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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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Cell cycle gene alterations in H1.2 knock-down cells.(A) Complementation of altered expression of cell cycle-related genes in H1.2 knocked-down cells by stable expression of recombinant HA-tagged, shRNA-resistant H1.2. Expression of the cell cycle genes indicated was measured by RT-qPCR with specific oligonucelotides in parental H1.2 KD and H1.2sh/rH1.2HA cells treated or not with Dox for 6 days. GAPDH expression was measured for normalization. Data is expressed as relative units specific gene/GAPDH. The values represent the mean and SD of a representative experiment performed in triplicate. (B) Changes in the accumulation or phosphorylation of cell cycle-related proteins upon inhibition of H1.2. Expression of the indicated proteins was measured by Western blot with specific antibodies in cells treated or not with Dox for 6 days. H1.2 knocked-down cells were compared to parental T47D or control cells. Tubulin antibody was used as loading control. (C) CDK2 activity in H1.2 depleted cells. CDK2 was immunoprecipitated with the specific antibody from cellular extracts of Dox-treated or untreated cells (H1.2 KD or control) and incubated with histone H1 and γ-ATP. Products were resolved in SDS-PAGE and detected by autoradiography. Immunoprecipitated material was measured by immunoblotting with the anti CDK2 antibody.
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pgen-1000227-g009: Cell cycle gene alterations in H1.2 knock-down cells.(A) Complementation of altered expression of cell cycle-related genes in H1.2 knocked-down cells by stable expression of recombinant HA-tagged, shRNA-resistant H1.2. Expression of the cell cycle genes indicated was measured by RT-qPCR with specific oligonucelotides in parental H1.2 KD and H1.2sh/rH1.2HA cells treated or not with Dox for 6 days. GAPDH expression was measured for normalization. Data is expressed as relative units specific gene/GAPDH. The values represent the mean and SD of a representative experiment performed in triplicate. (B) Changes in the accumulation or phosphorylation of cell cycle-related proteins upon inhibition of H1.2. Expression of the indicated proteins was measured by Western blot with specific antibodies in cells treated or not with Dox for 6 days. H1.2 knocked-down cells were compared to parental T47D or control cells. Tubulin antibody was used as loading control. (C) CDK2 activity in H1.2 depleted cells. CDK2 was immunoprecipitated with the specific antibody from cellular extracts of Dox-treated or untreated cells (H1.2 KD or control) and incubated with histone H1 and γ-ATP. Products were resolved in SDS-PAGE and detected by autoradiography. Immunoprecipitated material was measured by immunoblotting with the anti CDK2 antibody.

Mentions: Expression of some of the identified genes was further analyzed by conventional RT-qPCR with specific oligonucleotides. Interestingly, we detected induction of the cell cycle inhibitor CDKN1A (p21Cip1), which had not been observed using the previous methods. Some of the genes were repressed (CDC2, CDKN3, RFC3) or activated (CDKN1A) as early as 2 days after Dox addition. Other genes were not repressed until day 6 (CDC20) (Figure S4). Next, we tested whether inhibition of genes in H1.2 depleted cells could be reverted by stable reintroduction of recombinant H1.2 (Figure 9A). Inhibition of CDC2, CCNB2, CDC20, MAD2L1 and RFC3 was reverted by restoring H1.2 levels. Induction of CDKN1A (p21Cip1) was only partially reverted by reintroduction of H1.2.


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)

Cell cycle gene alterations in H1.2 knock-down cells.(A) Complementation of altered expression of cell cycle-related genes in H1.2 knocked-down cells by stable expression of recombinant HA-tagged, shRNA-resistant H1.2. Expression of the cell cycle genes indicated was measured by RT-qPCR with specific oligonucelotides in parental H1.2 KD and H1.2sh/rH1.2HA cells treated or not with Dox for 6 days. GAPDH expression was measured for normalization. Data is expressed as relative units specific gene/GAPDH. The values represent the mean and SD of a representative experiment performed in triplicate. (B) Changes in the accumulation or phosphorylation of cell cycle-related proteins upon inhibition of H1.2. Expression of the indicated proteins was measured by Western blot with specific antibodies in cells treated or not with Dox for 6 days. H1.2 knocked-down cells were compared to parental T47D or control cells. Tubulin antibody was used as loading control. (C) CDK2 activity in H1.2 depleted cells. CDK2 was immunoprecipitated with the specific antibody from cellular extracts of Dox-treated or untreated cells (H1.2 KD or control) and incubated with histone H1 and γ-ATP. Products were resolved in SDS-PAGE and detected by autoradiography. Immunoprecipitated material was measured by immunoblotting with the anti CDK2 antibody.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2563032&req=5

pgen-1000227-g009: Cell cycle gene alterations in H1.2 knock-down cells.(A) Complementation of altered expression of cell cycle-related genes in H1.2 knocked-down cells by stable expression of recombinant HA-tagged, shRNA-resistant H1.2. Expression of the cell cycle genes indicated was measured by RT-qPCR with specific oligonucelotides in parental H1.2 KD and H1.2sh/rH1.2HA cells treated or not with Dox for 6 days. GAPDH expression was measured for normalization. Data is expressed as relative units specific gene/GAPDH. The values represent the mean and SD of a representative experiment performed in triplicate. (B) Changes in the accumulation or phosphorylation of cell cycle-related proteins upon inhibition of H1.2. Expression of the indicated proteins was measured by Western blot with specific antibodies in cells treated or not with Dox for 6 days. H1.2 knocked-down cells were compared to parental T47D or control cells. Tubulin antibody was used as loading control. (C) CDK2 activity in H1.2 depleted cells. CDK2 was immunoprecipitated with the specific antibody from cellular extracts of Dox-treated or untreated cells (H1.2 KD or control) and incubated with histone H1 and γ-ATP. Products were resolved in SDS-PAGE and detected by autoradiography. Immunoprecipitated material was measured by immunoblotting with the anti CDK2 antibody.
Mentions: Expression of some of the identified genes was further analyzed by conventional RT-qPCR with specific oligonucleotides. Interestingly, we detected induction of the cell cycle inhibitor CDKN1A (p21Cip1), which had not been observed using the previous methods. Some of the genes were repressed (CDC2, CDKN3, RFC3) or activated (CDKN1A) as early as 2 days after Dox addition. Other genes were not repressed until day 6 (CDC20) (Figure S4). Next, we tested whether inhibition of genes in H1.2 depleted cells could be reverted by stable reintroduction of recombinant H1.2 (Figure 9A). Inhibition of CDC2, CCNB2, CDC20, MAD2L1 and RFC3 was reverted by restoring H1.2 levels. Induction of CDKN1A (p21Cip1) was only partially reverted by reintroduction of H1.2.

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