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Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis.

Singh R, Mortazavi A, Telu KH, Nagarajan P, Lucas DM, Thomas-Ahner JM, Clinton SK, Byrd JC, Freitas MA, Parthun MR - Nucleic Acids Res. (2013)

Bottom Line: Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant.To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown.Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5' untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

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Histone H2A 1C influences cell proliferation and carcinogenesis in U2OS cells. (A) U2OS cells were transfected with siRNAs targeting the indicated histone H2A loci. qRT pcr assays were used to measure each H2A mRNA before (blue bars) and after (red bars) transfection. (B) Cells were transfected with the indicated siRNAs (or mock transfected). U2OS cell proliferation was measured by counting cell numbers at the indicated times. (C) U2OS cells were transfected with the indicated siRNAs and plated in soft agar media. Plates were photographed following 5 days of growth. (D) U2OS cells treated with the indicated siRNAs were plated in soft agar in mitrotiter wells. Following 6 days, cell growth was quantitated by solubilizing the cells in the presence of Cytoselect (Cytoselect 96-well cell transformation assay kit, Cell Biolabs, Inc.) and measuring relative fluorescence intensity.
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gkt736-F3: Histone H2A 1C influences cell proliferation and carcinogenesis in U2OS cells. (A) U2OS cells were transfected with siRNAs targeting the indicated histone H2A loci. qRT pcr assays were used to measure each H2A mRNA before (blue bars) and after (red bars) transfection. (B) Cells were transfected with the indicated siRNAs (or mock transfected). U2OS cell proliferation was measured by counting cell numbers at the indicated times. (C) U2OS cells were transfected with the indicated siRNAs and plated in soft agar media. Plates were photographed following 5 days of growth. (D) U2OS cells treated with the indicated siRNAs were plated in soft agar in mitrotiter wells. Following 6 days, cell growth was quantitated by solubilizing the cells in the presence of Cytoselect (Cytoselect 96-well cell transformation assay kit, Cell Biolabs, Inc.) and measuring relative fluorescence intensity.

Mentions: To determine whether the effect of H2A isoform modulation on cell proliferation and tumorigenicity was cell-type specific, we repeated the analyses using a different cell line. As seen in Figure 3A, specific siRNA-based depletion of H2A isoforms was also achieved in U2OS cells. Strikingly, the effect of this H2A isoform knockdown on cell proliferation and growth in soft agar was similar to that seen with 293TN cells. Namely, siRNA knockdown of H2A 1C caused a significant increase in cell growth and a dramatic increase in the colony size observed during growth on soft agar, whereas siRNA knockdown of H2A 1B/E has a smaller effect on both phenotypes (Figure 3B–D).Figure 3.


Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis.

Singh R, Mortazavi A, Telu KH, Nagarajan P, Lucas DM, Thomas-Ahner JM, Clinton SK, Byrd JC, Freitas MA, Parthun MR - Nucleic Acids Res. (2013)

Histone H2A 1C influences cell proliferation and carcinogenesis in U2OS cells. (A) U2OS cells were transfected with siRNAs targeting the indicated histone H2A loci. qRT pcr assays were used to measure each H2A mRNA before (blue bars) and after (red bars) transfection. (B) Cells were transfected with the indicated siRNAs (or mock transfected). U2OS cell proliferation was measured by counting cell numbers at the indicated times. (C) U2OS cells were transfected with the indicated siRNAs and plated in soft agar media. Plates were photographed following 5 days of growth. (D) U2OS cells treated with the indicated siRNAs were plated in soft agar in mitrotiter wells. Following 6 days, cell growth was quantitated by solubilizing the cells in the presence of Cytoselect (Cytoselect 96-well cell transformation assay kit, Cell Biolabs, Inc.) and measuring relative fluorescence intensity.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814372&req=5

gkt736-F3: Histone H2A 1C influences cell proliferation and carcinogenesis in U2OS cells. (A) U2OS cells were transfected with siRNAs targeting the indicated histone H2A loci. qRT pcr assays were used to measure each H2A mRNA before (blue bars) and after (red bars) transfection. (B) Cells were transfected with the indicated siRNAs (or mock transfected). U2OS cell proliferation was measured by counting cell numbers at the indicated times. (C) U2OS cells were transfected with the indicated siRNAs and plated in soft agar media. Plates were photographed following 5 days of growth. (D) U2OS cells treated with the indicated siRNAs were plated in soft agar in mitrotiter wells. Following 6 days, cell growth was quantitated by solubilizing the cells in the presence of Cytoselect (Cytoselect 96-well cell transformation assay kit, Cell Biolabs, Inc.) and measuring relative fluorescence intensity.
Mentions: To determine whether the effect of H2A isoform modulation on cell proliferation and tumorigenicity was cell-type specific, we repeated the analyses using a different cell line. As seen in Figure 3A, specific siRNA-based depletion of H2A isoforms was also achieved in U2OS cells. Strikingly, the effect of this H2A isoform knockdown on cell proliferation and growth in soft agar was similar to that seen with 293TN cells. Namely, siRNA knockdown of H2A 1C caused a significant increase in cell growth and a dramatic increase in the colony size observed during growth on soft agar, whereas siRNA knockdown of H2A 1B/E has a smaller effect on both phenotypes (Figure 3B–D).Figure 3.

Bottom Line: Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant.To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown.Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5' untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

Show MeSH
Related in: MedlinePlus