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Heterochromatin protein 1 (HP1) proteins do not drive pericentromeric cohesin enrichment in human cells.

Serrano A, Rodríguez-Corsino M, Losada A - PLoS ONE (2009)

Bottom Line: In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region.To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA.Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Dynamics Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

ABSTRACT
Sister chromatid cohesion mediated by cohesin is essential for accurate chromosome segregation. Classical studies suggest that heterochromatin promotes cohesion, but whether this happens through regulation of cohesin remains to be determined. Heterochromatin protein 1 (HP1) is a major component of heterochromatin. In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region. To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA. We have also studied the consequences of treating human cells with drugs that change the histone modification profile of heterochromatin and thereby affect HP1 localization. Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis. However, depletion of HP1gamma leads to defects in mitotic progression.

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Related in: MedlinePlus

Knock down of HP1 proteins by siRNA in HeLa cells.(A) Extracts made from HeLa cells transfected with siRNAs specific to HP1alpha (lane 5), HP1beta (lane 10) or HP1gamma (lane 15) were analyzed by immunoblotting. To estimate the extent of the depletion of the corresponding isoform, increasing amounts of a control cell extract were loaded on the same gel (lanes 1–4, 5–9 and 11–14). The levels of the chromatin remodeller ISWI were analyzed as a loading control. (B&C) HeLa cells transfected as in A were analyzed by immunofluorescence with the indicated antibodies (green) and counterstained with DAPI (blue). In B, cells were not pre-extracted before fixation. In C, cells were pre-extracted before fixation to detect only the chromatin-bound population. Representative examples of mitotic cells are shown. Scale bars: 50 micrometers in B and 5 micrometers in C.
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pone-0005118-g003: Knock down of HP1 proteins by siRNA in HeLa cells.(A) Extracts made from HeLa cells transfected with siRNAs specific to HP1alpha (lane 5), HP1beta (lane 10) or HP1gamma (lane 15) were analyzed by immunoblotting. To estimate the extent of the depletion of the corresponding isoform, increasing amounts of a control cell extract were loaded on the same gel (lanes 1–4, 5–9 and 11–14). The levels of the chromatin remodeller ISWI were analyzed as a loading control. (B&C) HeLa cells transfected as in A were analyzed by immunofluorescence with the indicated antibodies (green) and counterstained with DAPI (blue). In B, cells were not pre-extracted before fixation. In C, cells were pre-extracted before fixation to detect only the chromatin-bound population. Representative examples of mitotic cells are shown. Scale bars: 50 micrometers in B and 5 micrometers in C.

Mentions: siRNA oligonucleotides directed against each of the three HP1 isoforms were introduced separately in HeLa cells. Quantitative immunoblotting performed 120 hours after transfection showed that, in all cases, the siRNA treatment reduced the cellular levels of the corresponding isoform around 90% (Figure 3A). This reduction was confirmed by immunofluorescence (Figure 3B). Importantly, disappearance of the chromatin-bound population of HP1 proteins was observed also in mitotic cells (Figure 3C and Figure S2). This is particularly significant since it is at this region that cohesin accumulates in mitosis.


Heterochromatin protein 1 (HP1) proteins do not drive pericentromeric cohesin enrichment in human cells.

Serrano A, Rodríguez-Corsino M, Losada A - PLoS ONE (2009)

Knock down of HP1 proteins by siRNA in HeLa cells.(A) Extracts made from HeLa cells transfected with siRNAs specific to HP1alpha (lane 5), HP1beta (lane 10) or HP1gamma (lane 15) were analyzed by immunoblotting. To estimate the extent of the depletion of the corresponding isoform, increasing amounts of a control cell extract were loaded on the same gel (lanes 1–4, 5–9 and 11–14). The levels of the chromatin remodeller ISWI were analyzed as a loading control. (B&C) HeLa cells transfected as in A were analyzed by immunofluorescence with the indicated antibodies (green) and counterstained with DAPI (blue). In B, cells were not pre-extracted before fixation. In C, cells were pre-extracted before fixation to detect only the chromatin-bound population. Representative examples of mitotic cells are shown. Scale bars: 50 micrometers in B and 5 micrometers in C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662427&req=5

pone-0005118-g003: Knock down of HP1 proteins by siRNA in HeLa cells.(A) Extracts made from HeLa cells transfected with siRNAs specific to HP1alpha (lane 5), HP1beta (lane 10) or HP1gamma (lane 15) were analyzed by immunoblotting. To estimate the extent of the depletion of the corresponding isoform, increasing amounts of a control cell extract were loaded on the same gel (lanes 1–4, 5–9 and 11–14). The levels of the chromatin remodeller ISWI were analyzed as a loading control. (B&C) HeLa cells transfected as in A were analyzed by immunofluorescence with the indicated antibodies (green) and counterstained with DAPI (blue). In B, cells were not pre-extracted before fixation. In C, cells were pre-extracted before fixation to detect only the chromatin-bound population. Representative examples of mitotic cells are shown. Scale bars: 50 micrometers in B and 5 micrometers in C.
Mentions: siRNA oligonucleotides directed against each of the three HP1 isoforms were introduced separately in HeLa cells. Quantitative immunoblotting performed 120 hours after transfection showed that, in all cases, the siRNA treatment reduced the cellular levels of the corresponding isoform around 90% (Figure 3A). This reduction was confirmed by immunofluorescence (Figure 3B). Importantly, disappearance of the chromatin-bound population of HP1 proteins was observed also in mitotic cells (Figure 3C and Figure S2). This is particularly significant since it is at this region that cohesin accumulates in mitosis.

Bottom Line: In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region.To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA.Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Dynamics Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

ABSTRACT
Sister chromatid cohesion mediated by cohesin is essential for accurate chromosome segregation. Classical studies suggest that heterochromatin promotes cohesion, but whether this happens through regulation of cohesin remains to be determined. Heterochromatin protein 1 (HP1) is a major component of heterochromatin. In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region. To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA. We have also studied the consequences of treating human cells with drugs that change the histone modification profile of heterochromatin and thereby affect HP1 localization. Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis. However, depletion of HP1gamma leads to defects in mitotic progression.

Show MeSH
Related in: MedlinePlus