EMBO Reports

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone‐to‐protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79‐methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked‐out (KO) in postnatal male germ cells, we found that Dot1l‐KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l‐KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l‐KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCF^FBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCF^FBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7–BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

The extensive diversity of membrane lipids is rarely appreciated by cell and molecular biologists. Although most researchers are familiar with the three main classes of lipids in animal cell membranes, few realize the enormous combinatorial structural diversity that exists within each lipid class, a diversity that enables functional specialization of lipids. In this brief review, we focus on one class of membrane lipids, the sphingolipids, which until not long ago were thought by many to be little more than structural components of biological membranes. Recent studies have placed sphingolipids—including ceramide, sphingosine and sphingosine‐1‐phosphate—at the centre of a number of important biological processes, specifically in signal transduction pathways, in which their levels change in a highly regulated temporal and spatial manner. We outline exciting progress in the biochemistry and cell biology of sphingolipids and focus on their functional diversity. This should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingolipids in regulating defined aspects of cell physiology.

The arginine methyltransferases CARM1 and PRMT1 associate with the p160 family of nuclear hormone receptor coactivators. This association enhances transcriptional activation by nuclear receptors. We describe a method for identifying arginine N‐methyltransferase substrates using arrayed high‐density protein membranes to perform solid‐phase supported enzyme reactions in the presence of the methyl donor S‐adenosyl‐L‐methionine. Using this screen, we identified distinct substrates for CARM1 and PRMT1. All PRMT1 substrates harbor the expected GGRGG methylation motif, whereas the peptide sequence comparisons of the CARM1 substrates revealed no such motif. The predominant CARM1 substrate identified in this screen was PABP1. We mapped the methylated region of this RNA binding molecule in vitro and demonstrate that PABP1 is indeed methylated in vivo. Prior to these findings, the only known substrate for CARM1 was histone H3. We broaden the number of CARM1 targets and suggest a role for CARM1 in regulating transcription/translation.

Phosphoinositide lipids (PPIn) are enriched in stearic‐ and arachidonic acids (38:4) but how this enrichment is established and maintained during phospholipase C (PLC) activation is unknown. Here we show that the metabolic fate of newly synthesized phosphatidic acid (PA), the lipid precursor of phosphatidylinositol (PI), is influenced by the fatty acyl‐CoA used with preferential routing of the arachidonoyl‐enriched species toward PI synthesis. Furthermore, during agonist stimulation the unsaturated forms of PI(4,5P)₂ are replenished significantly faster than the more saturated ones, suggesting a favored recycling of the unsaturated forms of the PLC‐generated hydrolytic products. Cytidine diphosphate diacylglycerol synthase 2 (CDS2) but not CDS1 was found to contribute to increased PI resynthesis during PLC activation. Lastly, while the lipid transfer protein, Nir2 is found to contribute to rapid PPIn resynthesis during PLC activation, the faster re‐synthesis of the 38:4 species does not depend on Nir2. Therefore, the fatty acid side‐chain composition of the lipid precursors used for PI synthesis is an important determinant of their metabolic fates, which also contributes to the maintenance of the unique fatty acid profile of PPIn lipids.

The ubiquitin–proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C‐terminus of Hsc70‐interacting protein (CHIP) with a U‐box domain is an E3 ubiquitin‐ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U‐box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C‐terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self‐ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as ‘a quality‐control E3’ that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.

Constitutive expression of Wnt1 and Wnt5a in HC11 mammary cells led to elevated TCF transcriptional activity. Intriguingly, Wnt‐expressing cells also displayed activation of ErbB1 and mitogen‐activated protein kinase (MAPK), in contrast to control HC11 cells, which did not. Furthermore, conditioned media harvested from Wnt‐expressing cells stimulated ErbB1 and the MAPK cascade when added to control cells. This process was rapid and could be blocked by an ErbB1 antibody that interferes with ligand binding and by matrix metalloproteinase (MMP) inhibitors. These results suggest that in mammary cells Wnt binding to its receptor, Frizzled (Fz), transactivates ErbB1, probably by MMP‐mediated release of soluble ErbB1 ligands. Importantly, Wnt‐transactivated ErbB1 was responsible for MAPK activation and the increased levels of cyclin D1 present in the Wnt‐expressing HC11 cells. Our finding that Wnts transactivate ErbB1 in addition to stimulating the prototypic β‐catenin/TCF pathway may help to explain why wnt1 is a potent oncogene in the mammary gland.