Selective Anchoring of DNA Methyltransferases 3A and 3B to Nucleosomes Containing Methylated DNA

Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.Proper DNA methylation patterns are essential for mammalian development and differentiation. More than three decades ago, de novo cytosine DNA methylation and its maintenance were proposed to exist in eukaryotic cells (29, 54); however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. DNA methyltransferases (DNMTs) DNMT1, DNMT3A, and DNMT3B primarily establish and maintain global DNA methylation patterns (39, 48). DNMT1 preferentially methylates hemimethylated DNA in vitro (7) and is tethered to replication foci during S phase (38). In contrast, DNMT3A and DNMT3B (DNMT3A/3B) have no preference for hemimethylated DNA (49) and are required for de novo methylation of genomic DNA (48). It has been thought that DNMT1 acts mainly as a “maintenance methyltransferase” during DNA synthesis and that DNMT3A and DNMT3B act as “de novo” enzymes. However, more recent studies indicate that DNMT1 may also be required for de novo methylation of genomic DNA (17, 30) and that DNMT3A/3B are also required for maintenance functions (11, 40, 55). Furthermore, the different DNMTs cooperate in maintaining the methylation of some regions of the genome, particularly repetitive elements (40, 53).Recruitment of individual DNMT enzymes to different regions of chromatin in vivo, particularly to gene regulatory regions, may require interaction with auxiliary factors (28, 36). DNMT1, which is diffusely localized throughout nuclei in non-S-phase cells (38), is targeted to replication foci by interacting with proliferating cell nuclear antigen (PCNA) (15) and also physically interacts with UHRF1 (ubiquitinlike, containing PHD and RING finger domains 1) that binds to hemimethylated DNA (3, 4, 8, 27, 62). DNMT3 enzymes are usually found localized to heterochromatin regions in most transient-expression assays (5, 12). As genomic DNA in chromatin is packaged into nucleosomes which might limit the accessibility of target sites to the enzymes, the interaction of DNMTs with nucleosomes in a chromatin context is important for the regulation of genomic methylation.Genetic and biochemical studies have provided many insights into the distinct and cooperative functions of the DNMT enzymes; however, few of these studies have addressed how they interact with chromatin in vivo. Recombinant DNMT1 and DNMT3 enzymes can methylate the CpG sites on nucleosomes assembled in vitro (26, 50, 56, 65). Recently DNMT3L has been found to connect DNMT3A2 to nucleosomes in embryonic stem cells (52). However, DNMT3L is expressed only during gametogenesis and embryonic stages (1, 9), suggesting that other mechanisms might be necessary for directing the enzyme to specific chromatin regions in somatic cells.In the present study, we investigated how different DNMT enzymes interact with chromatin at the nucleosomal level in somatic cell lines. Micrococcal nuclease (MNase) treatment of nuclei in a low-ionic-strength buffer digests nucleosomal linker DNA regions, thereby minimizing the disruption of protein complexes on the nucleosomes. We prepared nucleosomes from partial or maximum MNase-digested nuclei and resolved them on sucrose density gradients to analyze their interactions with chromatin proteins. The results indicate that while DNMT1 interacts primarily with linker DNA, DNMT3A/3B enzymes interact strongly with nucleosomes containing methylated repetitive elements and also containing methylated CpG islands (CGIs) and may not require additional proteins for this strong binding. These data are particularly intriguing in that they provide insights into the mechanisms of the interaction of DNMTs with chromatin and maintenance of DNA methylation in somatic cells.     

Gene markers for grain polyphenol oxidase activity in common wheat

Polyphenol oxidase (PPO) in grain is regarded as a major factor resulting in time-dependent darkening of wheat end products, particularly for Asian noodles and steamed bread. Breeding wheat cultivars with low PPO activity using efficient and reliable markers is one of the best ways to reduce the undesirable darkening. In the present study, we developed a gene-specific marker (PPO05) for low PPO activity from the sequence AY515506. This marker detected double PCR fragments (<750 and >750 bp) in the cultivars with low PPO activity and a single PCR fragment (<750 bp) in the cultivars with high PPO activity. Screening of this marker on 235 Chinese wheat micro-core collections showed that the double fragments were present in 113 genotypes and the single fragments in the remaining 122 genotypes. Statistic analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with single fragments. Through sequence analysis and blast search in NCBI, we found that the cultivars with the double fragments contained the PPO-2Ab allele, while the cultivars with the single fragments contained the PPO-2Aa allele. The PPO-2Ab and PPO-2Da alleles were associated with the low grain PPO activity and the PPO-2Aa and PPO-2Db alleles associated with the high PPO activity. The genotypes carrying both PPO-2Ab and PPO-2Da showed the lowest PPO activity, while the genotypes carrying both PPO-2Aa and PPO-2Db showed the highest PPO activity. Comparison of PPO05 and STS01 with the STS markers PPO18 and PPO29 showed that the larger and small fragments of PPO05 were equivalent to the 876- and 685-bp fragments of PPO18, respectively, and that STS01 was the complementary marker of PPO29. Thus, the STS markers PPO05 and STS01 along with PPO18 and PPO29 are the efficient and reliable markers for the evaluation of PPO activity and can be used in wheat breeding programs to improve the quality of noodles and other end products.     

一株珊瑚礁-海草床复合生态系统固氮菌的分离与鉴定

三亚珊瑚礁自然保护区部分珊瑚礁退化后逐渐演替为以泰来藻(Thalassia hemprichii)为优势种的海草床群落,采用选择性无氮培养基从泰来藻植株的根际,分离得到一株固氮菌,编号为G33-1,经形态学、生理生化鉴定和16SrDNA及固氮基因nifH的序列分析,初步鉴定为成团泛菌(Pantoea agglomerans)。该菌为革兰氏阴性菌,以周生鞭毛运动,呈直杆状,菌落圆形,半透明乳白色,比较湿润,有光泽,直径约1mm,低凸,光滑,边缘比较整齐。最适培养条件为:氯化钠浓度25‰,生长温度为37°C,起始pH值为8。与成团泛菌标准菌株(ATCC27155TM)相比较,在碳源利用、精氨酸双水解、苯丙氨酸脱胺酶、鸟氨酸脱胺酶、以及生长温度和盐度等方面都具有较高的相似性,以16SrDNA为基础构建的系统进化树分析结果,表明其与成团泛菌属Pantoeaag-glomeransWAB1870进化距离最近,相似性大于99%。此外,利用乙炔还原法对固氮活性进行测定,其具有较高的固氮活性,达299.16nmolC2H2/(mL.h)。     

Detection of Helicobacteraceae in intestinal biopsies of children with Crohn's disease

Background: Given that members of Helicobacteraceae family colonize the intestinal mucus layer, it has been hypothesized that they may play a role in Crohn’s disease. This study investigated the presence of Helicobacteraceae DNA in biopsies collected from children with Crohn’s disease and controls. Materials and Methods: The presence of Helicobacteraceae DNA was investigated in intestinal biopsies collected from 179 children undergoing colonoscopy (Crohn’s disease n = 77, controls n = 102) using a Helicobacteraceae‐specific PCR. Results: Members of the Helicobacteraceae were detected in 32/77 children with Crohn’s disease (41.5%) and 23/102 controls (22.5%). Statistical analysis showed the prevalence of Helicobacteraceae detected in patients to be significantly higher than that in controls (p = .0062). Analysis of non‐pylori Helicobacteraceae showed that their prevalence was also significantly higher in patients than in controls (p = .04). Helicobacter pylori was detected in 14.0% of the biopsies across all groups. Given that all children tested were negative for gastric H. pylori, this was a surprising finding. Phylogenetic analysis of H. pylori sequences detected in the biopsies showed that the H. pylori strains identified in the patients did not group with gastric H. pylori included in the analysis, but rather with other H. pylori strains detected in the intestine, gall bladder, and liver. Conclusions:  The higher prevalence of Helicobacteraceae DNA in Crohn’s disease patients would suggest that members of this family may be involved in this disease. In addition, phylogenetic analysis of H. pylori strains showed that extragastric sequences clustered together, indicating that different H. pylori strains may adapt to colonize extragastric niches.     

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus

Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the 'LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG(-/-)) cell line was used. Infection of UNG(-/-) and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG(-/-) cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.     

Artificial reproduction of the Yangtze field vole: in vitro embryo development and fertilization with fresh and freeze-thawed sperm

Several research groups are using the Yangtze field vole as a model for studying schistosome infection, but relatively little is known about the species's reproductive physiology. The authors examined the vole's in vivo and in vitro embryonic development as well as the efficacy of in vitro fertilization using either fresh or cryopreserved sperm to breed these rodents.