Fig. 4. Identification of two amino-acid residues in the HP1 box of TIF1ß, which are critical for the HP1-binding activity of TIF1ß. (A) A schematic representation of the conserved domains of TIF1ß. Numbers refer to amino-acid positions. An alignment of the HP1 interaction domains from TIF1ß, TIF1 and CAF-1 is shown. Invariant amino acids are shaded. Mutations introduced into the conserved hydrophobic residues of the TIF1ß HP1 box are indicated below the alignment. Database accession numbers: mouse TIF1ß (mTIF1ß, X99644); mouse TIF1 (mTIF1, S78219); human CAF-1 (hCAF-1, XP009408). (B) TIF1ßV488A/L490A has no HP1-binding activity in yeast. The indicated FLAG-epitope-tagged TIF1ß constructs, f:TIF1ß wildtype (f:TIF1ßWT) and f:TIF1ßV488A/L490A (f:TIF1ßVL/AA), were fused with the acidic activation domain (AAD) of VP16 and assayed for interaction with the `unfused' DNA-binding domain (DBD) of the oestrogen receptor ER or a DBD fusion containing HP1, HP1ß, HP1 or the KRAB transcriptional repression domain of KOX-1 in the yeast reporter strain PL3, which contains a URA3 reporter gene driven by three ER binding sites (Le Douarin et al., 1995b). Transformants were grown in liquid medium containing uracil. OMPdecase activities determined on each cell-free extracts are expressed in nmol substrate/min/mg protein. The values (±20%) are the average of at least three independent transformants. Note that expression of all fusion proteins was confirmed by western blotting. (C) TIF1ßV488A/L490A has no HP1 binding activity in mammalian cells. Whole cell extracts from COS-1 cells transfected with 5 µg of expression vector for unfused FLAG (control) or FLAG-TIF1ß were analyzed by western blotting either directly (input) or following immunoprecipitation with the M2 anti-FLAG antibody (FLAG IP). A western blot probed with a HP1 mAb is shown. Inputs correspond to 1/10 the amount of cell extract used for immunoprecipitation.

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