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

  • ️Fri Jan 01 2010

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

Joke G van Bemmel et al. PLoS One. 2010.

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Lamina associated domains in the Drosophila genome.

(A) Genome – NL interaction maps in Drosophila Kc cells and human Lung Fibroblasts along a 4 Mb region at respectively chromosome 2L and chromosome 18. Human data are from . Y-axes depict the log2 transformed Dam-LAM over Dam-only methylation ratio, smoothed by a running median of respectively 15 and 5 probes. Rectangles below each map represent calculated LAD positions for Drosophila (red) and human (blue). Grey rectangles at the bottom represent genes at the + and - strand. (B) Distribution of LAD sizes in Drosophila (red) and human cells (blue). Dashed lines mark the median LAD sizes. (C) Histogram of the number of genes per LAD. (D–F) Profiles across aligned LAD borders (824 borders, left and mirrored right borders combined). Running window median (red line) and a random subset of 2001 single genes (black dots in E and F). The region around each border from which data was taken ranges from the center of the inter-LAD region to the center of the LAD; this ensures that each data point is used only once. X-axis depicts the position relative to the nearest LAD border; positive coordinates inside, negative coordinates outside LADs. (D) Median gene coverage. (E) mRNA levels in A-values, (log2(Cy5)+log2(Cy3))/2 (F) Median RpII18 occupancy on entire genes as determined by DamID [data from30].

Figure 2
Figure 2. SU(HW) sites are enriched at LAD borders and within LADs.

(A) Binding maps of insulator proteins along a 1 Mb region on chromosome 2L. Y-axes depict the Dam-insulator over Dam-only methylation ratio (high values are truncated at 10). Grey rectangles represent LADs. (B) Theoretical profiles of features that are respectively enriched at LAD borders, enriched inside LADs or depleted from LADs (upper panels). Profiles of insulator and EcR binding peaks across aligned LAD borders. 824 borders, left and mirrored right borders combined (lower panels). The region around each border from which data was taken ranges from the center of the inter-LAD region to the center of the LAD; this ensures that each data point is used only once. X-axis depicts the position relative to the nearest LAD border; positive coordinates inside LADs and negative coordinates outside LADs. Y-axes depict the median number of binding peaks within a running window of 10 kb. Y-axes are scaled to frequencies within the plotted window, depending on the genome-wide frequency of the feature. (C) Percentage of insulator binding peaks within LADs (top) and percentage of inter-LAD peaks within a 10 kb region just outside LADs (bottom). Black horizontal lines represent the percentage expected by chance. **significantly enriched or *depleted compared to random permutation simulations; p<10−3. (D) Model of the chromatin organization in LADs, with SU(HW) binding mainly at LAD borders and inside LADs; and DWG, BEAF-32, GAF and CTCF preferentially located in inter-LAD regions.

Figure 3
Figure 3. SU(HW) distribution relative to LADs is sequence driven and linked to CP190 binding.

(A) Profiles of sequence motifs across aligned LAD borders. X-axis depicts the position relative to the nearest LAD border; positive coordinates inside LADs and negative coordinates outside LADs. Colored lines show the median frequency within a running window of 10 kb for sequence motifs, grey lines for DamID identified peaks. (B) Profiles of CP190 peaks across aligned LAD borders; all Cp190 peaks (1st panel), CP190 peaks without SU(HW) binding, defined as peaks with an average log2(SU(HW)binding ratio) <1.0 (2nd panel), CP190 peaks with SU(HW) binding, defined as CP190 peaks with an average log2(SU(HW)binding ratio) >1.5 (3rd panel).

Figure 4
Figure 4. 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).

Figure 5
Figure 5. SU(HW) is a local antagonist of genome – NL interactions.

(A–B) Median NL interaction (log2 Dam-LAM/Dam ratio) across LADs without SU(HW) peaks (190 borders, 1st panel), LADs with at least one SU(HW) peak (634 borders, 2nd panel, red triangle represents the presence of one or more SU(HW) peaks at any position inside LADs), the 25% of LADs with the highest SU(HW) peak density (206 borders, 3rd panel, red triangles represent high density of SU(HW) peaks). (A) After knockdown of SU(HW) (blue line), after control knockdown (grey line). (B) After overexpression of SU(HW) (orange line), after control overexpression (grey line). (C) Ave changes in NL interaction levels per LAD, for LADs without SU(HW) (grey), LADs with at least one SU(HW) peak (light blue or orange), the 25% of LADs with the highest SU(HW) peak density (dark blue or orange) after knockdown (blue, 1st panel) and overexpression of SU(HW) (orange, 2nd panel). * Wilcoxon test; p<10−3 (D) Median changes in NL interaction across aligned SU(HW) peaks (red triangle) inside LADs (bright lines), in border regions (pale lines) and outside LADs (grey lines) after knockdown of SU(HW) (blue, 1st panel) and after overexpression of SU(HW) (orange, 2nd panel). (E) Cis-spreading of SU(HW) DamID signals from aligned SU(HW) binding peaks (red triangle), inside LADs (red line) and outside LADs (grey line).

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