Sequencing of pediatric gliomas has identified missense mutations Lys27Met (K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B). We report that human diffuse intrinsic pontine gliomas (DIPGs) containing the K27M mutation display significantly lower overall amounts of H3 with trimethylated lysine 27 (H3K27me3) and that histone H3K27M transgenes are sufficient to reduce the amounts of H3K27me3 in vitro and in vivo. We find that H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-to-methionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. We propose that K-to-M substitutions may represent a mechanism to alter epigenetic states in a variety of pathologies.

Anorexia and weight loss are part of the wasting syndrome of late-stage cancer, are a major cause of morbidity and mortality in cancer, and are thought to be cytokine mediated. Macrophage inhibitory cytokine-1 (MIC-1) is produced by many cancers. Examination of sera from individuals with advanced prostate cancer showed a direct relationship between MIC-1 abundance and cancer-associated weight loss. In mice with xenografted prostate tumors, elevated MIC-1 levels were also associated with marked weight, fat and lean tissue loss that was mediated by decreased food intake and was reversed by administration of antibody to MIC-1. Additionally, normal mice given systemic MIC-1 and transgenic mice overexpressing MIC-1 showed hypophagia and reduced body weight. MIC-1 mediates its effects by central mechanisms that implicate the hypothalamic transforming growth factor-beta receptor II, extracellular signal-regulated kinases 1 and 2, signal transducer and activator of transcription-3, neuropeptide Y and pro-opiomelanocortin. Thus, MIC-1 is a newly defined central regulator of appetite and a potential target for the treatment of both cancer anorexia and weight loss, as well as of obesity.

OBJECTIVE: To investigate the development of high fat diet-induced obesity and leptin resistance. DESIGN: Two experiments were carried out in this study. Firstly, we fed the mice with a high-or low-fat diet for up to 19 weeks to examine a progressive development of high fat diet-induced obesity. Secondly, we examined peripheral and central exogenous leptin sensitivity in mice fed high-or low-fat diets for 1, 8 or 19 weeks. SUBJECTS: A total of 168 C57BLa6J mice (3 weeks old) were used in this study. MEASUREMENTS: In the ®rst experiment, we measured the body weight, energy intake, adipose tissue mass, tibia bone length, and plasma leptin in mice fed either a high-or low-fat diet for 1, 8, 15 and 19 weeks. In the second experiment, body weight change and cumulative energy intake were measured at 6 h intervals for 72 h after leptin injection in mice fed a high-or low-fat diet for 1, 8 or 19 weeks. RESULTS: The results from the ®rst experiment suggested that the development of high fat diet-induced obesity in mice could be divided into early, middle and late stages. Compared with the mice fed a low-fat diet, the mice fed a high-fat diet showed a gradually increased body weight ( 5.2%), fat storage (epididymal plus perirenal; 6.7%) and plasma leptin ( 18%) at 1 week; 11.4%, 68.1%, and 223%, respectively, at 8 weeks; and 30.5%, 141%, and 458%, respectively, at 19 weeks. Energy intake of high fat diet-fed mice was equal to that of low fat diet-fed controls for the ®rst 3 weeks; it fell below control levels over the next 5 week period, but began to increase gradually after 8 weeks of high-fat diet feeding and then increased dramatically from 15 weeks to be 14% higher than that of controls after 19 weeks. The results from our second experiment showed that: (1) after 1 week of feeding, the mice fed a highfat diet were sensitive to a 2 mgag (body weight) intraperitoneal (i.p.) injection of leptin, with no differences in body weight change or cumulative energy intake post-injection; (2) after 8 weeks of feeding, the mice fed a high-fat diet were insensitive to 2 mgag (body weight) i.p. leptin, but were sensitive to a 0.1 mg intracerebroventricular (i.c.v.) injection of leptin; (3) after 19 weeks of feeding, the mice fed a high-fat diet were insensitive to 0.1 mg i.c.v. leptin, but were sensitive to a high dose of 2 mg i.c.v. leptin. CONCLUSIONS: The present study demonstrated that the development of high fat diet-induced obesity (19 weeks) in C57 B1a6J mice could be divided into three stages: (1) an early stage in response to high-fat diet that mice were sensitive to exogenous leptin; (2) a reduced food intake stage when mice had an increase in leptin production and still retained central leptin sensitivity; and (3) an increased food intake stage, accompanied by a reduction of central leptin sensitivity.

Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at chromatin boundaries, play key roles in the structural and functional organization of chromosomes. We examined the binding of these two factors on the Kaposi's sarcoma-associated herpesvirus (KSHV) episome during latent infection and found a striking colocalization within the control region of the major latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF-binding site from the viral genome disrupted cohesin binding, and crippled colony formation in 293 cells. Clonal instability correlated with elevated expression of lytic cycle gene products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA depletion of RAD21 from latently infected cells caused an increase in K14 and ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian c-myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization.

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