TSPYL1 regulates steroidogenic gene expression and male factor fertility in mice.

OBJECTIVE: To determine the importance of testis-specific, Y-encoded-like 1 (TSPYL1) in survival and male factor fertility in mice. DESIGN: Experimental prospective study. SETTING: Research laboratories in a university medical faculty. ANIMALS: We generated Tspyl1 knockout (KO) mouse lines by CRISPR/Cas9. The lines were maintained by pairing heterozygous mice to provide wild-type control and KO males for comparison. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Mendelian ratio, body and testis weight, histology, sperm motility, mating tests, pregnancy outcome, transcript levels of genes for testosterone production, and serum testosterone level. RESULT(S): A variable percentage of Tspyl1 KO mice survived beyond weaning depending on the genetic background. Growth around weaning was retarded in KO mice, but the testes-to-body weight ratio remained normal and complete spermatogenesis was revealed in testis histology. Sperm was collected from the cauda epididymis, and a significantly smaller percentage of sperm was progressively motile (22.3% ± 18.3%, n = 14 samples) compared with wild type (58.9% ± 11.5%, 11 samples). All 11 KO mice tested had defective mounting behavior. From 11 KO males paired with a total of 88 females, only one litter was born, compared with 53 litters sired by 11 age-matched wild-type males. Expression of Star, Cyp11a1, Cyp17a1, Hsd3b6, and Hsd17b3 in the KO testis was significantly reduced, while serum testosterone level was within the normal range. CONCLUSION(S): TSPYL1 is critical for survival and reproductive success in mice. TSPYL1 enhances the expression of key steroidogenic genes in the mouse testis.

Lrrc7 mutant mice model developmental emotional dysregulation that can be alleviated by mGluR5 allosteric modulation.

LRRC7 has been identified as a candidate gene for severe childhood emotional dysregulation. Direct experimental evidence for a role of LRRC7 in the disease is needed, as is a better understanding of its impact on neuronal structure and signaling, and hence potential treatment targets. Here, we generated and analyzed an Lrrc7 mutant mouse line. Consistent with a critical role of LRRC7 in emotional regulation, mutant mice had inappropriate juvenile aggressive behavior and significant anxiety-like behavior and social dysfunction in adulthood. The pivotal role of mGluR5 signaling was demonstrated by rescue of behavioral defects with augmentation of mGluR5 receptor activity by 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB). Intra-peritoneal injection of CDPPB alleviated abnormal juvenile behavior, as well as anxiety-like behavior and hypersociability at adulthood. Furthermore, mutant primary neurons had impaired neurite outgrowth which was rescued by CDPPB treatment. In conclusion, Lrrc7 mutant mice provide a valuable tool to model childhood emotional dysregulation and persistent mental health comorbidities. Moreover, our data highlight an important role of LRRC7 in mGluR5 signaling, which is a potential new treatment target for anxiety and social dysfunction.

TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons.

Testis-specific protein, Y-encoded-like 2 (TSPYL2) is an X-linked gene in the locus for several neurodevelopmental disorders. We have previously shown that Tspyl2 knockout mice had impaired learning and sensorimotor gating, and TSPYL2 facilitates the expression of Grin2a and Grin2b through interaction with CREB-binding protein. To identify other genes regulated by TSPYL2, here, we showed that Tspyl2 knockout mice had an increased level of H3K27 trimethylation (H3K27me3) in the hippocampus, and TSPYL2 interacted with the H3K27 methyltransferase enhancer of zeste 2 (EZH2). We performed chromatin immunoprecipitation (ChIP)-sequencing in primary hippocampal neurons and divided all Refseq genes by k-mean clustering into four clusters from highest level of H3K27me3 to unmarked. We confirmed that mutant neurons had an increased level of H3K27me3 in cluster 1 genes, which consist of known EZH2 target genes important in development. We detected significantly reduced expression of genes including Gbx2 and Prss16 from cluster 1 and Acvrl1, Bdnf, Egr3, Grin2c, and Igf1 from cluster 2 in the mutant. In support of a dynamic role of EZH2 in repressing marked synaptic genes, the specific EZH2 inhibitor GSK126 significantly upregulated, while the demethylase inhibitor GSKJ4 downregulated the expression of Egr3 and Grin2c. GSK126 also upregulated the expression of Bdnf in mutant primary neurons. Finally, ChIP showed that hemagglutinin-tagged TSPYL2 co-existed with EZH2 in target promoters in neuroblastoma cells. Taken together, our data suggest that TSPYL2 is recruited to promoters of specific EZH2 target genes in neurons, and enhances their expression for proper neuronal maturation and function.

The nucleosome assembly protein TSPYL2 regulates the expression of NMDA receptor subunits GluN2A and GluN2B.

TSPYL2 is an X-linked gene encoding a nucleosome assembly protein. TSPYL2 interacts with calmodulin-associated serine/threonine kinase, which is implicated in X-linked mental retardation. As nucleosome assembly and chromatin remodeling are important in transcriptional regulation and neuronal function, we addressed the importance of TSPYL2 through analyzing Tspyl2 loss-of-function mice. We detected down-regulation of N-methyl-D-aspartate receptor subunits 2A and 2B (GluN2A and GluN2B) in the mutant hippocampus. Evidence from luciferase reporter assays and chromatin immunoprecipitation supported that TSPYL2 regulated the expression of Grin2a and Grin2b, the genes encoding GluN2A and GluN2B. We also detected an interaction between TSPYL2 and CBP, indicating that TSPYL2 may activate gene expression through binding CBP. In terms of functional outcome, Tspyl2 loss-of-function impaired long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, mutant mice showed a deficit in fear learning and memory. We conclude that TSPYL2 contributes to cognitive variability through regulating the expression of Grin2a and Grin2b.