FIGURE 3.

ASCIZ regulates Dynll1 as a ZnF transcription factor. A, firefly luciferase reporter activity of the human Dynll1 promoter (solid bars) or pGL3 empty vector (open bars) transiently transfected into early-passage primary WT and Asciz^−/− MEFs (three independent embryos per genotype). Values are means ± S.E. relative to Renilla luciferase as transfection control. B, luciferase reporter assays as in A using an immortalized Asciz^−/− MEF cell line transiently co-transfected with GFP-fusion constructs of the human 823-residue ASCIZ isoform, a ZnF-deleted ASCIZ fragment, 823-residue ASCIZ with all 20 SQ/TQ motifs mutated to AQ, or a ZnF-deleted and AQ-mutated fragment, as well as Dynll1-firefly luciferase and Renilla luciferase vectors. Values are means ± S.E. normalized to the empty GFP-only vector control (n = 3–4 independent experiments). C, ChIP assays using the ASCIZ antibody and real-time quantitative PCR analyses of the indicated regions (relative to the ATG start codon) of the Dynll1 promoter, and the corresponding Gapdh control from the same precipitations. Values are means ± S.E. of four different sets of WT and Asciz^−/− littermate early-passage MEFs. TSS, transcription start site. D, immunoblot analysis of early-passage WT and Asciz^−/− littermate primary MEFs, and the same Asciz^−/− MEFs after retroviral complementation with the empty vector, the human 823-residue ASCIZ isoform, a ZnF-deleted ASCIZ fragment, 823-residue ASCIZ with all 20 SQ/TQ motifs mutated to AQ, or a ZnF-deleted and AQ-mutated fragment. Virus-encoded GFP serves as an infection control.