An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.

A hypomorphic lsd1 allele results in heart development defects in mice.

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic Lsd1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Molecular analyses revealed hyperphosphorylation of E-cadherin in the hearts of mutant animals. These results identify a previously unknown role for Lsd1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.

Defective heart development in hypomorphic LSD1 mice.

Lysine-specific demethylase 1 (LSD1/AOF2/KDM1A), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. LSD1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that LSD1-interacting proteins regulate the activity and specificity of LSD1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic LSD1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Transcriptional profiling revealed altered expression of a limited subset of genes in the hearts. This includes an increase in calmodulin kinase (CK) 2ß, the regulatory subunit of the CK2 kinase, which correlates with E-cadherin hyperphosphorylation. These results identify a previously unknown role for LSD1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.Cell Research advance online publication 6 December 2011; doi:10.1038/cr.2011.194.

KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints.

Differential DNA methylation of the paternal and maternal alleles regulates the parental origin-specific expression of imprinted genes in mammals. The methylation imprints are established in male and female germ cells during gametogenesis, and the de novo DNA methyltransferase DNMT3A and its cofactor DNMT3L are required in this process. However, the mechanisms underlying locus- and parental-specific targeting of the de novo DNA methylation machinery in germline imprinting are poorly understood. Here we show that amine oxidase (flavin-containing) domain 1 (AOF1), a protein related to the lysine demethylase KDM1 (also known as LSD1), functions as a histone H3 lysine 4 (H3K4) demethylase and is required for de novo DNA methylation of some imprinted genes in oocytes. AOF1, now renamed lysine demethylase 1B (KDM1B) following a new nomenclature, is highly expressed in growing oocytes where genomic imprints are established. Targeted disruption of the gene encoding KDM1B had no effect on mouse development and oogenesis. However, oocytes from KDM1B-deficient females showed a substantial increase in H3K4 methylation and failed to set up the DNA methylation marks at four out of seven imprinted genes examined. Embryos derived from these oocytes showed biallelic expression or biallelic suppression of the affected genes and died before mid-gestation. Our results suggest that demethylation of H3K4 is critical for establishing the DNA methylation imprints during oogenesis.

The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation.

Histone methylation and DNA methylation cooperatively regulate chromatin structure and gene activity. How these two systems coordinate with each other remains unclear. Here we study the biological function of lysine-specific demethylase 1 (LSD1, also known as KDM1 and AOF2), which has been shown to demethylate histone H3 on lysine 4 (H3K4) and lysine 9 (H3K9). We show that LSD1 is required for gastrulation during mouse embryogenesis. Notably, targeted deletion of the gene encoding LSD1 (namely, Aof2) in embryonic stem (ES) cells induces progressive loss of DNA methylation. This loss correlates with a decrease in DNA methyltransferase 1 (Dnmt1) protein, as a result of reduced Dnmt1 stability. Dnmt1 protein is methylated in vivo, and its methylation is enhanced in the absence of LSD1. Furthermore, Dnmt1 can be methylated by Set7/9 (also known as KMT7) and demethylated by LSD1 in vitro. Our findings suggest that LSD1 demethylates and stabilizes Dnmt1, thus providing a previously unknown mechanistic link between the histone and DNA methylation systems.

The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

Mouse strain-specific coding polymorphism in the Slc4a2/Ae2 gene encodes Ae2c2 variants differing in isoform-specific dominant negative activity.

The Slc4a2/Ae2 gene encodes multiple polypeptides arising from alternate promoter usage. The Ae2c promoter gives rise to only one Ae2c transcript from the human Ae2 gene, but to two, alternatively spliced, Ae2c1 and Ae2c2 transcripts from the mouse and rat genes. Unlike in the rat, the mouse Ae2c2 transcript encodes a novel Ae2c2 polypeptide. Here we report that the Ae2c2 residue 9 can be either proline or serine in a mouse strain-specific manner. Both Ae2c2 polypeptides express low function in Xenopus oocytes secondary to reduced or absent surface expression. Ae2c2S, but not Ae2c2P, exerts a dominant negative effect when coexpressed with Ae2a polypeptide, has a less prominent effect when coexpressed with Ae2b1 or Ae2c1 polypeptides, but has no effect on the function of coexpressed Ae2b2 polypeptide. Coexpression of Ae2c2P does not reduce activity of any Ae2 polypeptide variant. Ae2c2S and Ae2c2P also express low functional activity in HEK-293 cells. Knowledge of strain-specific coding polymorphisms with potential functional consequences such as that of Ae2c2 should aid in interpretation of strain-specific phenotypes investigated in the mouse phenome project.

Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells.

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase-mediated disruption of this allele results in hemimethylation of approximately 20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.

Regulated secretion of glycosylated human ferritin from hepatocytes.

Serum ferritin has been used widely in clinical medicine chiefly as an indicator of iron stores and inflammation. Circulating ferritin also can have paracrine effects. Despite the clinical significance of serum ferritin, its secretion remains an enigma. The consensus view is that serum ferritin arises from tissue ferritins--principally ferritin light--which can be glycosylated. Ferritin heavy and light chains are cytosolic proteins that form cages of 24 subunits to store intracellular iron. We show that ferritin light is secreted when its expression is increased in stable, transfected HepG2 cells or adenovirus-infected HepG2 cells. Export occurs through the classical secretory pathway and some chains are N-glycosylated. Ferritins do not need to form cages prior to secretion. Secretion is blocked specifically, effectively, and rapidly by a factor in serum. The timing of this inhibition of ferritin secretion suggests that normally cytosolic ferritin L is targeted to the secretory pathway during translation despite the absence of a conventional signal sequence. Thus, secretion of glycosylated and unglycosylated ferritin is a regulated and not a stochastic process.

Ferritins can regulate the secretion of apolipoprotein B.

Apolipoprotein B is secreted with atherogenic lipids as lipoprotein particles from hepatocytes. Regulation of the secretion of apolipoprotein B is largely post-translational and reflects the balance between processes that leads to particle assembly or to intracellular degradation. Previously, we conducted a proteomic screen to find proteins that bind apolipoprotein B in rat liver microsomes. We identified ferritin heavy and light chains in this screen among other proteins and showed that the two ferritins bind apolipoprotein B directly in vitro. In hepatocytes and other cells, ferritin heavy and light chains form cytosolic cages that store iron. We now show that ferritin heavy or light chains post-translationally inhibit the secretion of apolipoprotein B without altering the export of other hepatic proteins including albumin, factor XIII, and apolipoprotein A-I. This inhibition of apolipoprotein B secretion is not due to diminished lipid synthesis and can be partially overcome by stimulating triglyceride synthesis. The block in apolipoprotein B secretion by ferritins leads to an increase in endoplasmic reticulum-associated degradation of the apolipoprotein. Thus, despite being cytosolic proteins without known chaperone activity, ferritins can specifically regulate the secretion of apolipoprotein B post-translationally. The metabolic pathways for iron storage and intercellular cholesterol and triglyceride transport could intersect.

The nonclassic secretion of thioredoxin is not sensitive to redox state.

Thioredoxin (Trx) is a cytosolic, redox-active protein that is secreted from many cells and has several extracellular functions. In activated lymphocytes, the pathway of secretion does not involve the Golgi apparatus. Levels of extracellular Trx are decreased by the antioxidant N-acetylcysteine. Hence, the secretion of Trx could be altered by the redox status of the cell or the protein. To study Trx mutants, we characterized the secretion of human Trx from Chinese hamster ovary cells. Secretion of human Trx is unaffected by brefeldin A, slow but efficient, and sensitive to low temperature and factors in serum. We demonstrate that N-acetylcysteine reduces the cellular level of Trx but not the proportion secreted; thus this chemical does not block the nonclassic pathway for Trx secretion. Furthermore, we find that mutations in either the active site or the dimerization site of Trx do not alter its secretion. Thus the nonclassic secretion of Trx is not dependent on the redox status of either the cell or the protein.

Improperly folded green fluorescent protein is secreted via a non-classical pathway.

The green fluorescent protein is a cytosolic protein frequently used as a molecular tag to study protein localization in intact cells. We discovered that this protein is secreted into the medium by several but not all cell lines through a non-classical secretory pathway that is insensitive to brefeldin A. Green fluorescent protein is secreted efficiently by Chinese hamster ovary cells, with 60% of synthesized proteins secreted over 8 hours. This pathway is sensitive to changes in temperature but not to factors in serum or chemicals known to affect other non-classical protein secretion pathways. Fluorescence is observed in cells expressing green fluorescent protein, indicating that some of the protein must be fully folded in the cytosol. However, secreted green fluorescent protein is not fluorescent and therefore not folded properly. Furthermore, cellular fluorescence does not change over 6 hours whereas a significant proportion of green fluorescent protein is secreted. Thus, nascent green fluorescent protein either is folded correctly or incorrectly, and the improperly folded molecules can be exported. Non-classical secretion might be a route by which cells remove an excess of improperly folded, cytosolic proteins.

A proteomic approach identifies proteins in hepatocytes that bind nascent apolipoprotein B.

The biogenesis of apolipoprotein B is quite complex in view of its huge size, hydrophobicity, obligate association with lipids such as cholesterol and triglycerides prior to secretion, and intracellular degradation of a substantial proportion of newly synthesized molecules. Multiple proteins likely serve roles as molecular chaperones to assist in folding, assembly with lipids, and regulation of the secretion of apolipoprotein B. In these studies, we developed a strategy to isolate proteins associated with apolipoprotein B in rat livers. The purification consisted of two stages: first, microsomes were prepared from rat liver and treated with chemical cross-linkers, and second, the solubilized proteins were co-immunoprecipitated with antibody against apolipoprotein B. We found that several proteins were cross-linked to apolipoprotein B. The proteins were digested with trypsin, and the released peptides were sequenced by tandem mass spectrometry. The sequences precisely matched 377 peptides in 99 unique proteins. We show that at least two of the identified proteins, ferritin heavy and light chains, can directly bind apolipoprotein B. These and possibly other proteins identified by this proteomic approach are novel candidates for proteins that affect apolipoprotein B during its biogenesis.