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A negative regulatory element (NRE1; position −794 to −774) was previously identified that mediates the downregulation of CYP1A1, including partial suppression of Ah receptor-dependent induction. The CCAAT-box binding protein, nuclear factor-Y (NF-Y), is a component of one of two protein complexes that specifically and competitively bind the CYP1A1 NRE1 in vitro with nearly equal affinity. The second complex involves an unidentified protein(s) called the negative regulatory factor (NRF). Competitive electrophoretic mobility shift assays (EMSA) revealed two distinct classes of NF-Y-binding CCAAT-box elements distinguished by their ability or inability to also bind NRF. To further explore the identity of NRE1-binding proteins, a purification scheme was developed culminating in NRE1-dependent DNA affinity chromatography and sequence analysis. An approximate 106-kDa protein was purified and shown to be α-actinin-4 (ACTN4), one of two ubiquitously expressed non-muscle actinins. Electrophoretic mobility shift assays combined with Western blot analysis and co-immunoprecipitation experiments suggested that ACTN4 is associated with the NF-Y complex, but not NRF. Attempts to demonstrate a role for NF-Y/ACTN4 in regulating CYP1A1 expression were unsuccessful, likely due to an inability to significantly change nuclear ACTN4 levels with phosphatidylinositol 3′-kinase agonists and antagonists. However, given ACTN4's known functions and the suspected functions of actin and actin-related proteins in chromatin remodeling and other nuclear events, ACTN4 may assist NF-Y in recruiting chromatin-remodeling complexes or may direct NF-Y/ACTN4-targeted genes to the nuclear matrix and active transcriptional complexes.