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Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth.

Sancho M, Diani E, Beato M, Jordan A - PLoS Genet. (2008)

Bottom Line: Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down.Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing.On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type.

View Article: PubMed Central - PubMed

Affiliation: Centre de Regulació Genòmica, Barcelona, Spain.

ABSTRACT
At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants.

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Inhibition of H1 variants causes different effects on cell proliferation and cell cycle progression.(A) T47D cells infected with the inducible shRNA-expression system against H1.2, H1.4 or empty plasmid were treated for 6 days with Dox or left untreated and observed at the optical microscopy. (B) In order to measure the effect of H1 depletion on cell proliferation, each H1 variant knock-down cell line (RFP and GFP-positive) was mixed 1∶1 with parental T47D cells (RFP and GFP-negative) and treated with Dox. Every three days, cells were split and the percentage of RFP/GFP-positive cells measured by FACS. Data is expressed as ratio of RFP/GFP-positive (KD) versus RFP/GFP-negative (wild-type) cells along time. Data corresponds to a representative experiment performed in duplicate. (C) Cell cycle profile after propidium iodide (PI) staining of H1 variant knock-down cell lines grown for six days in the presence or absence of Dox. Data is expressed as percentage of cells in G1, S and G2/M cell cycle phases. The values represent the mean and SD of a representative experiment performed in triplicate. (D) H1.4 knocked-down and control cells grown in the presence of Dox for 6 and 10 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death. (E) H1.2 KD cells grown for six days in the presence or absence of Dox and without serum for the last two days, were treated with serum and the cell cycle profile was analyzed at time points indicated. (F) Cell cycle profile of H1.2 KD cells in response to treatment with different inhibitors. Cells were treated or not with Dox for 6 days. Hydroxyurea (HU) or methyl methanosulfonate (MMS) were added at the indicated concentrations 12 h before analysis of the cell cycle profile by FACS measurement of PI-stained cells.
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pgen-1000227-g002: Inhibition of H1 variants causes different effects on cell proliferation and cell cycle progression.(A) T47D cells infected with the inducible shRNA-expression system against H1.2, H1.4 or empty plasmid were treated for 6 days with Dox or left untreated and observed at the optical microscopy. (B) In order to measure the effect of H1 depletion on cell proliferation, each H1 variant knock-down cell line (RFP and GFP-positive) was mixed 1∶1 with parental T47D cells (RFP and GFP-negative) and treated with Dox. Every three days, cells were split and the percentage of RFP/GFP-positive cells measured by FACS. Data is expressed as ratio of RFP/GFP-positive (KD) versus RFP/GFP-negative (wild-type) cells along time. Data corresponds to a representative experiment performed in duplicate. (C) Cell cycle profile after propidium iodide (PI) staining of H1 variant knock-down cell lines grown for six days in the presence or absence of Dox. Data is expressed as percentage of cells in G1, S and G2/M cell cycle phases. The values represent the mean and SD of a representative experiment performed in triplicate. (D) H1.4 knocked-down and control cells grown in the presence of Dox for 6 and 10 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death. (E) H1.2 KD cells grown for six days in the presence or absence of Dox and without serum for the last two days, were treated with serum and the cell cycle profile was analyzed at time points indicated. (F) Cell cycle profile of H1.2 KD cells in response to treatment with different inhibitors. Cells were treated or not with Dox for 6 days. Hydroxyurea (HU) or methyl methanosulfonate (MMS) were added at the indicated concentrations 12 h before analysis of the cell cycle profile by FACS measurement of PI-stained cells.

Mentions: Upon Dox treatment, we observed differences in growth rate among H1 variant knock-downs, in particular, H1.2 and H1.4 depleted cells failed to reach confluency (Figure 2A) and exhibited a slower growth rate. To quantify this we mixed, at a 1∶1 ratio, each of the shRNA expressing cell lines (RedFP and GFP-positive upon Dox treatment) with parental T47D cells (RedFP and GFP-negative), and we monitored by FACS the proportion between the two populations over time in culture in the presence of Dox (Figure 2B). Slow progression of H1.4 knocked-down cells was seen at six days after Dox addition and of H1.2 depleted cells at day 9. At day 12, H1.5 depleted cells were also less abundant than the parental ones. In addition, at day 6, H1.4 cells had changed morphology towards a necrotic phenotype (Figure 2A and Figure S2).


Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth.

Sancho M, Diani E, Beato M, Jordan A - PLoS Genet. (2008)

Inhibition of H1 variants causes different effects on cell proliferation and cell cycle progression.(A) T47D cells infected with the inducible shRNA-expression system against H1.2, H1.4 or empty plasmid were treated for 6 days with Dox or left untreated and observed at the optical microscopy. (B) In order to measure the effect of H1 depletion on cell proliferation, each H1 variant knock-down cell line (RFP and GFP-positive) was mixed 1∶1 with parental T47D cells (RFP and GFP-negative) and treated with Dox. Every three days, cells were split and the percentage of RFP/GFP-positive cells measured by FACS. Data is expressed as ratio of RFP/GFP-positive (KD) versus RFP/GFP-negative (wild-type) cells along time. Data corresponds to a representative experiment performed in duplicate. (C) Cell cycle profile after propidium iodide (PI) staining of H1 variant knock-down cell lines grown for six days in the presence or absence of Dox. Data is expressed as percentage of cells in G1, S and G2/M cell cycle phases. The values represent the mean and SD of a representative experiment performed in triplicate. (D) H1.4 knocked-down and control cells grown in the presence of Dox for 6 and 10 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death. (E) H1.2 KD cells grown for six days in the presence or absence of Dox and without serum for the last two days, were treated with serum and the cell cycle profile was analyzed at time points indicated. (F) Cell cycle profile of H1.2 KD cells in response to treatment with different inhibitors. Cells were treated or not with Dox for 6 days. Hydroxyurea (HU) or methyl methanosulfonate (MMS) were added at the indicated concentrations 12 h before analysis of the cell cycle profile by FACS measurement of PI-stained cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2563032&req=5

pgen-1000227-g002: Inhibition of H1 variants causes different effects on cell proliferation and cell cycle progression.(A) T47D cells infected with the inducible shRNA-expression system against H1.2, H1.4 or empty plasmid were treated for 6 days with Dox or left untreated and observed at the optical microscopy. (B) In order to measure the effect of H1 depletion on cell proliferation, each H1 variant knock-down cell line (RFP and GFP-positive) was mixed 1∶1 with parental T47D cells (RFP and GFP-negative) and treated with Dox. Every three days, cells were split and the percentage of RFP/GFP-positive cells measured by FACS. Data is expressed as ratio of RFP/GFP-positive (KD) versus RFP/GFP-negative (wild-type) cells along time. Data corresponds to a representative experiment performed in duplicate. (C) Cell cycle profile after propidium iodide (PI) staining of H1 variant knock-down cell lines grown for six days in the presence or absence of Dox. Data is expressed as percentage of cells in G1, S and G2/M cell cycle phases. The values represent the mean and SD of a representative experiment performed in triplicate. (D) H1.4 knocked-down and control cells grown in the presence of Dox for 6 and 10 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death. (E) H1.2 KD cells grown for six days in the presence or absence of Dox and without serum for the last two days, were treated with serum and the cell cycle profile was analyzed at time points indicated. (F) Cell cycle profile of H1.2 KD cells in response to treatment with different inhibitors. Cells were treated or not with Dox for 6 days. Hydroxyurea (HU) or methyl methanosulfonate (MMS) were added at the indicated concentrations 12 h before analysis of the cell cycle profile by FACS measurement of PI-stained cells.
Mentions: Upon Dox treatment, we observed differences in growth rate among H1 variant knock-downs, in particular, H1.2 and H1.4 depleted cells failed to reach confluency (Figure 2A) and exhibited a slower growth rate. To quantify this we mixed, at a 1∶1 ratio, each of the shRNA expressing cell lines (RedFP and GFP-positive upon Dox treatment) with parental T47D cells (RedFP and GFP-negative), and we monitored by FACS the proportion between the two populations over time in culture in the presence of Dox (Figure 2B). Slow progression of H1.4 knocked-down cells was seen at six days after Dox addition and of H1.2 depleted cells at day 9. At day 12, H1.5 depleted cells were also less abundant than the parental ones. In addition, at day 6, H1.4 cells had changed morphology towards a necrotic phenotype (Figure 2A and Figure S2).

Bottom Line: Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down.Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing.On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type.

View Article: PubMed Central - PubMed

Affiliation: Centre de Regulació Genòmica, Barcelona, Spain.

ABSTRACT
At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants.

Show MeSH
Related in: MedlinePlus