Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth.
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.
Affiliation: Centre de Regulació Genòmica, Barcelona, Spain.
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.
Related in: MedlinePlus
Mentions: To further explore the relationship between H1.2 depletion and the cell cycle gene expression profile, we tested expression of 84 cell cycle-related genes by RT-real time PCR using the SuperArray (Bioscience Corporation) platform (Figure 8). Fourteen genes were significantly repressed (more than 2-fold) when H1.2 was depleted after Dox addition for 6 days in serum-starved cells (q≤0.5). This number was increased to 24 repressed genes when considering ≥1.5-fold and q≤0.1, and included 10 genes already identified in the customized microarray experiments: BIRC5, CDKN3, CDC20, CCNB2, CDC2, CCNB1, MAD2L1, CKS2, BCL2 and RBBP8. Among the newly identified genes were CDK2, PCNA, MCM2, MCM3, MCM5, CDKN2A (p16INK4A) and CDKN2B (p15INK4B). Only two genes were activated ≥2-fold: RBL2 and CCNG2. In the same experiment, cells kept ±Dox (6 days) and serum-starved (last two days) were further incubated with 10% serum for an additional 12 hours period and RNA was extracted. Differences in expression of the repressed genes were even higher, confirming that expression of this subset of genes was affected as a consequence of H1.2 depletion (Figure 8).