Targeted Deficiency of the Transcriptional Activator Hnf1α Alters Subnuclear Positioning of Its Genomic Targets (original) (raw)
Figure 2
Methylated histone H3 marks exhibit a non-random subnuclear distribution.
(A) Dual immunofluorescence confocal analysis of domains enriched in H3-Lys4me2, H3-Lys9me3, and H3-Lys27me3 (red) compared with RNA polymerase II (green) in interphase wild-type hepatocyte nuclei. Adjacent Venn diagrams display percentages of colocalization (mean±SEM from 20 nuclei) of signals exceeding the 75th percentile of nuclear signal intensity in wild-type nuclei. (B) Triple immunofluorescence of RNA polymerase II (red), lamin A/C (green) and either H3-Lys4me2 or H3-Lys27me3 (blue) in hepatocytes. Insets below show peripheral nuclear segments at higher magnification. (C) Erosion analysis of the nuclear distribution of RNA polymerase II, H3-Lys4me2, or H3-Lys27me3. Nuclei were subdivided into 5 concentric zones, and total nuclear fluorescence intensities were determined for each epitope using non-thresholded images. The graphs indicate the percentages of total nuclear fluorescence intensities observed in each zone (mean±SEM). The values were normalized to the relative nuclear areas occupied by the different zones, so that a value of 1 was obtained if the percentage is as expected in case of unbiased distribution. At least 20 nuclei were analyzed in each case. Significance values for the comparison between the 5 zones for each of the 3 epitopes were obtained by ANOVA.