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The insulator protein SU(HW) fine-tunes nuclear lamina interactions of the Drosophila genome.

van Bemmel JG, Pagie L, Braunschweig U, Brugman W, Meuleman W, Kerkhoven RM, van Steensel B - PLoS ONE (2010)

Bottom Line: Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression.By knockdown and overexpression studies we demonstrate that SU(HW) weakens genome - NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation.Our results provide insights into the evolution of LAD organization and identify SU(HW) as a fine-tuner of genome - NL interactions.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs) in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that the Drosophila genome is also organized in discrete LADs, which are about five times smaller than human LADs but contain on average a similar number of genes. Systematic comparison to new and published insulator binding maps shows that only SU(HW) binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW) weakens genome - NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation. Our results provide insights into the evolution of LAD organization and identify SU(HW) as a fine-tuner of genome - NL interactions.

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Related in: MedlinePlus

SU(HW) alone is not essential for demarcation of LAD borders.Genome - NL interaction maps after knockdown and overexpression of SU(HW). (A) Western blot analysis of SU(HW) expression levels after knockdown (ctrl: control RNAi; kd1 and kd2: SU(HW) RNAi with two independent dsRNA fragments) and after overexpression (ctrl: control vector oe: overexression by transfection of SU(HW) under an Act5C promoter). 1st lane in each panel: transfected with Dam-LAM, 2nd lane with Dam-only. (B–C) Median NL interaction (log2 Dam-LAM/Dam ratio) across all aligned LAD borders (824 borders, 1st panel); border regions with SU(HW) present (220 borders, 2nd panel, red triangle represents SU(HW) at the borders), borders without SUH(HW) present (604 borders, 3rd panel). (B) Knockdown of SU(HW) (blue line) and control knockdown (grey line). (C) Overexpression of SU(HW) (orange line), and corresponding control (grey line).
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pone-0015013-g004: SU(HW) alone is not essential for demarcation of LAD borders.Genome - NL interaction maps after knockdown and overexpression of SU(HW). (A) Western blot analysis of SU(HW) expression levels after knockdown (ctrl: control RNAi; kd1 and kd2: SU(HW) RNAi with two independent dsRNA fragments) and after overexpression (ctrl: control vector oe: overexression by transfection of SU(HW) under an Act5C promoter). 1st lane in each panel: transfected with Dam-LAM, 2nd lane with Dam-only. (B–C) Median NL interaction (log2 Dam-LAM/Dam ratio) across all aligned LAD borders (824 borders, 1st panel); border regions with SU(HW) present (220 borders, 2nd panel, red triangle represents SU(HW) at the borders), borders without SUH(HW) present (604 borders, 3rd panel). (B) Knockdown of SU(HW) (blue line) and control knockdown (grey line). (C) Overexpression of SU(HW) (orange line), and corresponding control (grey line).

Mentions: For RNAi we used two different, non-overlapping, double-stranded RNA (dsRNA) fragments to exclude off-target effects. Treatment with a dsRNA fragment derived from the unrelated white gene served as a control. Western blot analysis showed that both su(Hw) dsRNA fragments caused efficient knockdown of the SU(HW) protein (Fig. 4a, 1st panel). Knockdown of SU(HW) had no effect on the doubling time of the cells (data not shown), ruling out secondary effects of an altered cell cycle on the DamID pattern. Elevated levels of SU(HW) were obtained by co-transfection of the DamID plasmids with a vector that drives expression of SU(HW) from an Act5C promoter. Western blot analysis showed only a slight increase in expression of SU(HW), presumably because only a minority of cells is transfected (Fig. 4a, 2nd panel). However, because the overexpression vector is co-transfected with the DamID vector, overexpression may be expected to be more prominent in cells that express Dam-LAM. We generated DamID maps of NL association for each treatment in two independent experiments.


The insulator protein SU(HW) fine-tunes nuclear lamina interactions of the Drosophila genome.

van Bemmel JG, Pagie L, Braunschweig U, Brugman W, Meuleman W, Kerkhoven RM, van Steensel B - PLoS ONE (2010)

SU(HW) alone is not essential for demarcation of LAD borders.Genome - NL interaction maps after knockdown and overexpression of SU(HW). (A) Western blot analysis of SU(HW) expression levels after knockdown (ctrl: control RNAi; kd1 and kd2: SU(HW) RNAi with two independent dsRNA fragments) and after overexpression (ctrl: control vector oe: overexression by transfection of SU(HW) under an Act5C promoter). 1st lane in each panel: transfected with Dam-LAM, 2nd lane with Dam-only. (B–C) Median NL interaction (log2 Dam-LAM/Dam ratio) across all aligned LAD borders (824 borders, 1st panel); border regions with SU(HW) present (220 borders, 2nd panel, red triangle represents SU(HW) at the borders), borders without SUH(HW) present (604 borders, 3rd panel). (B) Knockdown of SU(HW) (blue line) and control knockdown (grey line). (C) Overexpression of SU(HW) (orange line), and corresponding control (grey line).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991331&req=5

pone-0015013-g004: SU(HW) alone is not essential for demarcation of LAD borders.Genome - NL interaction maps after knockdown and overexpression of SU(HW). (A) Western blot analysis of SU(HW) expression levels after knockdown (ctrl: control RNAi; kd1 and kd2: SU(HW) RNAi with two independent dsRNA fragments) and after overexpression (ctrl: control vector oe: overexression by transfection of SU(HW) under an Act5C promoter). 1st lane in each panel: transfected with Dam-LAM, 2nd lane with Dam-only. (B–C) Median NL interaction (log2 Dam-LAM/Dam ratio) across all aligned LAD borders (824 borders, 1st panel); border regions with SU(HW) present (220 borders, 2nd panel, red triangle represents SU(HW) at the borders), borders without SUH(HW) present (604 borders, 3rd panel). (B) Knockdown of SU(HW) (blue line) and control knockdown (grey line). (C) Overexpression of SU(HW) (orange line), and corresponding control (grey line).
Mentions: For RNAi we used two different, non-overlapping, double-stranded RNA (dsRNA) fragments to exclude off-target effects. Treatment with a dsRNA fragment derived from the unrelated white gene served as a control. Western blot analysis showed that both su(Hw) dsRNA fragments caused efficient knockdown of the SU(HW) protein (Fig. 4a, 1st panel). Knockdown of SU(HW) had no effect on the doubling time of the cells (data not shown), ruling out secondary effects of an altered cell cycle on the DamID pattern. Elevated levels of SU(HW) were obtained by co-transfection of the DamID plasmids with a vector that drives expression of SU(HW) from an Act5C promoter. Western blot analysis showed only a slight increase in expression of SU(HW), presumably because only a minority of cells is transfected (Fig. 4a, 2nd panel). However, because the overexpression vector is co-transfected with the DamID vector, overexpression may be expected to be more prominent in cells that express Dam-LAM. We generated DamID maps of NL association for each treatment in two independent experiments.

Bottom Line: Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression.By knockdown and overexpression studies we demonstrate that SU(HW) weakens genome - NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation.Our results provide insights into the evolution of LAD organization and identify SU(HW) as a fine-tuner of genome - NL interactions.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs) in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that the Drosophila genome is also organized in discrete LADs, which are about five times smaller than human LADs but contain on average a similar number of genes. Systematic comparison to new and published insulator binding maps shows that only SU(HW) binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW) weakens genome - NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation. Our results provide insights into the evolution of LAD organization and identify SU(HW) as a fine-tuner of genome - NL interactions.

Show MeSH
Related in: MedlinePlus