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TAF4b is a gonadal-enriched subunit of the TFIID complex that is required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro. Moreover, co-immunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a post-meiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis. [ABSTRACT FROM AUTHOR]

Palmitate attenuates insulin secretion and reduces the viability of insulinproducing cells. Previous studies identified the aberrant palmitoylation or mispalmitoylation of proteins as one mechanism by which palmitate causes β-cell damage. In this report, we identify a role for lysosomal protein degradation as a mechanism by which β cells defend themselves against excess palmitate. The cation-independent mannose 6-phosphate receptor (CI-MPR) is responsible for the trafficking of mannose 6-phosphate-tagged proteins to lysosomes via Golgi sorting and from extracellular locations through endocytosis. RINm5F cells, which are highly sensitive to palmitate, lack CI-MPR. The reconstitution of CI-MPR expression attenuates the induction of endoplasmic reticulum (ER) stress and the toxic effects of palmitate on RINm5F cell viability. INS832/13 cells express CI-MPR and are resistant to the palmitate-mediated loss of cell viability. The reduction of CI-MPR expression increases the sensitivity of INS832/13 cells to the toxic effects of palmitate treatment. The inhibition of lysosomal acid hydrolase activity by weak base treatment of islets under glucolipotoxic conditions causes islet degeneration that is prevented by the inhibition of protein palmitoylation. These findings indicate that defects in lysosomal function lead to the enhanced sensitivity of insulin-producing cells to palmitate and support a role for normal lysosomal function in the protection of β cells from excess palmitate. [ABSTRACT FROM AUTHOR]

The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through networks of interactions with other nuclear proteins, control patterns of gene expression during development. For example, the network interactions of the pituitary-specific HD protein Pit-1 control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. Inactivating mutations in Pit-1 disrupt these processes, giving rise to the syndrome of combined pituitary hormone deficiency. Pit-1 interacts with CCAAT/enhancer-binding protein alpha (C/EBPα) to regulate prolactin transcription. Here, we used the combination of biochemical analysis and live-cell microscopy to show that two different point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions between Pit-1 and C/EBPα in different ways. The results showed that the first α-helix of the POU-S domain is critical for the assembly of Pit-1 with C/EBPα, and they showed that DNA-binding activity conferred by the HD is critical for the final intranuclear positioning of the metastable complex. This likely reflects more general mechanisms that govern cell-type-specific transcriptional control, and the results from the analysis of the point mutations could indicate an important link between the mislocalization of transcriptional complexes and disease processes. [ABSTRACT FROM AUTHOR]