Alpha actinins (ACTNs) are known for their ability to modulate cytoskeletal organization and cell motility by cross-linking actin filaments. We show here that ACTN4 harbors a functional LXXLL receptor interaction motif, interacts with nuclear receptors in vitro and in mammalian cells, and potently activates transcription mediated by nuclear receptors. Whereas overexpression of ACTN4 potentiates estrogen receptor ␣ (ER␣)-mediated transcription in transient transfection reporter assays, knockdown of ACTN4 decreases it. In contrast, histone deacetylase 7 (HDAC7) inhibits estrogen receptor ␣ (ER␣)-mediated transcription. Moreover, the ACTN4 mutant lacking the CaM (calmodulin)-like domain that is required for its interaction with HDAC7 fails to activate transcription by ER␣. Chromatin immunoprecipitation (ChIP) assays demonstrate that maximal associations of ACTN4 and HDAC7 with the pS2 promoter are mutually exclusive. Knockdown of ACTN4 significantly decreases the expression of ER␣ target genes including pS2 and PR and also affects cell proliferation of MCF-7 breast cancer cells with or without hormone, whereas knockdown of HDAC7 exhibits opposite effects. Interestingly, overexpression of wild-type ACTN4, but not the mutants defective in interacting with ER␣ or HDAC7, results in an increase in pS2 and PR mRNA accumulation in a hormone-dependent manner. In summary, we have identified ACTN4 as a novel, atypical coactivator that regulates transcription networks to control cell growth.The alpha actinins (ACTNs) 2 belong to a family of cytoskeletal proteins that bind actin filaments to maintain cytoskeletal structure and cell morphology (1). Among the four members of the family, ACTN2 and ACTN3 are expressed primarily in muscle, while ACTN1 and ACTN4 are ubiquitously expressed (2). All four members of the actinin family share high sequence homology with conserved functional domains including an N-terminal actin-binding domain containing two highly conserved calponin homology (CH1 and CH2) domains, a central domain consisting of four spectrin repeats (SR), two EF hand calcium-binding domains and a C-terminal calmodulin (CaM)-like domain (3). Although predominantly localized in the cytoskeleton, ACTN4 is also found in the nucleus of certain cell types and is capable of translocating to the nucleus in response to extracellular stimuli (4). In addition to its role in the cytosol, we have recently identified a novel function for ACTN4 in transcriptional regulation by myocyte enhancer factor 2 (MEF2) (5). Another study has also demonstrated an association between ACTN4 and nuclear factor B (NF-B) (6). These findings suggest that ACTN4 may play an unexpected role in transcriptional regulation.Nuclear hormone receptors, including vitamin D receptor (VDR) and steroid hormone receptors such as estrogen receptors (ER) are ligand-activated transcription factors that control aspects of homeostasis, cell differentiation, proliferation, and development (7-9). Transcriptional regulation by nuclear receptors is thought to occur through t...