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Effects of HBO1 Overexpression on Chromatin Protein Levels in Cell Cycle Staging

This study explores the role of HBO1 and other replication factors (MCM3, MCM5, ORC1, Cdt1, Cdc6) in different stages of the cell cycle (G1/S, G2/M) using whole cellular extracts and chromatin fractions from CCL-156 cells. We analyzed mRNA and protein levels in response to Actinomycin D treatment and HBO1 overexpression, employing FACS for cell staging and Western blotting for protein detection. Notably, HBO1 expression levels varied, indicating its significant regulatory function in chromatin-associated processes during cell cycle progression.

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Effects of HBO1 Overexpression on Chromatin Protein Levels in Cell Cycle Staging

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  1. D Whole cellular extracts Chromatin fractions - + + - Myc-HBO1 - + + - control 1.2 1.1 0.98 0.97 1.1 - 3.5 MCM3 MCM5 ORC1 Cdt1 Cdc6 Myc-HBO1 HBO1 Supplementary Figure 3 A B G1/S G2/M MW(bp) Chromatin fractions Whole cellular extracts HBO1 100 2n 4n Asy. G1/S G2/M Asy. G1/S G2/M G1/S staged (hydroxyurea) ORC1 HBO1 100 Cdt1 Cdt1 200 ORC1 MCM2 100 Cdc6 2n 4n G2/M staged (nocodazole) 300 ORC2 Actin C Chromatin fractions Whole cellular extracts Actinomycin D + + - - Cdt1 HBO1 ORC1 ORC2 Cdc6 MCM3 Actin Levels of mRNA and protein bound to bulk chromatin at different stages of the cell cycle, in response to actinomycin D treatment, and upon HBO1 overexpression. (A) Expression level of HBO1, MCM2, ORC1, and Cdt1 mRNAs in CCL-156 cells staged in early S phase (hydroxyurea block) or at the G2/M transition (nocodozole block). Efficient staging was confirmed by FACS analysis. (B) Western blot analysis of HBO1, Cdt1, Cdc6, ORC1, and ORC2 expression in CCL-156 cells staged in early S phase and at the G2/M transition. (C) Expression level and chromatin association of HBO1 and replication factors in the absence or presence of actinomycin D (actD). Note that, for unknown reasons, soluble HBO1 is degraded following ActD treatment, whereas the level of chromatin-bound HBO1 is unaffected. (D) Expression level and chromatin association of HBO1 and replication factors under conditions of HBO1 overexpression. Values for each protein indicate fold-difference upon HBO1 overexpression

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