL and if ORAI1 is involved remains unknown. To address this, PMNL were isolated at 3 d of calving from dairy cows diagnosed as clinically healthy (n = 20, CONTROL) or with plasma concentration of calcium < 2.0 mmol/L as a criterion for diagnosis of subclinical hypocalcemia (n = 20, HYPOCAL). PMNL isolated from both groups of cows were treated with or without the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin, Ca2+ ionophore Ionomycin, and ORAI1 blocker 2APB. The intracellular Ca2+ concentration, ORAI1 abundance, ROS, phagocytosis rate, migration, and adhering capacity of treated PMNL were evaluated. Some of the in vitro assays also included use of small interfering ORAI1 RNA (siORAI1), 100 nM 1,25(OH)2D3, or 100 nM parathyroid hormone (PTH). Intracellular Ca2+ concentration was markedly lower in HYPOCAL. In addition, ORAI1 was detected in PMNL plasma membrane via FACS and was markedly lower in cows with HYPOCAL. Migration, adhesion capacity, and phagocytosis rate of PMNL were lower in response to HYPOCAL. Furthermore, plasma and PMNL concentration of nucleosome assembly protein (NAP2) and pro-platelet basic protein (CXCL7) was markedly lower with HYPOCAL. All these changes were associated with lower ROS production by PMNL. Thapsigargin and ionomycin treatment in vitro increased ORAI1 expression, migration of PMNL, adhering capacity, phagocytosis rate, and ROS production; conversely, those effects were abrogated by siORAI1 and ORAI1 inhibitor 2APB treatment. Also cytosolic Ca2+ concentration and ORAI1 abundance were increased by 1,25(OH)2D3 and PTH supplementation. Overall, the data indicate that failure of PMNL to uptake Ca2+ due to downregulation of ORAI1 during subclinical hypocalcemia is a factor contributing to impaired PMNL function. In addition, plasma PTH or 1,25(OH)2D3 could regulate ORAI1 and also participate in the regulation of PMNL activity.

NAP Family CG5017 Chaperone Pleiotropically Regulates Human AHR Target Genes Expression in Drosophila Testis

To study the regulatory mechanism of the Aryl hydrocarbon receptor (AHR), target genes of transcription are necessary for understanding the normal developmental and pathological processes. Here, we examined the effects of human AHR ligands on male fecundity. To induce ectopic human AhR gene expression, we used Drosophila melanogaster transformed with human AhR under the control of a yeast UAS promoter element capable of activation in the two-component UAS-GAL4 system. We found that exogenous AHR ligands decrease the number of Drosophila gonadal Tj-positive cells. We also found both an increase and decrease of AHR target gene expression, including in genes that control homeostasis and testis development. This suggests that gonadal AHR activation may affect the expression of gene networks that control sperm production and could be critical for fertility not just in Drosophila but also in humans. Finally, we found that the activation of the expression for some AHR target genes depends on the expression of testis-specific chaperone CG5017 in gonadal cells. Since CG5017 belongs to the nucleosome assembly protein (NAP) family and may participate in epigenetic regulation, we propose that this nucleotropic chaperone is essential to provide the human AHR with access to only the defined set of its target genes during spermatogenesis.