SLC1A5 provides glutamine and asparagine necessary for bone development in mice.

Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here, we identify SLC1A5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show SLC1A5 acts cell autonomously to regulate protein synthesis and osteoblast differentiation. SLC1A5 provides both glutamine and asparagine which are essential for osteoblast differentiation. Mechanistically, glutamine and to a lesser extent asparagine support amino acid biosynthesis. Thus, osteoblasts depend on Slc1a5 to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development.

Compound NSC84167 selectively targets NRF2-activated pancreatic cancer by inhibiting asparagine synthesis pathway.

Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = -0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7-8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.

Elevated Asparagine Biosynthesis Drives Brain Tumor Stem Cell Metabolic Plasticity and Resistance to Oxidative Stress.

Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited. IMPLICATIONS: This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network.

ZBTB1 Regulates Asparagine Synthesis and Leukemia Cell Response to L-Asparaginase.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value.

SOX12 promotes colorectal cancer cell proliferation and metastasis by regulating asparagine synthesis.

The sex-determining region Y (SRY)-box (SOX) family has a crucial role in carcinogenesis and cancer progression. However, the role of SOX12 and the mechanism by which it is dysregulated in colorectal cancer (CRC) remain unclear. Here we analyzed SOX12 expression patterns in two independent CRC cohorts (cohort I, n = 390; cohort II, n = 363) and found that SOX12 was significantly upregulated in CRC, indicating a poor prognosis in CRC patients. Overexpression of SOX12 promoted CRC cell proliferation and metastasis, whereas downregulation of SOX12 hampered CRC aggressiveness. Mechanistically, SOX12 facilitated asparagine synthesis by transactivating glutaminase (GLS), glutamic oxaloacetic transaminase 2 (GOT2), and asparagine synthetase (ASNS). Downregulation of GLS, GOT2, and ASNS blocked SOX12-mediated CRC cell proliferation and metastasis, whereas ectopic expression of GLS, GOT2, and ASNS attenuated the SOX12 knockdown-induced suppression of CRC progression. In addition, serial deletion, site-directed mutagenesis, luciferase reporter, and chromatin immunoprecipitation (ChIP) assays indicated that hypoxia-inducible factor 1α (HIF-1α) directly binds to the SOX12 promoter and induces SOX12 expression. Administration of L-asparaginase decreased SOX12-mediated tumor growth and metastasis. In human CRC samples, SOX12 expression positively correlated with GLS, GOT2, ASNS, and HIF-1α expression. Based on these results, SOX12 may serve as a prognostic biomarker and L-asparaginase represents a potential novel therapeutic agent for CRC.

Effects of nitrogen combined with zinc application on glutamate, glutamine, aspartate and asparagine accumulation in two winter wheat cultivars.

Zinc (Zn) deficiency remarkably depresses the protein concentration in the grain of winter wheat. Cultivar 'Pingan 8' showed lower Zn concentrations in the grain than did cultivar 'Yangao 006' after nitrogen (N) combined with Zn application. However, little is known about how amino acids are influenced by Zn combined with N application or about the differences in amino acid accumulation between the two winter wheat cultivars. A pot experiment was conducted to characterize amino acid accumulation in the low Zn-accumulating cultivar 'Pingan 8' and the high Zn-accumulating cultivar 'Yangao 006' at various growth stages (seedling, jointing, grain filling and maturity) as influenced by N and Zn supply. The N (N0.2) combined with Zn (Zn10) application significantly increased grain yields and the concentrations of N, Zn and crude protein in the grain of both wheat cultivars. N combined with Zn application significantly increased the concentrations of glutamate (Glu) and asparagine (Asn) but decreased the concentrations of glutamine (Gln) and aspartate (Asp) in cultivar 'Yangao 006'; the N combined with Zn application decreased the concentrations of Glu and Gln but increased the concentrations of Asp and Asn in cultivar 'Pingan 8' at the jointing, grain filling and mature stages. Correlation analysis results showed that there were significant relationships between grain yields, spike number, grain number and Zn, N, crude protein, Glu, Gln, Asp and Asn concentrations in the shoots and grain of winter wheat at different growth stages. These results demonstrate that N combined with Zn application enhanced protein synthesis by altering amino acid accumulation in both winter wheat cultivars. Cultivar 'Pingan 8' had lower Gln, Asp and Asn concentrations and higher Glu concentrations than did cultivar 'Yangao 006' after the N0.05 treatment but had higher Glu, Gln, Asp, and Asn concentrations and lower Glu concentrations than did cultivar 'Yangao 006' after the N0.2 treatment. These results revealed that the difference in amino acid concentrations between the two cultivars was related to the N application level.

Characterization of a novel variant in siblings with Asparagine Synthetase Deficiency.

Asparagine Synthetase Deficiency (ASD) is a recently described inborn error of metabolism caused by bi-allelic pathogenic variants in the asparagine synthetase (ASNS) gene. ASD typically presents congenitally with microcephaly and severe, often medically refractory, epilepsy. Development is generally severely affected at birth. Tone is abnormal with axial hypotonia and progressive appendicular spasticity. Hyperekplexia has been reported. Neuroimaging typically demonstrates gyral simplification, abnormal myelination, and progressive cerebral atrophy. The present report describes two siblings from consanguineous parents with a homozygous Arg49Gln variant associated with a milder form of ASD that is characterized by later onset of symptoms. Both siblings had a period of normal development before onset of seizures, and development regression. Primary fibroblast studies of the siblings and their parents document that homozygosity for Arg49Gln blocks cell growth in the absence of extracellular asparagine. Functional studies with these cells suggest no impact of the Arg49Gln variant on basal ASNS mRNA or protein levels, nor on regulation of the gene itself. Molecular modelling of the ASNS protein structure indicates that the Arg49Gln variant lies near the substrate binding site for glutamine. Collectively, the results suggest that the Arg49Gln variant affects the enzymatic function of ASNS. The clinical, cellular, and molecular observations from these siblings expand the known phenotypic spectrum of ASD.

Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme.

The biosynthesis of branched alkoxy groups, such as the unique t-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metabolites contain an isopropyl group proposed to be formed using CysS, a cobalamin-dependent radical S-adenosylmethionine (SAM) methyltransferase. Here, we reconstitute the CysS-catalyzed reaction, on p-aminobenzoate thioester substrates, and demonstrate that it not only catalyzes sequential methylations of a methyl group to form ethyl and isopropyl groups but remarkably also sec-butyl and t-butyl groups. To our knowledge, this is the first in vitro reconstitution of a cobalamin-dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t-butyl group.

Nitrile-synthesizing enzyme: Gene cloning, overexpression and application for the production of useful compounds.

One of the nitrile-synthesizing enzymes, ß-cyano-L-alanine synthase, catalyzes ß-cyano-L-alanine (ß-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the ß-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of ß-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine. This strategy can be used for the synthesis of labeled amino acids by using a carbon-labeled KCN as a substrate, resulting in an application for positron emission tomography.

Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes.

Asparagine synthetase1, but not asparagine synthetase2, is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots.

Asparagine is synthesized from glutamine by the reaction of asparagine synthetase (AS) and is the major nitrogen form in both xylem and phloem sap in rice (Oryza sativa L.). There are two genes encoding AS, OsAS1 and OsAS2, in rice, but the functions of individual AS isoenzymes are largely unknown. Cell type- and NH4(+)-inducible expression of OsAS1 as well as analyses of knockout mutants were carried out in this study to characterize AS1. OsAS1 was mainly expressed in the roots, with in situ hybridization showing that the corresponding mRNA was specifically accumulated in the three cell layers of the root surface (epidermis, exodermis and sclerenchyma) in an NH4(+)-dependent manner. Conversely, OsAS2 mRNA was abundant in leaf blades and sheathes of rice. Although OsAS2 mRNA was detectable in the roots, its content decreased when NH4(+) was supplied. Retrotransposon-mediated knockout mutants lacking AS1 showed slight stimulation of shoot length and slight reduction in root length at the seedling stage. On the other hand, the mutation caused an approximately 80-90% reduction in free asparagine content in both roots and xylem sap. These results suggest that AS1 is responsible for the synthesis of asparagine in rice roots following the supply of NH4(+). Characteristics of the NH4(+)-dependent increase and the root surface cell-specific expression of OsAS1 gene are very similar to our previous results on cytosolic glutamine synthetase1;2 and NADH-glutamate synthase1 in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4(+) in rice roots.

Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis.

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNA(Asn) may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA(Asn). We report the 3.7-Šresolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA(Asn) molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNA(Asn) formation evolutionarily preceded the direct attachment of Asn to tRNA(Asn), our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.

Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis.

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA(Asn). The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA(Asn). S. aureus thus encodes both the direct and indirect routes for Asn-tRNA(Asn) formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.

Genetic variation and possible SNP markers for breeding wheat with low-grain asparagine, the major precursor for acrylamide formation in heat-processed products.

BACKGROUND: In products made from wheat (Triticum aestivum) flour, acrylamide formation is almost exclusively determined by the level of free asparagine in the grain. Genetic variability for grain asparagine content was evaluated in order to assess the potential for acrylamide mitigation by breeding. RESULTS: Free asparagine levels in the grains of 92 varieties varied from 137 to 471 mg kg⁻¹, representing an approximate threefold difference between the low- and high-asparagine genotypes. Heritability was low, with a value of 32%, indicating that breeding cultivars with inherently low grain asparagine would be a challenge. A genome-wide scan with single-nucleotide polymorphism (SNP) markers identified nine SNPs that were significantly (P < 0.001) associated with variation in free asparagine. The significant SNPs were localized on chromosome 5A, and explained between 14% and 24% of the observed variation. These putative SNPs are candidates for further studies to develop molecular markers. CONCLUSION: Significant genetic variation exists for reducing acrylamide precursors in wheat flour, indicating that breeding and genetics could play an important role in mitigating the acrylamide risk in wheat products. The study identified a region on chromosome 5A that could provide a basis for further research to develop functional markers.

Two-codon T-box riboswitch binding two tRNAs.

T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as single-specificity riboswitches that can bind specific tRNA anticodons through codon-anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum, the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNA(Asn) and tRNA(Glu). To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that "flexible" T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.

Molecular characterization of PVAS3: an asparagine synthetase gene from common bean prevailing in developing organs.

In common bean, asparagine synthetase (AS; EC 6.3.5.4) is encoded by three members of a multigene family called PVAS1, PVAS2 and PVAS3. Two of these genes, PVAS1 and PVAS2, have been extensively studied, but little is known about PVAS3, remaining unclear whether PVAS3 function is redundant to the other AS or if it plays a specific role in Phaseolus vulgaris metabolism. In this work, we used a molecular approach to characterize PVAS3 expression and to gain some knowledge about its physiological function. We showed that, in contrast to PVAS1 and PVAS2, PVAS3 was expressed in all organs analyzed. Interestingly, PVAS3 was the AS gene most highly expressed in nodules, leaves and pods at the earliest stages of development, and its expression decreased as these organs developed. Expression of PVAS3 parallels the accumulation of AS protein and the asparagine content during the earliest stages of nodule, leaf and pod development, suggesting an important role for PVAS3 in the synthesis of asparagine in that period. Furthermore, PVAS3 was not repressed by light, as most class-II AS genes. Surprisingly, fertilization of nodulated plants with nitrate or ammonium, conditions that induce PVAS1 and PVAS2 and the shift from ureides to amide synthesis, repressed the expression of PVAS3 in nodules and roots. The possible implications of this regulation are discussed.

Asparagine synthetase: regulation by cell stress and involvement in tumor biology.

Asparagine synthetase (ASNS) catalyzes the conversion of aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. The enzyme is ubiquitous in its organ distribution in mammals, but basal expression is relatively low in tissues other than the exocrine pancreas. Human ASNS activity is highly regulated in response to cell stress, primarily by increased transcription from a single gene located on chromosome 7. Among the genomic elements that control ASNS transcription is the C/EBP-ATF response element (CARE) within the promoter. Protein limitation or an imbalanced dietary amino acid composition activate the ASNS gene through the amino acid response (AAR), a process that is replicated in cell culture through limitation for any single essential amino acid. Endoplasmic reticulum stress also increases ASNS transcription through the PERK-eIF2-ATF4 arm of the unfolded protein response (UPR). Both the AAR and UPR lead to increased synthesis of ATF4, which binds to the CARE and induces ASNS transcription. Elevated expression of ASNS protein is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia and may be a predictive factor in drug sensitivity for certain solid tumors as well. Activation of the GCN2-eIF2-ATF4 signaling pathway, leading to increased ASNS expression appears to be a component of solid tumor adaptation to nutrient deprivation and/or hypoxia. Identifying the roles of ASNS in fetal development, tissue differentiation, and tumor growth may reveal that ASNS function extends beyond asparagine biosynthesis.

Cell cycle-dependent recruitment of polycomb proteins to the ASNS promoter counteracts C/ebp-mediated transcriptional activation in Bombyx mori.

Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori.