FIGURE 4.

Htz1^Ac is regulated in asf1Δ cells by an indirect mechanism. A, each Htz1^Ac (but not total Htz1.HA₃) is decreased in asf1Δ cells. Rpn8 serves as a loading control. B, asf1Δ reduces each Htz1^Ac but has no effect on the amount of total Htz1 associated with chromatin. WT and asf1Δ cells were spheroblasted (total, T); fractionated into cytoplasm (Cy), nucleus (N), and chromatin (Ch); and immunoblotted as indicated. Appropriate localization of H2B and Rpn8 indicates efficient fractionation (as in Fig. 1C). C, schematic depicts the various roles of Asf1, a histone H3-H4 donor to various acetyltransferase complexes (substrates indicated), CAF/proliferating cell nuclear antigen (PCNA) at replication forks (for replication-dependent deposition (RDD)), and HIR in actively transcribed regions (for replication-independent deposition (RID)). D, glucose (final concentration, 2%) was added to repress GAL1_p-driven transcription in GAL1_p.ASF1.HA₃ or GAL1_p.ASF1.HA₃.PEST strains, with samples taken for WCE isolation and immunoblotting at times indicated. Rpn8 serves as a loading control. E, GAL1_p.ASF1.HA₃ or GAL1_p.ASF1.HA₃.PEST strains were maintained in glucose (G, 2%) or galactose/raffinose (GR, 2%/1%) containing medium for >24 h before the samples were collected for WCE isolation and immunoblotting. Rpn8 serves as a loading control.