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Association between active genes occurs at nuclear speckles and is modulated by chromatin environment.

Brown JM, Green J, das Neves RP, Wallace HA, Smith AJ, Hughes J, Gray N, Taylor S, Wood WG, Higgs DR, Iborra FJ, Buckle VJ - J. Cell Biol. (2008)

Bottom Line: Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear.Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies.Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, England, UK.

ABSTRACT
Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse alpha-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human alpha-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.

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Chromosomal environments differ between erythroid-specific genes. (a) Representative intermediate erythroblast nuclei after DNA-FISH of four 2-Mb BAC contigs. The nuclear periphery is outlined in red. The contig centered on the SLC4A1 gene on HSA17 shows marked decondensation of the chromatin region. The contig centered on the HBB gene on HSA11 does not exhibit the same levels of decondensation. The contig covering the mouse α-globin genes on MMU11 again does not display the levels of decondensation seen around SLC4A1. The contig covering the terminal 2 Mb of 16p13.3, surrounding the human α-globin genes, has been published previously (Brown et al., 2006) and is included here to demonstrate the marked difference in chromatin decondensation around the human and mouse α-globin genes. Note that the telomeric chromatin around HBA is more linearly extended than that around interstitial SLC4A1. (b) Maximum diameter measurements of BAC contig FISH signals in erythroblast and lymphoblast nuclei for SLC4A1, HBB, and mouse α-globin. These measurements minimize differences in signal size because they cannot take account of folding in the discontinuous SLC4A1 signal, yet there are significant differences between values for SLC4A1 and HBB and also for SLC4A1 and mouse α-globin. Values represent the mean ± SD (error bars) of measurements taken from one to two hybridizations for each contig probe set and cell type. Bar, 3 μm.
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fig2: Chromosomal environments differ between erythroid-specific genes. (a) Representative intermediate erythroblast nuclei after DNA-FISH of four 2-Mb BAC contigs. The nuclear periphery is outlined in red. The contig centered on the SLC4A1 gene on HSA17 shows marked decondensation of the chromatin region. The contig centered on the HBB gene on HSA11 does not exhibit the same levels of decondensation. The contig covering the mouse α-globin genes on MMU11 again does not display the levels of decondensation seen around SLC4A1. The contig covering the terminal 2 Mb of 16p13.3, surrounding the human α-globin genes, has been published previously (Brown et al., 2006) and is included here to demonstrate the marked difference in chromatin decondensation around the human and mouse α-globin genes. Note that the telomeric chromatin around HBA is more linearly extended than that around interstitial SLC4A1. (b) Maximum diameter measurements of BAC contig FISH signals in erythroblast and lymphoblast nuclei for SLC4A1, HBB, and mouse α-globin. These measurements minimize differences in signal size because they cannot take account of folding in the discontinuous SLC4A1 signal, yet there are significant differences between values for SLC4A1 and HBB and also for SLC4A1 and mouse α-globin. Values represent the mean ± SD (error bars) of measurements taken from one to two hybridizations for each contig probe set and cell type. Bar, 3 μm.

Mentions: We established bacterial artificial chromosome (BAC) probe pools covering 2 Mb centered around SLC4A1 on HSA17 and β-globin on HSA11 and examined the appearance of these contigs by FISH when hybridized to erythroblast and lymphoblast nuclei. The SLC4A1 region was decondensed in around two thirds of cases in both cell types and frequently appeared as an expanded cloud of signal (decondensed in erythroblasts, 62%, n = 105; decondensed in lymphoblasts, 71%, n = 50; Fig. 2 a). In contrast, the FISH signal for the β-globin region on HSA11 was most often condensed (decondensed in erythroblasts, 24%, n = 100; decondensed in lymphoblasts, 21%, n = 100; Fig. 2 a) and was never observed decondensed to the same degree as seen for the SLC4A1 region. To quantify the degree of decondensation, we measured the diameter of each contig signal at its widest point and found significant differences between values for SLC4A1 and β-globin in both erythroblasts (P = 2.51 × 10−29) and lymphoblasts (P = 9.87 × 10−26; n = 88/54 for SLC4A1 and n = 50/50 for β-globin in erythroblasts/lymphoblasts; Fig. 2 b).


Association between active genes occurs at nuclear speckles and is modulated by chromatin environment.

Brown JM, Green J, das Neves RP, Wallace HA, Smith AJ, Hughes J, Gray N, Taylor S, Wood WG, Higgs DR, Iborra FJ, Buckle VJ - J. Cell Biol. (2008)

Chromosomal environments differ between erythroid-specific genes. (a) Representative intermediate erythroblast nuclei after DNA-FISH of four 2-Mb BAC contigs. The nuclear periphery is outlined in red. The contig centered on the SLC4A1 gene on HSA17 shows marked decondensation of the chromatin region. The contig centered on the HBB gene on HSA11 does not exhibit the same levels of decondensation. The contig covering the mouse α-globin genes on MMU11 again does not display the levels of decondensation seen around SLC4A1. The contig covering the terminal 2 Mb of 16p13.3, surrounding the human α-globin genes, has been published previously (Brown et al., 2006) and is included here to demonstrate the marked difference in chromatin decondensation around the human and mouse α-globin genes. Note that the telomeric chromatin around HBA is more linearly extended than that around interstitial SLC4A1. (b) Maximum diameter measurements of BAC contig FISH signals in erythroblast and lymphoblast nuclei for SLC4A1, HBB, and mouse α-globin. These measurements minimize differences in signal size because they cannot take account of folding in the discontinuous SLC4A1 signal, yet there are significant differences between values for SLC4A1 and HBB and also for SLC4A1 and mouse α-globin. Values represent the mean ± SD (error bars) of measurements taken from one to two hybridizations for each contig probe set and cell type. Bar, 3 μm.
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fig2: Chromosomal environments differ between erythroid-specific genes. (a) Representative intermediate erythroblast nuclei after DNA-FISH of four 2-Mb BAC contigs. The nuclear periphery is outlined in red. The contig centered on the SLC4A1 gene on HSA17 shows marked decondensation of the chromatin region. The contig centered on the HBB gene on HSA11 does not exhibit the same levels of decondensation. The contig covering the mouse α-globin genes on MMU11 again does not display the levels of decondensation seen around SLC4A1. The contig covering the terminal 2 Mb of 16p13.3, surrounding the human α-globin genes, has been published previously (Brown et al., 2006) and is included here to demonstrate the marked difference in chromatin decondensation around the human and mouse α-globin genes. Note that the telomeric chromatin around HBA is more linearly extended than that around interstitial SLC4A1. (b) Maximum diameter measurements of BAC contig FISH signals in erythroblast and lymphoblast nuclei for SLC4A1, HBB, and mouse α-globin. These measurements minimize differences in signal size because they cannot take account of folding in the discontinuous SLC4A1 signal, yet there are significant differences between values for SLC4A1 and HBB and also for SLC4A1 and mouse α-globin. Values represent the mean ± SD (error bars) of measurements taken from one to two hybridizations for each contig probe set and cell type. Bar, 3 μm.
Mentions: We established bacterial artificial chromosome (BAC) probe pools covering 2 Mb centered around SLC4A1 on HSA17 and β-globin on HSA11 and examined the appearance of these contigs by FISH when hybridized to erythroblast and lymphoblast nuclei. The SLC4A1 region was decondensed in around two thirds of cases in both cell types and frequently appeared as an expanded cloud of signal (decondensed in erythroblasts, 62%, n = 105; decondensed in lymphoblasts, 71%, n = 50; Fig. 2 a). In contrast, the FISH signal for the β-globin region on HSA11 was most often condensed (decondensed in erythroblasts, 24%, n = 100; decondensed in lymphoblasts, 21%, n = 100; Fig. 2 a) and was never observed decondensed to the same degree as seen for the SLC4A1 region. To quantify the degree of decondensation, we measured the diameter of each contig signal at its widest point and found significant differences between values for SLC4A1 and β-globin in both erythroblasts (P = 2.51 × 10−29) and lymphoblasts (P = 9.87 × 10−26; n = 88/54 for SLC4A1 and n = 50/50 for β-globin in erythroblasts/lymphoblasts; Fig. 2 b).

Bottom Line: Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear.Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies.Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, England, UK.

ABSTRACT
Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse alpha-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human alpha-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.

Show MeSH
Related in: MedlinePlus