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Ten‐eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1‐upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast‐like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5‐hmC) profiling and found that 5‐hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4–monomethylated, where the H3K27‐acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast‐like HCC., Ten‐eleven translocation 1 (TET1), a methylcytosine dioxygenase of the DNA demethylation pathway, is overexpressed in the subgroup of hepatocellular carcinoma with the hepatoblast‐like expression pattern. Comprehensive epigenome profilings revealed the ectopic enhancer activation of HMGA2 through cytosine hydroxymethylation, H3K27 acetylation, H3K4 monomethylation, and promoter‐enhancer interaction in a TET1‐dependent manner.

Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated “primed” state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis., How enhancers transition from a hypoacetylated primed state to a hyperacetylated-active state in response to differentiation stimuli is incompletely understood. Here the authors show that SETD5 forms a complex with NCoR-HDAC3 co-repressor to prevent histone acetylation of master adipogenic gene enhancers, while SETD5 degradation triggers enhancer hyperacetylation and transition to active state.

Retinal stem cell differentiation is orchestrated by transcription cascades, and is prone to be influenced by stress factors. While threshold radiation dose for human mental retardation is reported, the effects on retinal transcription factors are unknown. To elucidate the effects of low-dose radiation on retinal differentiation, stem cells were irradiated. Genome-wide gene expression and histone modification were observed. After a month, the expression of PAX6, an essential transcription factor for retinal ganglion cell, was increased by 30 mGy but decreased by 180 mGy. POU5F1, POU2F1, SOX2, and PAX6 were predicted to be involved in regulation of PAX6. Further, upstream of POU5F1/POU2F1/ SOX2 was detected. Altered Rho family could be responsible for activity of SRF/STAT3/RELA, which were upstream of POU2F1/SOX2. These cascades could contribute to the regulation of PAX6 and unstable retinal development under stressful environment. Identification of the hierarchical regulations in the differentiation will help elucidate the retinal cell maintenance.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by excessive pulmonary vascular remodeling. However, despite advances in therapeutic strategies, patients with PAH bearing mutations in the bone morphogenetic protein receptor type 2 (

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by excessive pulmonary vascular remodeling. However, despite advances in therapeutic strategies, patients with PAH bearing mutations in the bone morphogenetic protein receptor type 2 (BMPR2)-encoding gene present severe phenotypes and outcomes. We sought to investigate the effect of PER-like kinase (PERK), which participates in one of three major pathways associated with the unfolded protein response (UPR), on PAH pathophysiology in BMPR2 heterozygous mice. BMPR2 heterozygosity in pulmonary artery smooth muscle cells (PASMCs) decreased the abundance of the antiapoptotic microRNA miR124-3p through the arm of the UPR mediated by PERK. Hypoxia promoted the accumulation of unfolded proteins in BMPR2 heterozygous PASMCs, resulting in increased PERK signaling, cell viability, cellular proliferation, and glycolysis. Proteomic analyses revealed that PERK ablation suppressed PDGFRβ-STAT1 signaling and glycolysis in hypoxic BMPR2 heterozygous PASMCs. Furthermore, PERK ablation or PERK inhibition ameliorated pulmonary vascular remodeling in the Sugen/chronic hypoxia model of PAH, irrespective of BMPR2 status. Hence, these findings suggest that PERK inhibition is a promising therapeutic strategy for patients with PAH with or without BMPR2 mutation.

Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease arising in the peripartum period. 16 kDa-prolactin (16K PRL) and its subproduct microRNA (miR)-146a-5p are candidate biomarkers for PPCM. However, we showed that miR-146a-5p is not well upregulated in the sera of PPCM patients. Here, we found that miR-320a/b were the most specific miRNAs that suppressed the expression of protein kinase RNA-like endoplasmic reticulum kinase (PERK). We transfected neonatal rat cardiomyocytes with plasmids containing full-length prolactin (23K PRL) or 16K PRL in vitro. We found that 23K PRL, but not 16K PRL, upregulated PERK signaling, and hypoxia promoted this effect. During the perinatal period, cardiomyocyte-specific PERK homogenous knockout (CM-KO) mice showed PPCM phenotypes after consecutive deliveries. Downregulation of PERK or JAK/STAT signaling and upregulation of apoptosis were observed in CM-KO mouse hearts. These results demonstrated that interruption of 23K PRL-PERK interaction by miR-320a/b could be a therapeutic strategy for PPCM. Funding Statement: This study was supported by the Japan Society for the Promotion of Science KAKENHI grant number JP18K16756 and JP20K17210 (T.S.) and by the research grant from Public Trust Cardiovascular Research Fund (T.S.). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The institutional review board of National Cerebral and Cardiovascular Center in Osaka approved this study. Eligible women were enrolled after providing written informed consent. Animal Ethics Approval Statement: All experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly adhered to the guidelines for animal experiments of the University of Tokyo.

Peripartum cardiomyopathy (PPCM) is a life-threatening heart failure occurring in the peripartum period. Although mal-angiogenesis, induced by the 16-kDa N-terminal prolactin fragment (16 K PRL), is involved in the pathogenesis, the effect of full-length prolactin (23 K PRL) is poorly understood. We transfected neonate rat cardiomyocytes with plasmids containing 23 K PRL or 16 K PRL in vitro and found that 23 K PRL, but not 16 K PRL, upregulated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling, and hypoxia promoted this effect. During the perinatal period, cardiomyocyte-specific PERK homogenous knockout (CM-KO) mice showed PPCM phenotypes after consecutive deliveries. Downregulation of PERK or JAK/STAT signaling and upregulation of apoptosis were observed in CM-KO mouse hearts. Moreover, in bromocriptine-treated CM-KO mice, cardiac function did not improve and cardiomyocyte apoptosis was not suppressed during the peripartum period. These results demonstrate that interaction between 23 K PRL and PERK signaling is cardioprotective during the peripartum term.

Tubulointerstitial fibrosis has been recently reported to be caused by the collapse of the epigenetic regulation of kidney diseases. We examined whether pharmacological inhibition of histone modification is effective against renal fibrosis. DZNep (3-deazaneplanocin A) was originally developed as an anti-cancer drug to inhibit the repressive histone mark, H3K27me3. We used a model of chronic tubulointerstitial fibrosis induced by unilateral ischaemia/reperfusion and administered DZNep intravenously to the mice for 8 weeks. We found DZNep contributes to the reduction of tubulointerstitial fibrosis. We selected only tubular cells from in vivo samples using laser-capture microdissection because epigenetic regulation is specific to the cell types, and we focused on the changes in the tubular cells. We performed a genome-wide analysis of tubular cells using high-throughput sequencing (RNA-seq) to identify novel epigenetic factors associated with renal fibrosis. We found that pro-fibrotic genes such as COL3A1 (collagen type 3a1) and TIMP2 (tissue inhibitor of metalloproteinase 2) were suppressed by DZNep in vivo. In addition, pro-fibrotic genes such as COL4A1 (collagen type 4a1), TIMP2 and MMP14 were down-regulated by DZNep in vitro. In conclusion, we found that pharmacological epigenetic modification by DZNep decreased the expression levels of fibrogenic genes in tubular cells and inhibited tubulointerstitial fibrosis.

Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.

Type I interferons (IFNs) play important roles in antitumor immunity. We generated IFN-α-producing cells by genetically engineering induced pluripotent stem cell (iPSC)-derived proliferating myeloid cells (iPSC-pMCs). Local administration of IFN-α-producing iPSC-pMCs (IFN-α-iPSC-pMCs) alters the tumor microenvironment and propagates the molecular signature associated with type I IFN. The gene-modified cell actively influences host XCR1+ dendritic cells to enhance CD8+ T cell priming, resulting in CXCR3-dependent and STING-IRF3 pathway-independent systemic tumor control. Administration of IFN-α-iPSC-pMCs in combination with immune checkpoint blockade overcomes resistance to single-treatment modalities and generates long-lasting antitumor immunity. These preclinical data suggest that IFN-α-iPSC-pMCs might constitute effective immune-stimulating agents for cancer that are refractory to checkpoint blockade.

SUMMARYLysine 9 di-methylation and lysine 27 tri-methylation of histone H3 (H3K9me2 and H3K27me3) are generally linked to gene repression. However, the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. We found that tumor necrosis factor (TNF)-α rapidly induces the co-occupancy of lysine demethylases 7A (KDM7A) and 6A (UTX) with nuclear factor kappa-B (NF-κB) recruited elements in human endothelial cells. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and both are required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods demonstrated increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A- and UTX-recruitment. Simultaneous inhibition of KDM7A and UTX significantly reduced leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of endothelial cells.HIGHLIGHTSKDM7A and UTX cooperatively control NF-κB-dependent transcription in vascular endothelial cells.Demethylation of repressive histone marks by KDM7A and UTX is critical for early inflammatory responses.KDM7A and UTX are associated with TNF-α-induced looping of super enhancers.Pharmacological inhibition of KDM7A and UTX reduces leukocyte adhesive interactions with endothelial cells in mice.

We have fully integrated public chromatin chromatin immunoprecipitation sequencing (ChIP‐seq) and DNase‐seq data (n > 70,000) derived from six representative model organisms (human, mouse, rat, fruit fly, nematode, and budding yeast), and have devised a data‐mining platform—designated ChIP‐Atlas (http://chip-atlas.org). ChIP‐Atlas is able to show alignment and peak‐call results for all public ChIP‐seq and DNase‐seq data archived in the NCBI Sequence Read Archive (SRA), which encompasses data derived from GEO, ArrayExpress, DDBJ, ENCODE, Roadmap Epigenomics, and the scientific literature. All peak‐call data are integrated to visualize multiple histone modifications and binding sites of transcriptional regulators (TRs) at given genomic loci. The integrated data can be further analyzed to show TR–gene and TR–TR interactions, as well as to examine enrichment of protein binding for given multiple genomic coordinates or gene names. ChIP‐Atlas is superior to other platforms in terms of data number and functionality for data mining across thousands of ChIP‐seq experiments, and it provides insight into gene regulatory networks and epigenetic mechanisms.

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.

Noncoding regions of the human genome possess enhancer activity and harbor risk loci for heritable diseases. Whereas the binding profiles of multiple transcription factors (TFs) have been investigated, integrative analysis with the large body of public data available so as to provide an overview of the function of such noncoding regions has remained a challenge. Here we have fully integrated public ChIP-seq and DNase-seq data (n ~ 70,000), including those for 743 human transcription factors (TFs) with 97 million binding sites, and have devised a data-mining platform —designated ChIP-Atlas—to identify significant TF-genome, TF-gene, and TF-TF interactions. Using this platform, we found that TFs enriched at macrophage or T-cell enhancers also accumulated around risk loci for autoimmune diseases, whereas those enriched at hepatocyte or macrophage enhancers were preferentially detected at loci associated with HDL-cholesterol levels. Of note, we identified “hotspots” around such risk loci that accumulated multiple TFs and are therefore candidates for causal variants. Integrative analysis of public chromatin-profiling data is thus able to identify TFs and tissues associated with heritable disorders.

Long non‐coding RNAs (lncRNAs) play important roles in many cellular pathways, but their contribution to the defense of eukaryotic cells against pathogens remains poorly understood. A new study from Imamura et al in The EMBO Journal reports that Salmonella infection in human cells impacts nuclear RNA decay, which in turn drives the accumulation of otherwise unstable nuclear lncRNAs, some of which may have protective effects against this common bacterial pathogen. These unexpected findings demand more efforts to fully decrypt the molecular functions of lncRNAs in innate and adaptive immunity.

Brown fat dissipates energy as heat and protects against obesity. Here, we identified nuclear factor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide open chromatin analysis of murine brown and white fat followed by motif analysis of brown-fat-specific open chromatin regions. NFIA and the master transcriptional regulator of adipogenesis, PPARγ, co-localize at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARγ, leading to increased chromatin accessibility and active transcription. Introduction of NFIA into myoblasts results in brown adipocyte differentiation. Conversely, the brown fat of NFIA knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. Finally, expression of NFIA and the brown-fat-specific genes is positively correlated in human brown fat. These results indicate that NFIA activates the cell-type-specific enhancers and facilitates the binding of PPARγ for controlling the brown fat gene program.

Chronic tubulointerstitial hypoxia plays an important role as the final common pathway to end‐stage renal disease. HIF‐1 (hypoxia‐inducible factor‐1) is a master transcriptional factor under hypoxia, regulating downstream target genes. Genome‐wide analysis of HIF‐1 binding sites using high‐throughput sequencers has clarified various kinds of downstream targets and made it possible to demonstrate the novel roles of HIF‐1. Our aim of this study is to identify novel HIF‐1 downstream epigenetic targets which may play important roles in the kidney. Immortalized tubular cell lines (HK2; human kidney‐2) and primary cultured cells (RPTEC; renal proximal tubular cell lines) were exposed to 1% hypoxia for 24–72 h. We performed RNA‐seq to clarify the expression of mRNA and long non‐coding RNA (lncRNA). We also examined ChIP‐seq to identify HIF‐1 binding sites under hypoxia. RNA‐seq identified 44 lncRNAs which are up‐regulated under hypoxic condition in both cells. ChIP‐seq analysis demonstrated that HIF‐1 also binds to the lncRNAs under hypoxia. The expression of novel lncRNA, DARS‐AS1 (aspartyl‐tRNA synthetase anti‐sense 1), is up‐regulated only under hypoxia and HIF‐1 binds to its promoter region, which includes two hypoxia‐responsive elements. Its expression is also up‐regulated with cobalt chloride exposure, while it is not under hypoxia when HIF‐1 is knocked down by siRNA. To clarify the biological roles of DARS‐AS1, we measured the activity of caspase 3/7 using anti‐sense oligo of DARS‐AS1. Knockdown of DARS‐AS1 deteriorated apoptotic cell death. In conclusion, we identified the novel lncRNAs regulated by HIF‐1 under hypoxia and clarified that DARS‐AS1 plays an important role in inhibiting apoptotic cell death in renal tubular cells.

Summary: Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification. : Kondo et al. find that extracellular acidic pH induces different cellular responses than hypoxia and nutrient starvation. SREBP2 is a key transcriptional regulator of cholesterol biosynthetic genes and ACSS2 in response to extracellular acidification. SREBP2 target genes increase tumor growth in low pH and correlate with decreased survival in patients. Keywords: epigenetics, extracellular low pH, sterol regulatory element-binding protein 2, acyl-CoA synthetase short-chain family member 2, cancer metabolism, tumor microenvironment, nutrient starvation, hypoxia, lacate

A novel ab initio parameter-tuning-free system to identify transcriptional factor (TF) binding motifs (TFBMs) in genome DNA sequences was developed. It is based on the comparison of two types of frequency distributions with respect to the TFBM candidates in the target DNA sequences and the non-candidates in the background sequence, with the latter generated by utilizing the intergenic sequences. For benchmark tests, we used DNA sequence datasets extracted by ChIP-on-chip and ChIP-seq techniques and identified 65 yeast and four mammalian TFBMs, with the latter including gaps. The accuracy of our system was compared with those of other available programs (i.e. MEME, Weeder, BioProspector, MDscan and DME) and was the best among them, even without tuning of the parameter set for each TFBM and pre-treatment/editing of the target DNA sequences. Moreover, with respect to some TFs for which the identified motifs are inconsistent with those in the references, our results were revealed to be correct, by comparing them with other existing experimental data. Thus, our identification system does not need any other biological information except for gene positions, and is also expected to be applicable to genome DNA sequences to identify unknown TFBMs as well as known ones.

Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions., Author summary Differentiated cells possess unique characteristics to maintain vital activities. However, cells occasionally show abnormal behavior in pathological settings due to dysregulated gene expression. Endothelial-to-mesenchymal transition (EndMT) is a phenomenon in which endothelial cells lose their characteristics and acquire mesenchymal-like properties. Although EndMT is observed in various diseases including cancer, and augments fibrosis and vascular defects, the mechanism of EndMT induction is not fully understood. Here, we show that EndMT is triggered via reduced expression of ERG and FLI1, which have recently been recognized as pivotal transcription factors in endothelial cells (ECs). Mechanistically, ERG and FLI1 activate EC-specific genes and repress mesenchymal-like genes via epigenetic regulation to prevent EndMT. Furthermore, we demonstrate that microRNA-126, which is specifically expressed in ECs, is the key downstream target of ERG/FLI1 for regulating EndMT. Finally, we show that ERG and FLI1 expression is decreased in ECs within tumors, suggesting that EndMT is induced in the tumor microenvironment. Collectively, these findings indicate that loss of ERG and FLI1 leads to the aberrant behavior of ECs in pathological conditions.

Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.

Finding of transcription factor binding motifs (TFBMs) is essential for identifying the detailed transcriptional regulation networks. In this study, we developed a novel algorithm for identifying the TFBMs by using the genome DNA sequence. We compared the accuracy of our algorithm with that of the conventional ones by exploiting ChIP-on-chip genome DNA sequence fragments, as test data, which were extracted by treatments to induce specific TFBMs of S. cerevisiae and human estrogen receptor (ER) expressed in human MCF-7 breast cancer cells. As a result of our calculation of the S. cerevisiae data, our algorithm identified 69 % known TFBMs from 65 datasets, each of which is corresponding to distinct TFBMs. In contrast, the conventional algorithms identified 63 % (BioProspector), 49 % (MDscan), and 52 % (MEME); here, the default parameter set was used for each identification system. Also, for ER elements (ERE), our algorithm exactly identified the position-specific scoring matrix (PSSM) using the human ER data, whereas the conventional algorithms did not. If single sequence repeats (SSLs) are removed from the ER data, BioProspector identified the PSSM of the human ERE. Accordingly, our system is expected to identify unknown TFBMs as well as known ones using genome DNA sequences without editing/tuning the parameter set used in our identification system. Further, identification of co-regulated known/unknown transcription factors is now going on by using experimental data obtained by DNA microarray techniques and the cap analysis gene expression (CAGE) systems.