TI - Different Isoforms of Methylated Histone H3 Are Distributed Non-Randomly in Nuclear Space . AB - To explore possible relationships between Hnf1alpha -dependent gene activity , site -specific histone modifications and nuclear organization , we first assessed subnuclear distributions of histone modifications in primary hepatocytes and pancreatic islet cells . Both of these cell types are largely quiescent under normal conditions . The results showed that H3-Lys4me2-rich subnuclear regions displayed a high degree of colocalization with regions that are enriched in RNA polymerase II PHOSphorylated on serine 5 of the C terminal repeat , the predominant polymerase form in the transcriptional initiation complex [42] ( hereafter referred to as RNA polymerase II ) ( Figure 2A ) . In sharp contrast , gene -silencing marks H3-Lys9me3 and Lys27me3 were more abundant in regions that were not enriched in RNA polymerase II (Figure 2A) . These subnuclear distributions were independent of the fixative and processing methods used , and were observed with different H3-Lys27me3 antibodies ( Figure S1A , B ) . Furthermore , the H3-Lys27me3 immunostaining pattern was distinct from that of Histone H3 and other modifications including H3-Lys4me2 , H3-Lys27me1 , H3-Lys27me2 , H3-Lys9me3 , as well as the DNA stain TO-PRO-3 , indicating that it does not merely reflect chromatin density ( Figure S1C-F , Figure S2 , and not shown ) . We next examined the radial distribution of histone modifications . H3-Lys27me3 was markedly enriched whereas H3-Lys4me2 displayed relative depletion in the immediate vicinity of the inner nuclear membrane , as shown by co-immunostaining of Lamin A/C (Figure 2B) . Erosion analyses using non-thresholded images furthermore revealed markedly different radial enrichment patterns for RNA polymerase II , H3-Lys4me2 and H3-Lys27me3 (Figure 2C) . Thus , RNA polymerase II and H3-Lys4me2 were significantly depleted in peripheral nuclear zones compared to more interior nuclear regions ( Figure 2C , ANOVA p values 54x10-40 and 89x10-23 ) . In contrast , H3-Lys27me3 was significantly enriched in the outermost zones , compared to more internal regions ( Figure 2C , ANOVA p value 87x10-18 ) . These results are largely consistent with recent studies describing distinct nuclear patterns of histone modifications in cultured cell lines [43] , but extend it by showing that H3-Lys4me2 exhibits preferential colocalization with RNA polymerase II in central nuclear domains , while H3-Lys27me3 is particularly abundant in peripheral domains lacking enrichment in RNA polymerase II , H3-Lys4me2 or H3-Lys9me3 .