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. 2008 May 23;4(5):e1000079.
doi: 10.1371/journal.pgen.1000079.

Targeted deficiency of the transcriptional activator Hnf1alpha alters subnuclear positioning of its genomic targets

Reini F Luco  1 , Miguel A Maestro, Nicolas Sadoni, Daniele Zink, Jorge Ferrer
Affiliations

Targeted deficiency of the transcriptional activator Hnf1alpha alters subnuclear positioning of its genomic targets

Reini F Luco et al. PLoS Genet. .

Abstract

DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary beta-cells and hepatocytes freshly isolated from mice lacking Hnf1alpha, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a-/- cells inactive endogenous Hnf1alpha-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1alpha-targets in Hnf1a-/- cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Inactive Hnf1α targets in Hnf1a−/− cells exhibit local enrichment of H3-Lys27me3, decreased H3-Lys4me2, and reduced DNAse I sensitivity.
(A) RNA expression of tissue-specific Hnf1α targets (Afm, Cyp2j5, Pah, Kif12) and control genes (Tbp, Actb) in Hnf1a+/+ and Hnf1a−/− liver and islets. (B) ChIP analysis of Hnf1α occupancy in the promoter region of tissue-specific targets and a control gene (Nanog) in Hnf1a+/+ and Hnf1a−/− hepatocytes (Afm, Cyp2j5 and Pah, black and white bars respectively) and MIN6 beta-cells (Kif12, black bars). Results are normalized by Tbp enrichment. *p<0.05 and **p<0.01 relative to Nanog. (C–H) ChIP analysis of histone modifications in Hnf1α-targets and control genes in Hnf1a+/+ and Hnf1a−/− hepatocytes. For all genes the 5′ flanking regions are analyzed, except in D,G, where the entire Cyp2j5 locus is analyzed. Blue horizontal lines indicate amplicon positions, grey boxes are exons, red lines depict computationally predicted high-affinity HNF1 binding sites, and an arrow indicates the transcription start site. Graphs depict mean±SEM of the ratio of the percent input immunoprecipitated with anti-methyl specific H3 relative to anti-H3 antibodies in 3 independent experiments. Black bars represent Hnf1a+/+ and white bars Hnf1a−/− hepatocytes. *p<0.05 and **p<0.01 relative to Hnf1a+/+ hepatocytes. (I) General DNAse sensitivity of Cyp2j5. Representative PCR analysis of Cyp2j5 and Actb 5′ flanking and coding sequence regions after digestion of Hnf1a+/+ and Hnf1a−/− nuclei with increasing amounts of DNAse I. Results show reduced DNAse I sensitivity in Hnf1a−/− nuclei in the Cyp2j5 region, but not in the Actb control locus.
Figure 2
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.
Figure 3
Figure 3. Hnf1α-dependent Cyp2j5 activity correlates with differential positioning in RNA polymerase II and histone code domains.
(A–D) Representative confocal immuno-FISH analysis in Hnf1a+/+ and Hnf1a−/− hepatocytes of the Cyp2j5 locus (red) with RNA polymerase II (RNA Pol II, green) and either H3-Lys4me2 (A,B) or H3-Lys27me3 (C,D) (blue). The framed regions containing Cyp2j5 FISH signals are shown at higher magnification on the right of each panel with omission of blue or green channels (E–N) Quantitative analysis of histone marks and RNA polymerase II in Cyp2j5 (J–N) and Ly9 control (E–I) loci in Hnf1a+/+ and Hnf1a−/− hepatocytes. For each condition, non-thresholded fluorescence intensities of histone marks and RNA polymerase II were measured at 70–200 FISH signals, and each value was divided by its nuclear median intensity in the same channel. The graphs thus depict the average of such normalized signal values±SEM, except in I,N which shows mean±SEM of H3-Lys27me3/RNA polymerase II ratios (mK27/Pol II) calculated for each allele. (O–P) Classification of Cyp2j5 (P) and Ly9 control (O) alleles into 4 categories according to the simultaneous enrichment (+) or non-enrichment (−) of RNA polymerase II (RNA Pol II) and H3-Lys27me3 (K27me3) in Hnf1a+/+ (black bars) and Hnf1a−/− (white bars) hepatocytes. Each allele was scored as enriched (+) or non-enriched (−) based on whether or not the signal intensity exceeded the 75th percentile of nuclear signals. Alternate thresholds such as the nuclear median yielded comparably significant results (see text). Results are expressed as % of all alleles for each genotype. *p<0.05 and **p<0.01 relative to Hnf1a+/+ cells using Mann-Whitney or Fisher's exact test as appropriate.
Figure 4
Figure 4. Hnf1α determines the radial nuclear position of its target loci.
DNA-FISH erosion analysis of the radial positioning of the Ly9 control locus (A,C) and the Hnf1α-dependent loci Cyp2j5 (B) and Kif12 (D) in Hnf1a+/+ (black bars) and Hnf1a−/− (white bars) hepatocytes (A,B) and islet-cells (C,D). The nucleus was divided in 5 concentric zones and the percentage of FISH signals present in each zone was determined for each locus and genotype. The graph depicts the mean±SEM. *p<0.05 and **p<0.01 relative to Hnf1a+/+ cells.
Figure 5
Figure 5. Spatial resolution of Hnf1α-dependent repositioning.
(A) Two-color DNA FISH detection of adjacent loci Cyp2j5 (red) and 68H9 (green) in Hnf1a+/+ hepatocytes. (B) Schematic representation of the relative positions and distances (Kb) of BACs located centromeric (12L1 and 263F12) and telomeric (68H9 and 114C9) to Cyp2j5. Hnf1α-dependent genes in the region are drawn schematically in red. Note that no spliced transcript has been mapped to the region encompassed by BACs 68H9 and 114C9. (C–G) Classification of 12L1 (C), 263F12 (D), Cyp2j5 (E), 68H9 (F), and 114C9 (G) alleles into 4 categories according to the simultaneous enrichment (+) or non-enrichment (−) of RNA polymerase II (RNA Pol II) and H3-Lys27me3 (K27me3) in Hnf1a+/+ (black bars) and Hnf1a−/− (white bars) hepatocytes as described in Figure 3O–P. (H–L) Comparison of distances between the indicated BAC clone FISH signals in Hnf1a+/+ and Hnf1a−/− hepatocytes. For each comparison, the upper panels show the mean±SEM interlocus (signal center to center) distances in µm, with significance values calculated with the Mann-Whitney test. Lower panels show the percentage of non overlapping alleles, defined as those with signal center distances exceeding 0.4 µm, with significance values assessed with Fisher's exact test. *p<0.05, **p<0.01 relative to Hnf1a+/+ cells. More than 100 nuclei were analyzed in each case.
Figure 6
Figure 6. Summary model. Hnf1a−/−.
In the absence of Hnf1α, target nucleosomes exhibit increased trimethylated H3-Lys27, and are more likely to be located in peripheral nuclear domains with condensed chromatin enriched in methylated H3-Lys27. Hnf1a+/+: In wild-type cells, Hnf1α binding recruits complexes that lead to site-specific histone acetylation, H3-Lys4 methylation, and chromatin remodeling, while preventing trimethylation of H3-Lys27. This chromatin configuration is associated with relocalization to transcriptionally active, more centrally located nuclear subregions enriched in RNA polymerase II and H3-Lys4me2. We propose that activator-dependent local chromatin changes may be instrumental in gene positioning.
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References

    1. Kadonaga JT. Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors. Cell. 2004;116:247–257. - PubMed
    1. Kosak ST, Groudine M. Form follows function: the genomic organization of cellular differentiation. Genes Dev. 2004;18:1371–1384. - PubMed
    1. van Driel R, Fransz PF, Verschure PJ. The eukaryotic genome: a system regulated at different hierarchical levels. J Cell Sci. 2003;116:4067–4075. - PubMed
    1. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293:1074–1080. - PubMed
    1. Schubeler D, MacAlpine DM, Scalzo D, Wirbelauer C, Kooperberg C, et al. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 2004;18:1263–1271. - PMC - PubMed

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