The polycomb repressive complex (PRC) 1 protein Ring1B is an ubiquitin ligase that modifies nucleosomal histone H2A, a modification which plays a critical role in regulation of gene expression. We have shown that self-ubiquitination of Ring1B generates multiply branched, "noncanonical" polyubiquitin chains that do not target the ligase for degradation, but rather stimulate its activity toward histone H2A. This finding implies that Ring1B is targeted by a heterologous E3. In this study, we identified E6-AP (E6-associated protein) as a ligase that targets Ring1B for "canonical" ubiquitination and subsequent degradation. We further demonstrated that both the self-ubiquitination of Ring1B and its modification by E6-AP target the same lysines, suggesting that the fate of Ring1B is tightly regulated (e.g., activation vs. degradation) by the type of chains and the ligase that catalyzes their formation. As expected, inactivation of E6-AP affects downstream effectors: Ring1B and ubiquitinated H2A levels are increased accompanied by repressed expression of HoxB9, a PRC1 target gene. Consistent with these findings, E6-AP knockout mice display an elevated level of Ring1B and ubiquitinated histone H2A in various tissues, including cerebellar Purkinje neurons, which may have implications to the pathogenesis of Angelman syndrome, a neurodevelopmental disorder caused by deficiency of E6-AP in the brain.polycomb complexes | ubiquitin-proteasome system

Imprinting of the PWS/AS 2.4 Mb domain in the human is controlled by a paternally active imprinting center (PWS-IC). PWS-IC on the maternal allele is methylated and inactivated by an 880-bp sequence (AS-IC) located 30 kb upstream. In this communication, we report the identification of 7 cis acting elements within AS-IC. The elements: DMR, DNS, 2 OCTA sequences, SOX, E1, and E2 bind specific proteins that form at least 2 protein complexes. Using variants of an imprinted transgene, mutated at the elements each at a time, we show that (i) all 7 elements are involved in the methylation and inactivation of the maternal PWS-IC; (ii) the OCTA and SOX elements that bind a protein complex, and the E1 and E2 elements, function in establishing the primary imprint that constitutes an active and unmethylated AS-IC in the oocyte; (iii) DNS and DMR bind a multiprotein complex that may facilitate interaction between AS-IC and PWS-IC, mediating the inactivation in cis of PWS-IC; and (iv) all 7 elements participate in maintaining an unmethylated PWS-IC in the oocyte, which is essential for its maternal methylation later in development. Altogether, the above observations imply that the cis acting elements on AS-IC display diverse functions in establishing the imprints at both AS-IC and PWS-IC in the oocyte. A postulated epigenetic mark imprints the PWS-IC in the oocyte and maintains its inactive status during development before it is translated into maternal methylation.cis elements ͉ DNA methylation ͉ imprinting ͉ protein factors

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Background: The multidrug resistance gene MDR1 is highly expressed in hematopoietic stem cells and may protect them from toxic insults, therefore alterations in MDR1 may predispose to acute myeloid leukemia (AML). Glutathione-S-transferase (GST) null alleles may also influence leukemia predisposition. We therefore studied patients (pts) with AML and controls of two ethnic groups for polymorphisms (SNPs) in these genes. Our patients are younger (mean, 46.1 yrs) than the mean of AML pts in the literature, Arab pts being significantly younger (41.4 yrs) than Jewish pts (48.7 yrs) (p<0.002). We asked whether genetic background predisposes to AML in these young pts. Methods: We studied 84 Jewish pts and 42 Arab pts with de novo AML and 109 Jewish controls and 91 Arab controls. We studied three SNPs in the MDR1 gene: T-129C (which may reduce gene expression), G2677T (leading to an ala893ser substitution, which increases duodenal MDR1 activity) and C3435T, which is a synonymous substitution. We also studied GST null alleles (GST T1 and M1). Polymorphisms were studied using PCR-based techniques. Survival was analyzed using Kaplan-Meier analysis. SNP genotype frequencies were analyzed using Chi Square and Student’s t-test. Results: G2677T was found to be associated with AML: the T allele (serine) was more frequent in Arab controls than in Arab pts. Thus, it seems to exert a protective effect against developing AML among Arabs (p<0.01). For both ethnic groups combined, the TT genotype at nt 2677 was associated with a significantly older age at diagnosis, compared to the GG/TG genotypes (mean 57 versus 46 yrs, p<0.03). In contrast, none of the other individual MDR1 polymorphisms were significantly associated with a protective effect in either ethnic group, nor were they found to be associated with any difference in age at diagnosis. Using the PHASE 2.1 program to create possible combinations of pairs of SNPs and all 3 SNPs combined for each ethnic group, we found a statistically significant different distribution of combinations between Arab AML pts and Arab controls for all possible combinations (p< 0.01–0.001), and between Arab and Jewish controls (p<0.01–0.001). In contrast, no such difference was found between Jewish AML pts and Jewish controls (p<0.2–1). Statistically significant differences between Arab and Jewish AML pts were found for 2 combinations: G2677T and C3435T together and all 3 SNPs combined (p<0.01 and p<0.025, respectively). Survival was not influenced by any of the genotype profiles of MDR1 SNPs in either ethnic group. For GST alleles, we did not find a difference in the frequency of either GST T1 null, GST M1 null or double null genotypes when comparing AML pts to controls of the same ethnic groups. Conclusions: We conclude that MDR1 polymorphisms may protect against de novo AML in Arabs, most significantly the 2677 polymorphic T allele. This effect was not found for Jews. The T allele of 2677 was also associated with an older age at diagnosis in both ethnic groups. GST null genotypes did not predispose to the development of de novo AML in the ethnic groups studied. Neither MDR1 nor GST polymorphisms influenced the outcome of AML in our patient population.