Infection perturbs Bach2- and Bach1-dependent erythroid lineage 'choice' to cause anemia.

Elucidation of how the differentiation of hematopoietic stem and progenitor cells (HSPCs) is reconfigured in response to the environment is critical for understanding the biology and disorder of hematopoiesis. Here we found that the transcription factors (TFs) Bach2 and Bach1 promoted erythropoiesis by regulating heme metabolism in committed erythroid cells to sustain erythroblast maturation and by reinforcing erythroid commitment at the erythro-myeloid bifurcation step. Bach TFs repressed expression of the gene encoding the transcription factor C/EBPß, as well as that of its target genes encoding molecules important for myelopoiesis and inflammation; they achieved the latter by binding to their regulatory regions also bound by C/EBPß. Lipopolysaccharide diminished the expression of Bach TFs in progenitor cells and promoted myeloid differentiation. Overexpression of Bach2 in HSPCs promoted erythroid development and inhibited myelopoiesis. Knockdown of BACH1 or BACH2 in human CD34+ HSPCs impaired erythroid differentiation in vitro. Thus, Bach TFs accelerate erythroid commitment by suppressing the myeloid program at steady state. Anemia of inflammation and myelodysplastic syndrome might involve reduced activity of Bach TFs.

Heme-mediated SPI-C induction promotes monocyte differentiation into iron-recycling macrophages.

Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80(+)VCAM1(+) bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis.

Accelerated plasma-cell differentiation in Bach2-deficient mouse B cells is caused by altered IRF4 functions.

Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

The transcription repressors Bach2 and Bach1 promote B cell development by repressing the myeloid program.

Mature lymphoid cells express the transcription repressor Bach2, which imposes regulation on humoral and cellular immunity. Here we found critical roles for Bach2 in the development of cells of the B lineage, commencing from the common lymphoid progenitor (CLP) stage, with Bach1 as an auxiliary. Overexpression of Bach2 in pre-pro-B cells deficient in the transcription factor EBF1 and single-cell analysis of CLPs revealed that Bach2 and Bach1 repressed the expression of genes important for myeloid cells ('myeloid genes'). Bach2 and Bach1 bound to presumptive regulatory regions of the myeloid genes. Bach2(hi) CLPs showed resistance to myeloid differentiation even when cultured under myeloid conditions. Our results suggest that Bach2 functions with Bach1 and EBF1 to promote B cell development by repressing myeloid genes in CLPs.

BTB and CNC homology 1 deficiency disrupts intestinal IgA secretion through regulation of polymeric immunoglobulin receptor expression.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1-/-) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1-/- mice. Furthermore, plasma IgA was increased in Bach1-/- mice, but fecal IgA was decreased, indicating that Bach1-/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, the overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.NEW & NOTEWORTHY The transcriptional repressor Bach1 has been implicated in diverse intestinal functions, but the effects of Bach1 on IgA-mediated mucosal immunity remain unclear. We demonstrate here that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen, although the proportions of IgA-producing cells were not altered. Furthermore, Bach1 regulates the expression of pIgR, which plays an important role in the transepithelial transport of IgA, at the posttranscriptional level.

Exploring novel functions of BACH2 in the acquisition of antigen-specific antibodies.

BACH2 [BTB (broad-complex, tramtrak and bric à brac) and CNC (cap 'n' collar) homolog 2] is known as a transcriptional repressor and broadly functions in regulating immune cell differentiation. Here, we focus on BACH2 function in B cells, where BACH2 was first shown to play an important role in the immune system. In B cells, BACH2 orchestrates the gene regulatory network that promotes class switch and affinity maturation of antibodies and simultaneously represses plasma-cell differentiation. In this context, BACH2 regulates gene expression by modulating chromatin organization, cooperatively with other transcription factors and chromatin regulators, such as IRF4 (interferon regulatory factor 4) and PC4 (positive coactivator 4), respectively. In addition, our recent observation raises the possibility that BACH2 has diverse functions, such as those in gene activation. Since dysfunction of BACH2 leads to the onset of human immune deficiencies, revealing new functions of BACH2 may give a cue to solve how BACH2 contributes to preventing these diseases.

Bach1 derepression is neuroprotective in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of nigrostriatal dopaminergic neurons. Mounting evidence suggests that Nrf2 is a promising target for neuroprotective interventions in PD. However, electrophilic chemical properties of the canonical Nrf2-based drugs cause irreversible alkylation of cysteine residues on cellular proteins resulting in side effects. Bach1 is a known transcriptional repressor of the Nrf2 pathway. We report that Bach1 levels are up-regulated in PD postmortem brains and preclinical models. Bach1 knockout (KO) mice were protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity and associated oxidative damage and neuroinflammation. Functional genomic analysis demonstrated that the neuroprotective effects in Bach1 KO mice was due to up-regulation of Bach1-targeted pathways that are associated with both Nrf2-dependent antioxidant response element (ARE) and Nrf2-independent non-ARE genes. Using a proprietary translational technology platform, a drug library screen identified a substituted benzimidazole as a Bach1 inhibitor that was validated as a nonelectrophile. Oral administration of the Bach1 inhibitor attenuated MPTP neurotoxicity in pre- and posttreatment paradigms. Bach1 inhibitor-induced neuroprotection was associated with the up-regulation of Bach1-targeted pathways in concurrence with the results from Bach1 KO mice. Our results suggest that genetic deletion as well as pharmacologic inhibition of Bach1 by a nonelectrophilic inhibitor is a promising therapeutic approach for PD.

TANK Binding Kinase 1 Promotes BACH1 Degradation through Both Phosphorylation-Dependent and -Independent Mechanisms without Relying on Heme and FBXO22.

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.

Iron in Cancer Progression: Does BACH1 Promote Metastasis by Altering Iron Homeostasis?

The transcription factor BACH1, which is regulated by direct binding of prosthetic group heme, promotes epithelial-mesenchymal transition (EMT) and drives metastasis of diverse types of cancer cells. De-regulated target genes of BACH1 in cancer cells include those for glycolysis, oxidative phosphorylation, epithelial cell adhesion, and mesodermal cell motility. In addition, the canonical target genes of BACH1 include genes for the regulation of iron homeostasis. Importantly, cancer cells are addicted to iron. We summarize known functions of BACH1 in cancer and discuss how BACH1 may affect iron homeostasis in cancer cells to support their progression by increasing mobile iron within cells. The dependency on BACH1 for cancer progression may also confer upon cancer cells susceptibility to iron-dependent cell death ferroptosis. Finally, we discuss that the human transcription factors provide research opportunities for better understanding of cancer cell properties.

[Gene regulatory networks operated by iron-heme metabolism: focusing on the erythroid differentiation system].

Recently, attention has been focused on how metabolites regulate cellular differentiation. Heme synthesis is greatly enhanced during erythroid differentiation. Heme is used for hemoglobin synthesis, which is needed for oxygen transport by red blood cells. Additionally, several pieces of evidence revealed that heme may control gene expression to fulfill erythroblast terminal maturation. For instance, heme binds to and inactivates the transcription factor BTB Domain And CNC Homolog 1 (BACH1), thereby inducing the globin gene expression, which is the repressive target of BACH1. Moreover, heme induces autophagy/mitophagy-related gene expressions, which are the targets of the transcription factor GATA1 and may promote erythrocyte maturation by itself. Heme may directly bind to guanine tetramer (G-quadruplex) regions of genomic DNA, thereby regulating nearby gene expressions. Here, we present an overview of the mechanism by which iron-heme metabolism regulates gene regulatory networks by focusing on the erythroid differentiation system. Additionally, we discuss the prospects of future research regarding iron-heme metabolism.

The transcription factor BACH1 at the crossroads of cancer biology: From epithelial-mesenchymal transition to ferroptosis.

The progression of cancer involves not only the gradual evolution of cells by mutations in DNA but also alterations in the gene expression induced by those mutations and input from the surrounding microenvironment. Such alterations contribute to cancer cells' abilities to reprogram metabolic pathways and undergo epithelial-to-mesenchymal transition (EMT), which facilitate the survival of cancer cells and their metastasis to other organs. Recently, BTB and CNC homology 1 (BACH1), a heme-regulated transcription factor that represses genes involved in iron and heme metabolism in normal cells, was shown to shape the metabolism and metastatic potential of cancer cells. The growing list of BACH1 target genes in cancer cells reveals that BACH1 promotes metastasis by regulating various sets of genes beyond iron metabolism. BACH1 represses the expression of genes that mediate cell-cell adhesion and oxidative phosphorylation but activates the expression of genes required for glycolysis, cell motility, and matrix protein degradation. Furthermore, BACH1 represses FOXA1 gene encoding an activator of epithelial genes and activates SNAI2 encoding a repressor of epithelial genes, forming a feedforward loop of EMT. By synthesizing these observations, we propose a "two-faced BACH1 model", which accounts for the dynamic switching between metastasis and stress resistance along with cancer progression. We discuss here the possibility that BACH1-mediated promotion of cancer also brings increased sensitivity to iron-dependent cell death (ferroptosis) through crosstalk of BACH1 target genes, imposing programmed vulnerability upon cancer cells. We also discuss the future directions of this field, including the dynamics and plasticity of EMT.

IRF2BP2 is a novel HNF4α co-repressor: Its role in gluconeogenic gene regulation via biochemically labile interaction.

Hepatocyte nuclear factor 4α (HNF4α) has essential roles in controlling the expression of a variety of genes involved in key metabolic pathways, including gluconeogenesis in the liver. The mechanistic and physiological significance of peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) for HNF4α-mediated transcriptional activation models for gluconeogenic genes is well characterized. However, the transcriptional repression of HNF4α for those genes remains to be examined. In this study, we applied novel proteomic techniques to evaluate the interactions of HNF4α, including those with biochemically labile binding proteins. Based upon our experiments, we identified interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a novel HNF4α co-repressor. This interaction could not be detected by conventional immunoprecipitation. IRF2BP2 repressed the transcriptional activity of HNF4α dependent on its E3 ubiquitin ligase activity. Deficiency of the IRF2BP2 gene in HepG2 cells induced gluconeogenic genes comparable to that of forskolin-treated wild-type HepG2 cells. Together, these results suggest that IRF2BP2 represents a novel class of nuclear receptor co-regulator.

Ferroptosis is controlled by the coordinated transcriptional regulation of glutathione and labile iron metabolism by the transcription factor BACH1.

Ferroptosis is an iron-dependent programmed cell death event, whose regulation and physiological significance remain to be elucidated. Analyzing transcriptional responses of mouse embryonic fibroblasts exposed to the ferroptosis inducer erastin, here we found that a set of genes related to oxidative stress protection is induced upon ferroptosis. We considered that up-regulation of these genes attenuates ferroptosis induction and found that the transcription factor BTB domain and CNC homolog 1 (BACH1), a regulator in heme and iron metabolism, promotes ferroptosis by repressing the transcription of a subset of the erastin-induced protective genes. We noted that these genes are involved in the synthesis of GSH or metabolism of intracellular labile iron and include glutamate-cysteine ligase modifier subunit (Gclm), solute carrier family 7 member 11 (Slc7a11), ferritin heavy chain 1 (Fth1), ferritin light chain 1 (Ftl1), and solute carrier family 40 member 1 (Slc40a1). Ferroptosis has also been previously shown to induce cardiomyopathy, and here we observed that Bach1-/- mice are more resistant to myocardial infarction than WT mice and that the severity of ischemic injury is decreased by the iron-chelator deferasirox, which suppressed ferroptosis. Our findings suggest that BACH1 represses genes that combat labile iron-induced oxidative stress, and ferroptosis is stimulated at the transcriptional level by BACH1 upon disruption of the balance between the transcriptional induction of protective genes and accumulation of iron-mediated damage. We propose that BACH1 controls the threshold of ferroptosis induction and may represent a therapeutic target for alleviating ferroptosis-related diseases, including myocardial infarction.

Bach1 plays an important role in angiogenesis through regulation of oxidative stress.

Bach1 is a known transcriptional repressor of the heme oxygenase-1 (HO-1) gene. The purpose of this study was to determine whether angiogenesis is accelerated by genetic ablation of Bach1 in a mouse ischemic hindlimb model. Hindlimb ischemia was surgically induced in wild-type (WT) mice, Bach1-deficient (Bach1-/-) mice, apolipoprotein E-deficient (ApoE-/-) mice, and Bach1/ApoE double-knockout (Bach1-/-/ApoE-/-) mice. Blood flow recovery after hindlimb ischemia showed significant improvement in Bach1-/- mice compared with that in WT mice. Bach1-/-/ApoE-/- mice showed significantly improved blood flow recovery compared with that in ApoE-/- mice to the level of that in WT mice. Migration of endothelial cells in ApoE-/- mice was significantly decreased compared with that in WT mice. Migration of endothelial cells significantly increased in Bach1-/-/ApoE-/- mice compared with that in ApoE-/- mice to the level of that in WT mice. The expression levels of HO-1, peroxisome proliferator-activated receptor γ co-activator-1α, angiopoietin 1, and fibroblast growth factor 2 in endothelial cells isolated from Bach1-/-/ApoE-/- mice were significantly higher than those in ApoE-/- mice. Oxidative stress assessed by anti-acrolein antibody staining in ischemic tissues and urinary 8-isoPGF2α excretion were significantly increased in ApoE-/- mice compared with those in WT and Bach1-/- mice. Oxidative stress was reduced in Bach1-/-/ApoE-/- mice compared with that in ApoE-/- mice. These findings suggest that genetic ablation of Bach1 plays an important role in ischemia-induced angiogenesis under the condition of increased oxidative stress. Bach1 could be a potential therapeutic target to reduce oxidative stress and potentially improve angiogenesis for patients with peripheral arterial disease.

Study Profile of the Tohoku Medical Megabank Community-Based Cohort Study.

BACKGROUND: We established a community-based cohort study to assess the long-term impact of the Great East Japan Earthquake on disaster victims and gene-environment interactions on the incidence of major diseases, such as cancer and cardiovascular diseases. METHODS: We asked participants to join our cohort in the health check-up settings and assessment center based settings. Inclusion criteria were aged 20 years or over and living in Miyagi or Iwate Prefecture. We obtained information on lifestyle, effect of disaster, blood, and urine information (Type 1 survey), and some detailed measurements (Type 2 survey), such as carotid echography and calcaneal ultrasound bone mineral density. All participants agreed to measure genome information and to distribute their information widely. RESULTS: As a result, 87,865 gave their informed consent to join our study. Participation rate at health check-up site was about 70%. The participants in the Type 1 survey were more likely to have psychological distress than those in the Type 2 survey, and women were more likely to have psychological distress than men. Additionally, coastal residents were more likely to have higher degrees of psychological distress than inland residents, regardless of sex. CONCLUSION: This cohort comprised a large sample size and it contains information on the natural disaster, genome information, and metabolome information. This cohort also had several detailed measurements. Using this cohort enabled us to clarify the long-term effect of the disaster and also to establish personalized prevention based on genome, metabolome, and other omics information.

Comparative proteomic analysis to identify the novel target gene of angiotensin II in adrenocortical H295R cells.

Angiotensin II (Ang II) is a well-known peptide that maintains the balance of electrolytes in the higher vertebrates. Ang II stimulation in the adrenal gland induces the synthesis of mineralocorticoids, mainly aldosterone, through the up-regulation of aldosterone synthase (CYP11B2) gene expression. Additionally, it has been reported that Ang II activates multiple signaling pathways such as mitogen-activated protein kinase (MAPK) and Ca2+ signaling. Although Ang II has various effects on the cellular signaling in the adrenal cells, its biological significance, except for the aldosterone synthesis, is still unclear. In this study, we attempted to search the novel target gene(s) of Ang II in the human adrenal H295R cells using a proteomic approach combined with stable isotopic labeling using amino acid in cell culture (SILAC). Interestingly, we found that Ang II stimulation elevated the expression of phosphofructokinase type platelet (PFKP) in both protein and mRNA levels. Moreover, transactivation of PFKP by Ang II was dependent on extracellular-signal-regulated kinase (ERK) 1/2 activation. Finally, we observed that Ang II treatment facilitated glucose uptake in the H295R cells. Taken together, we here identified PFKP as a novel target gene of Ang II, indicating that Ang II not only stimulates steroidogenesis but also affects glucose metabolism.

Target of rapamycin-signaling modulates starch accumulation via glycogenin phosphorylation status in the unicellular red alga Cyanidioschyzon merolae.

The target of rapamycin (TOR) signaling pathway is involved in starch accumulation in various eukaryotic organisms; however, the molecular mechanism behind this phenomenon in eukaryotes has not been elucidated. We report a regulatory mechanism of starch accumulation by TOR in the unicellular red alga, Cyanidioschyzon merolae. The starch content in C. merolae after TOR-inactivation by rapamycin, a TOR-specific inhibitor, was increased by approximately 10-fold in comparison with its drug vehicle, dimethyl sulfoxide. However, our previous transcriptome analysis showed that the expression level of genes related to carbohydrate metabolism was unaffected by rapamycin, indicating that starch accumulation is regulated at post-transcriptional levels. In this study, we performed a phosphoproteome analysis using liquid chromatography-tandem mass spectrometry to investigate potential post-transcriptional modifications, and identified 52 proteins as candidate TOR substrates. Among the possible substrates, we focused on the function of CmGLG1, because its phosphorylation at the Ser613 residue was decreased after rapamycin treatment, and overexpression of CmGLG1 resulted in a 4.7-fold higher starch content. CmGLG1 is similar to the priming protein, glycogenin, which is required for the initiation of starch/glycogen synthesis, and a budding yeast complementation assay demonstrated that CmGLG1 can functionally substitute for glycogenin. We found an approximately 60% reduction in the starch content in a phospho-mimicking CmGLG1 overexpression strain, in which Ser613 was substituted with aspartic acid, in comparison with the wild-type CmGLG1 overexpression cells. Our results indicate that TOR modulates starch accumulation by changing the phosphorylation status of the CmGLG1 Ser613 residue in C. merolae.