Limits...
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.

Show MeSH

Related in: MedlinePlus

H1.2 knock-down causes a reduction in nucleosome spacing.(A) Nuclei from H1 variant knock-down cells treated or not with Dox for 6 days were treated with MNase and the profile of bulk chromatin was analyzed in gel electrophoresis to calculate the nucleosome repeat length (NRL). (B) Plot of nucleosome number versus DNA length for MNase-digested bulk chromatin of H1.2 knocked-down cells ±Dox. This plot has been used to calculate the corresponding NRL. Data from 5 independent experiments has been pooled. Average±SD is shown. Data from untreated and treated samples differed significantly in a t-Student test (95% confidence interval with a p value of 0.0001). Open triangles denote untreated, and solid square symbols correspond to cells treated for 6 days with Dox. (C) Complementation of reduced nucleosome spacing in H1.2 knocked-down cells by stable expression of shRNA-resistant H1.2 (rH1.2HA).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2563032&req=5

pgen-1000227-g005: H1.2 knock-down causes a reduction in nucleosome spacing.(A) Nuclei from H1 variant knock-down cells treated or not with Dox for 6 days were treated with MNase and the profile of bulk chromatin was analyzed in gel electrophoresis to calculate the nucleosome repeat length (NRL). (B) Plot of nucleosome number versus DNA length for MNase-digested bulk chromatin of H1.2 knocked-down cells ±Dox. This plot has been used to calculate the corresponding NRL. Data from 5 independent experiments has been pooled. Average±SD is shown. Data from untreated and treated samples differed significantly in a t-Student test (95% confidence interval with a p value of 0.0001). Open triangles denote untreated, and solid square symbols correspond to cells treated for 6 days with Dox. (C) Complementation of reduced nucleosome spacing in H1.2 knocked-down cells by stable expression of shRNA-resistant H1.2 (rH1.2HA).

Mentions: Reduced H1 content (down to 50%) in triple-H1 mouse embryonic stem (ES) cells leads to reduced nucleosome spacing (nucleosome repeat length), as measured after micrococcal nuclease (MNase) digestion of nuclear bulk chromatin [12]. We investigated nucleosome spacing of chromatin from the different H1 variant T47D knock-downs by MNase digestion of isolated nuclei (Figure 5A). H1.2 depletion, but not depletion of other H1 variants, caused a striking reduction in the spacing between nucleosomes. The calculated nucleosome repeat length (NRL) was decreased from approximately 184.7 to 173.5 upon H1.2 depletion (Figure 5B, obtained from five independent experiments). This was surprising as H1.2 only represents approximately 23% of the total H1 content in T47D cells (Figure S1). As H1.2 depletion reached 90–95% and compensatory increases in the expression of other H1 variants were not detected, we estimate that total H1 content was reduced by approximately 20%. This was confirmed by Coomassie staining of H1 preparations from H1.2 KD cells treated or not with Dox (Figure S1). Stable expression of HA-tagged, shRNA-resistant H1.2 in the H1.2 KD cell line reverted the reduction in nucleosome spacing caused by endogenous H1.2 depletion (Figure 5C).


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)

H1.2 knock-down causes a reduction in nucleosome spacing.(A) Nuclei from H1 variant knock-down cells treated or not with Dox for 6 days were treated with MNase and the profile of bulk chromatin was analyzed in gel electrophoresis to calculate the nucleosome repeat length (NRL). (B) Plot of nucleosome number versus DNA length for MNase-digested bulk chromatin of H1.2 knocked-down cells ±Dox. This plot has been used to calculate the corresponding NRL. Data from 5 independent experiments has been pooled. Average±SD is shown. Data from untreated and treated samples differed significantly in a t-Student test (95% confidence interval with a p value of 0.0001). Open triangles denote untreated, and solid square symbols correspond to cells treated for 6 days with Dox. (C) Complementation of reduced nucleosome spacing in H1.2 knocked-down cells by stable expression of shRNA-resistant H1.2 (rH1.2HA).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2563032&req=5

pgen-1000227-g005: H1.2 knock-down causes a reduction in nucleosome spacing.(A) Nuclei from H1 variant knock-down cells treated or not with Dox for 6 days were treated with MNase and the profile of bulk chromatin was analyzed in gel electrophoresis to calculate the nucleosome repeat length (NRL). (B) Plot of nucleosome number versus DNA length for MNase-digested bulk chromatin of H1.2 knocked-down cells ±Dox. This plot has been used to calculate the corresponding NRL. Data from 5 independent experiments has been pooled. Average±SD is shown. Data from untreated and treated samples differed significantly in a t-Student test (95% confidence interval with a p value of 0.0001). Open triangles denote untreated, and solid square symbols correspond to cells treated for 6 days with Dox. (C) Complementation of reduced nucleosome spacing in H1.2 knocked-down cells by stable expression of shRNA-resistant H1.2 (rH1.2HA).
Mentions: Reduced H1 content (down to 50%) in triple-H1 mouse embryonic stem (ES) cells leads to reduced nucleosome spacing (nucleosome repeat length), as measured after micrococcal nuclease (MNase) digestion of nuclear bulk chromatin [12]. We investigated nucleosome spacing of chromatin from the different H1 variant T47D knock-downs by MNase digestion of isolated nuclei (Figure 5A). H1.2 depletion, but not depletion of other H1 variants, caused a striking reduction in the spacing between nucleosomes. The calculated nucleosome repeat length (NRL) was decreased from approximately 184.7 to 173.5 upon H1.2 depletion (Figure 5B, obtained from five independent experiments). This was surprising as H1.2 only represents approximately 23% of the total H1 content in T47D cells (Figure S1). As H1.2 depletion reached 90–95% and compensatory increases in the expression of other H1 variants were not detected, we estimate that total H1 content was reduced by approximately 20%. This was confirmed by Coomassie staining of H1 preparations from H1.2 KD cells treated or not with Dox (Figure S1). Stable expression of HA-tagged, shRNA-resistant H1.2 in the H1.2 KD cell line reverted the reduction in nucleosome spacing caused by endogenous H1.2 depletion (Figure 5C).

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