Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases.

In mammals, three major DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. Dnmt3-like (Dnmt3l), which is a member of the Dnmt3 family but does not possess DNA methylation activity, was reported to be indispensable for global methylation in germ cells. Once the DNA methylation patterns are established, maintenance-type DNA methyltransferase Dnmt1 faithfully propagates them to the next generation via replication. All Dnmts possess multiple domains. For instance, Dnmt3a and Dnmt3b each contain a Pro-Trp-Trp-Pro (PWWP) domain that recognizes the histone H3K36me2/3 mark, an Atrx-Dnmt3-Dnmt3l (ADD) domain that recognizes unmodified histone H3 tail, and a catalytic domain that methylates CpG sites. Dnmt1 contains an N-terminal independently folded domain (NTD) that interacts with a variety of regulatory factors, a replication foci-targeting sequence (RFTS) domain that recognizes the histone H3K9me3 mark and H3 ubiquitylation, a CXXC domain that recognizes unmodified CpG DNA, two tandem Bromo-Adjacent-homology (BAH1 and BAH2) domains that read the H4K20me3 mark with BAH1, and a catalytic domain that preferentially methylates hemimethylated CpG sites. In this chapter, the structures and functions of these domains are described.

Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Nucleosome compaction facilitates HP1γ binding to methylated H3K9.

The α, ß and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1's chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg(2+) or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.

Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases.

In mammals, three DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. Dnmt3-like (Dnmt3l), which is a member of the Dnmt3 family but does not possess DNA methylation activity, was reported to be indispensable for global methylation in germ cells. Once the DNA methylation patterns are established, maintenance-type DNA methyltransferase Dnmt1 faithfully propagates them to the next generation via replication. All Dnmts possess multiple domains, and in this chapter, the structures and functions of these domains are described.

Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4.

The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine.

Epigenetics and regeneration.

During newt lens regeneration a unique transdifferentiation event occurs. In this process, dorsal iris pigmented epithelial cells transdifferentiate into lens cells. This system should provide a new insight into cellular plasticity in basic and applied research. Recently, a series of approaches to study epigenetic reprogramming during transdifferentiation have been performed. In this review, we introduce the regulation of dynamic regulation of core-histone modifications and the emergence of an oocyte-type linker histone during transdifferentiation. Finally, we show supporting evidence that there are common strategies of reprogramming between newt somatic cell in transdifferentiation and oocytes after somatic cell nuclear transfer.

Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity.

BACKGROUND: The human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch. METHODS: First, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous ß-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed. RESULTS: We found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo. CONCLUSION: The HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

[A case of 5-year survival with combination chemotherapy for advanced colonic cancer with multiple lung and liver metastases and peritoneal dissemination].

A 74-year-old man was admitted to the hospital because of abdominal fullness and constipation. Endoscopy and abdominal computed tomography (CT) scans showed an advanced sigmoid colonic cancer with multiple lung and liver metastases and peritoneal dissemination. Sigmoid resection was performed to relieve ileus. Postoperative chemotherapy was administered for metastatic lesions. We administered systemic chemotherapy with tegafur-uraci(l UFT)/UZEL( 10 months), oxaliplatin with fluorouracil (5-FU) and folinic acid (FOLFOX; 11 courses), irinotecan with 5-FU and folinic acid (FOLFIRI; 13 courses), irinotecan plus S-1 (IRIS; 23 courses), bevacizumab (32 courses), cetuximab (29 courses), and peritoneal infusion of paclitaxel( 65 courses). The patient survived for 5 years after the initial treatment.

A case of acute cholecystitis with abnormally high CA19-9.

BACKGROUND: CA19-9 is strongly expressed in malignant tumors of the digestive system and is widely used as a marker for gastrointestinal cancer. In this report, we describe a case of acute cholecystitis in which CA19-9 was markedly elevated. CASE PRESENTATION: A 53-year-old man was admitted to our hospital with a diagnosis of acute cholecystitis after being referred to our hospital with a chief complaint of fever and right hypochondrial pain. CA19-9 was abnormally high at 17,539.1 U/ml. Although the possibility of malignancy was considered, there was no obvious malignant lesion on imaging; the patient was diagnosed with cholecystitis, and laparoscopic cholecystectomy was performed the day after admission. The surgical specimen showed no malignant findings either grossly or in the final pathological examination. There were no complications in the patient's postoperative course, and he was discharged from the hospital on the third postoperative day. CA19-9 level quickly returned to within normal range after surgery. CONCLUSIONS: In acute cholecystitis, CA19-9 levels exceeding 10,000 U/ml are very rare. We report a case of acute cholecystitis without malignant findings despite a high CA19-9 level.

Enhanced survival of hypoimmunogenic otic progenitors following intracochlear xenotransplantation: repercussions for stem cell therapy in hearing loss models.

Stem cell replacement holds the potential for sensorineural hearing loss (SNHL) treatment. However, its translation into clinical practice requires strategies for improving stem cell survival following intracochlear transplantation. Considering recent findings showing that the inner ear contains a resident population of immune cells, we hypothesized that immune evasion would improve the survival and residence time of transplanted stem cells in the cochlea, potentially leading to better outcomes. To test this, we leveraged genetic engineering techniques to develop hypoimmunogenic human-induced pluripotent stem cells (hi-iPSC), which lack human leukocyte antigen expression. We found that gene editing does not affect the biological properties of hi-iPSCs, including their capacity to differentiate into otic neural progenitors (ONPs). Compared to wild-type ONPs, more hypoimmunogenic ONPs (derived from hi-iPSCs) were found in the inner ear of immunocompetent mice ten days following cochlear xenotransplantation. This approach may open a new avenue for experimental and clinical SNHL treatments.

Down-regulation of the tumor suppressor C-terminal Src kinase (Csk)-binding protein (Cbp)/PAG1 is mediated by epigenetic histone modifications via the mitogen-activated protein kinase (MAPK)/phosphatidylinositol 3-kinase (PI3K) pathway.

The transmembrane adaptor protein Cbp (or PAG1) functions as a suppressor of Src-mediated tumor progression by promoting the inactivation of Src. The expression of Cbp is down-regulated in Src-transformed cells and in various human cancer cells, suggesting a potential role for Cbp as a tumor suppressor. However, the mechanisms underlying the down-regulation of Cbp remain unknown. The present study shows that Cbp expression is down-regulated by epigenetic histone modifications via the MAPK/PI3K pathway. In mouse embryonic fibroblasts, transformation by oncogenic Src and Ras induced a marked down-regulation of Cbp expression. The levels of Cbp expression were inversely correlated with the activity of MEK and Akt, and Cbp down-regulation was suppressed by inhibiting MEK and PI3K. Src transformation did not affect the stability of Cbp mRNA, the transcriptional activity of the cbp promoter, or the DNA methylation status of the cbp promoter CpG islands. However, Cbp expression was restored by treatment with histone deacetylase (HDAC) inhibitors and by siRNA-mediated knockdown of HDAC1/2. Src transformation significantly decreased the acetylation levels of histone H4 and increased the trimethylation levels of histone H3 lysine 27 in the cbp promoter. EGF-induced Cbp down-regulation was also suppressed by inhibiting MEK and HDAC. Furthermore, the inhibition of MEK or HDAC restored Cbp expression in human cancer cells harboring Cbp down-regulation through promoter hypomethylation. These findings suggest that Cbp down-regulation is primarily mediated by epigenetic histone modifications via oncogenic MAPK/PI3K pathways in a subset of cancer cells.

Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a.

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues 1 to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys51, Lys53, Arg177 and Arg179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1-211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.

Safety and efficacy of bidirectional chemotherapy for treatment of patients with peritoneal dissemination from gastric cancer: Selection for cytoreductive surgery.

There is no standard treatment for peritoneal carcinomatosis (PC) from gastric cancer. New bidirectional chemotherapy (neoadjuvant intraperitoneal-systemic chemotherapy protocol (NIPS)) was developed. The aim of the present study was to assess the safety and efficacy of NIPS and to show the selection for cytoreductive surgery on PC from gastric cancer. Seventy-nine patients with PC from gastric cancer were treated with NIPS. A peritoneal port system was introduced into the abdominal cavity. The peritoneal wash cytological examination through a port was done before and after NIPS. The patients were treated with oral TS-1 twice a daily for 21 days, followed by a 1-week rest. On day 1, 8, and 15 from the start of oral TS-1 administration, 30 mg/m(2) of Docetaxel and 30 mg/m(2) of cisplatinum with 500 ml of saline were introduced into the peritoneal cavity through the port. A median course of oral TS-1 was 2.1 course and a median time of IP chemoterapy was 5.8. Peritoneal free cancer cells (PFCCs) had been detected in 65 (82.2%) patients before NIPS, and the positive cytology changed to be negative in 41 (63.0%) patients after NIPS. After NIPS, 41 patients underwent laparotomy, and complete cytoreduction was done in 32 (78%) patients. Complete cytoreduction was done in 27 (51.9%) of 52 patients with negative cytology but in only 4 (14.8%) of 27 patients with positive cytology (P < 0.001). Patients with negative cytology after NIPS survived significantly longer than those with positive cytology. The adverse effects after NIPS were mild and there was no treatment-related deaths. The grade 3/4 hematological adverse effects were found in 2 (2.6%) patients. Grade 3 renal toxicity and port site infection was found in three patients, respectively. NIPS using a port system is a safe and effective treatment for PC. Peritoneal wash cytology through a port system is a good indicator to select the patients to perform cytoreductive surgery.

[A case of effective treatment with UFT/LV chemotherapy for para-aortic lymph node metastasis from colonic cancer 8 years after colectomy].

A 77-year-old woman was admitted to our hospital because of para-aortic lymph node swelling pointed out by abdominal CT scan. She had a previous history of colectomy with a diagnosis of ascending colonic cancer 8 years later. Pathological examination was a moderately-differentiated adenocarcinoma with lymph node metastasis (Stage IIIa). PET scan demonstrated hot spots of para-aortic and left supra-clavicular lesions. The serum CEA and CA19-9 levels regained the normal value. No malignancy was recognized by endoscopic examinations of upper and lower gastrointestinal tract. We supported that swelling of lymph nodes were due to lymphoma. Laparotomic biopsy of para-aortic lymph node was done after 6 months. Histologically metastatic adenocarcinoma was recognized. We performed systemic chemotherapy of UFT and LV on recurrent colonic cancer. After 9 courses, spots of para-aortic and left supra-clavicular lesions disappeared on the PET scan. Lymph node metastasis was not found by CT scan 20 months after beginning chemotherapy. Thus, we consider that this therapy was recommendable for the treatment of an older adult patient with recurrent colonic cancer.

[A resected case of effective treatment with S-1/gemcitabine and Paclitaxel combination chemotherapy for advanced pancreatic cancer with peritoneal and liver metastases].

A 58-year-old woman was admitted to the hospital because of abdominal fullness and anorexia. Abdominal CT scan revealed pancreatic cancer with massive ascites showing peritoneal dissemination, combined with lymph node and liver metastases. Cytology of ascites showed malignancy. Abnormally high values of CA19-9 (1,908 U/mL) and CA125 (545 U/mL) were detected in serum. Histologically, metastatic adenocarcinoma was recognized by laparoscopic biopsy of peritoneal dissemination. We performed systemic chemotherapy of S-1, gemcitabine and peritoneal infusion of paclitaxel for nonresected pancreatic cancer. After 5 courses, ascites disappeared and the serum CA19-9 and CA125 levels regained their normal value. Peritoneal seeding was not found by second laparoscopic examination 20 months after beginning chemotherapy. Thus, we consider the patient had an effective response, and performed distal pancreatectomy and proximal resection of the stomach. Histological examination of the primary lesion revealed no cancer cells where fibrosis presented.

Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression.

The pluripotential cell-specific gene Nanog encodes a homeodomain-bearing transcription factor required for maintaining the undifferentiated state of stem cells. However, the molecular mechanisms that regulate Nanog gene expression are largely unknown. To address this important issue, we used luciferase assays to monitor the relative activities of deletion fragments from the 5'-flanking region of the gene. An adjacent pair of highly conserved Octamer- and Sox-binding sites was found to be essential for activating pluripotential state-specific gene expression. Furthermore, the 5'-end fragment encompassing the Octamer/Sox element was sufficient for inducing the proper expression of a green fluorescent protein reporter gene even in human embryonic stem (ES) cells. The potential of OCT4 and SOX2 to bind to this element was verified by electrophoretic mobility shift assays with extracts from F9 embryonal carcinoma cells and embryonic germ cells derived from embryonic day 12.5 embryos. However, in ES cell extracts, a complex of OCT4 with an undefined factor preferentially bound to the Octamer/Sox element. Thus, Nanog transcription may be regulated through an interaction between Oct4 and Sox2 or a novel pluripotential cell-specific Sox element-binding factor which is prominent in ES cells.

Histone code modifications on pluripotential nuclei of reprogrammed somatic cells.

Following hybridization with embryonic stem (ES) cells, somatic genomes are epigenetically reprogrammed and acquire pluripotency. This results in the transcription of somatic genome-derived tissue-specific genes upon differentiation. During nuclear reprogramming, it is expected that DNA and chromatin modifications, believed to function in cell-type-specific epigenotype memory, should be significantly modified. Indeed, current evidence indicates that acetylation and methylation of histone H3 and H4 amino termini play a major role in the regulation of gene activity through the modulation of chromatin conformation. Here, we show that the reprogrammed somatic genome of ES hybrid cells becomes hyperacetylated at H3 and H4, while lysine 4 (K4) of H3 becomes globally hyper-di- and -tri-methylated. In the Oct4 promoter region, histones H3 and H4 are acetylated and H3-K4 is highly tri-methylated on both the ES and reprogrammed somatic genomes, which correlates with gene activation and DNA demethylation. However, H3-K4 is also di- and tri-methylated in the promoter regions of Neurofilament-M (Nfm), Nfl, and Thy-1, which are all silent in both ES and hybrid cells. Thus, H3-K4 di- and tri-methylation of reprogrammed somatic genomes is independent of gene activity and represents one of the major events that occurs during somatic genome reprogramming towards a transcriptional activation-permissive state.

Pluripotential competence of cells associated with Nanog activity.

Nanog is a novel pluripotential cell-specific gene that plays a crucial role in maintaining the undifferentiated state of early postimplantation embryos and embryonic stem (ES) cells. We have explored the expression pattern and function of Nanog and a Nanog-homologue, Nanog-ps1.Nanog-ps1 was mapped on Chromosome 7 and shown to be a pseudogene. Immunocytochemical analysis in vivo showed that the NANOG protein was absent in unfertilized oocytes, and was detected in cells of morula-stage embryos, the inner cell mass of blastocysts and the epiblast of E6.5 and E7.5 embryos, but not in primordial germ cells of early postimplantation embryos. In monkey and human ES cells, NANOG expression was restricted to undifferentiated cells. Furthermore, reactivation of the somatic cell-derived Nanog was tightly linked with nuclear reprogramming induced by cell hybridization with ES cells and by nuclear transplantation into enucleated oocytes. Notably, mouse Nanog (+/-) ES cells, which produced approximately half the amount of NANOG produced by wild-type ES cells, readily differentiated to multi-lineage cells in culture medium including LIF. The labile undifferentiated state was fully rescued by constitutive expression of exogenous Nanog. Thus, the activity of Nanog is tightly correlated with an undifferentiated state of cells even in nuclear reprogrammed somatic cells. Nanog may function as a key regulator for sustaining pluripotency in a dose-dependent manner.

[A case of nonresected gastric cancer with peritoneal dissemination maintained on TS-1, cisplatin (CDDP) and docetaxel combination chemotherapy with good QOL].

A 64-year-old woman was admitted to the hospital for abdominal fullness and constipation. In the pelvic cavity, an abdominal CT scan revealed massive ascites showing malignancy on histological examination. Upper GI endoscopy revealed type 3 gastric cancer from the anglus to the cardia. A barium-enema showed a stenotic lesion at the sigmoid colon due to peritoneal dissemination. An abnormally high CA125(1,400 mg/ml) level was detected in serum. We performed systemic chemotherapy of TS-1, CDDP and peritoneal infusion of docetaxel on the nonresected gastric cancer with peritoneal dissemination. After 2 cycles, cytology of ascites revealed no malignancy, and the serum CA125 value regained its normal level. After 3 cycles, the killer cell effect was recognized by laparoscopic examination and the stenotic change of sigmoid colon had almost disappeared. The patient clinically achieved good QOL by this method, which was very effective for nonresected gastric cancer with peritoneal dissemination.

Nanog expression in mouse germ cell development.

Nanog is a newly identified transcriptional factor bearing a homeodomain and expressed in pluripotential cells of preimplantation and early postimplantation embryos, and embryonic stem (ES) and embryonic germ (EG) cells. Knockout experiments indicate that Nanog functions as a key player in maintaining the pluripotency of stem cells. Importantly, Nanog expression is highly expressed in primordial germ cells (PGCs) of E11.5 and E12.5 mouse embryos. However, its temporal and spatial expression pattern and function in germ cells are largely unknown. To address these issues, whole embryos and cryosections of embryos were immunostained with anti-NANOG and anti-STELLA/PGC7 antibodies. NANOG expression, repressed in colonized PGCs of E7.25-E7.5 embryos, became detectable in migrating PGCs of E7.75-E8.0 embryos. Both male and female PGCs migrating in E9.5 and E10.5 embryos and colonizing the genital ridges of E11.5 and E12.5 embryos were positive for NANOG immunostaining, while the NANOG expression pattern differed between the sexes in the later developmental stage. In female gonadal PGCs of E13.5 and E14.5 embryos, NANOG became undetectable in germ cells positive for the synaptonemal complex-specific protein SCP3, while in male PGCs of E14.5-E16.5 embryos, the number of NANOG-positive germ cells drastically decreased during the mitotic arrest. No germ cells positive for NANOG were detectable in testes and ovaries of adult mice. Thus, in germ cell development, NANOG is expressed in proliferating germ cells, in which nuclear reprogramming is progressing.