Elsevier

Neurochemistry International

Volume 54, Issues 3–4, March–April 2009, Pages 245-252
Neurochemistry International

NAC1, a POZ/BTB protein that functions as a corepressor

https://doi.org/10.1016/j.neuint.2008.12.008Get rights and content

Abstract

We now demonstrate that NAC1 acts as a corepressor for other POZ/BTB proteins. NAC1 is a POZ/BTB motif containing transcriptional repressor protein. In a mammalian two hybrid assay in neuronal (N2A) cells and non-neuronal (HEK 293T) cells, VP16 activation domain tagged NAC1 resulted in significant reversal of transcriptional inhibition with the Gal4-ZID, Gal4-BCL6, Gal4-ZF5, and kelch proteins Gal4-MAYVEN and Gal4-NRP/B fusion proteins. We also observed similar results with another corepressor, BCoR Gal4 fusion protein. NAC1 potentiated ZF5 mediated repression in Gal4-DBD fusion transient assays. GST pulldown assays further confirmed protein–protein interactions between these proteins and NAC1. Both the NAC1 isoforms demonstrated selective interaction through the POZ/BTB domain but not with the non-POZ/BTB region. Endogenous NAC1 and BCL6 physically associated in CNS regions. Strikingly, NAC1 did not interact with the pro-myelocytic leukemia zinc finger protein (PLZF), another POZ/BTB protein that is not found in the adult brain. Therefore, we conclude that NAC1 functions as a corepressor for POZ/BTB proteins expressed in the mature CNS.

Introduction

NAC1, is a POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex) domain containing transcriptional repressor protein. NAC1 can mediate both homomeric as well as heteromeric interactions like other POZ/BTB family proteins. Several functional roles have been reported for the POZ/BTB domain, including transcriptional repression (Ahmad et al., 2003, Melnick et al., 2005), cytoskeleton regulation (Kang et al., 2004, Bomont et al., 2000), ion channel tetramerization (Minor et al., 2000, Kreusch et al., 1998) and protein ubiquitination/degradation (Kobayashi et al., 2004, Pintard et al., 2003, Geyer et al., 2003). Recently, POZ/BTB proteins have been identified as the interaction partners of Cullin (Cul)3 Skp1-Cullin-F-box(SCF)-like E3 ubiquitin ligase complex, with the POZ/BTB domain mediating recruitment of the substrate recognition modules to the Cul3 component of the SCF-like complex (Krek, 2003, Willems et al., 2004, Pintard et al., 2004). Many POZ/BTB proteins contain zinc finger motifs that bind to DNA (Albagli et al., 1995, Collins et al., 2001, Stogios et al., 2005). NAC1 is a unique POZ/BTB transcriptional repressor because it lacks zinc finger motifs or kelch repeats in either of its two isoforms (Mackler et al., 2000, Korutla et al., 2002).

NAC1 was originally isolated as a cocaine regulated protein (Cha et al., 1997), from the nucleus accumbens that influences the response to addictive drugs (Koob et al., 2004, Nestler and Aghajanian, 1997). NAC1 protein is restricted to the cell nucleus and transports into the cytoplasm when the cells are differentiated into the neuronal phenotype (Mackler et al., 2000, Korutla et al., 2005a, Korutla et al., 2005b). The POZ/BTB domain of NAC1 is responsible for a large portion of the repressive function of the protein, although the non-POZ/BTB region can also mediate repression independently (Mackler et al., 2000). We reported the association of NAC1 with HDACs (Korutla et al., 2005a, Korutla et al., 2005b) and CoREST (Korutla et al., 2007), which is known to be in a complex with mSin3a and histone deacetylases (Grimes et al., 2000, You et al., 2001). Previous studies reported that the POZ/BTB domain of BCL6, ZID and PLZF interacted with corepressors NCoR and SMRT, which are associated with a large transcriptional regulatory complex that includes mSin3a and the histone deacetylases (Huynh and Bardwell, 1998, Deltour et al., 1999). BCL6 interacting corepressor (BCoR) interacted selectively with the POZ/BTB domain of BCL6 (Huynh et al., 2000).

The kelch proteins consist of a POZ/BTB domain and have specific tandem repeats. These proteins are conserved in a wide range of organisms, from viruses to mammals and have diverse functions in cell morphology, cell organization and gene expression, and function in protein–protein interactions (Albagli et al., 1995, Avraham et al., 2002). Several proteins with kelch domains are expressed in the brain and reported to be important for neuronal functions. MAYVEN and Actifilin are kelch repeat containing proteins that bind to f-actin and are expressed in neurons (Soltysik-Espanola et al., 1999, Chen et al., 2002). Recently, the role of MAYVEN in the cytoskeletal rearrangement leading to the process extension of oligodendrocytes was reported (Jiang et al., 2005). Another kelch containing protein, NRP/B, is involved in neurite outgrowth and neuronal differentiation (Kim et al., 1998).

The goal of this study was to identify the proteins that interact with NAC1. Here we are reporting that NAC1 functions as a corepressor for other POZ/BTB proteins. Our results along with the observation that NAC1 does not contain any obvious DNA binding ability, indicate NAC1 functions as a corepressor protein that may contribute differently than other family members to cellular function.

Section snippets

Cell culture

Murine neuroblastoma cells, clone N2A (ATCC # CCL131), were grown in monolayer cultures in minimum essential medium supplemented with 10% fetal calf serum (FCS). HEK 293T cells were grown in Dulbecco's modified Eagle medium supplemented with 10% FCS. Transfection assays in N2A cells and HEK 293T cells were performed in 20–30% confluent cultures in 24 well plates/48 well plates.

Plasmids

Expression constructs containing herpes virus VP16 AD were made by inserting full-length lNAC1 into a pVP16 vector

Identification of POZ/BTB proteins interacting with NAC1 in mammalian two hybrid assays and GST pulldowns

We performed mammalian two hybrid assays in neuronal (N2A cells), and non-neuronal (HEK 293T) cells with VP16-NAC1 and the Gal4 DNA binding domain fused to POZ/BTB cDNAs ZID, BCL6, and ZF5. When the VP16 activation domain fused to NAC1 was transiently transfected into cells together with other Gal4 cDNAs, this resulted in significant reversal of transcriptional inhibition observed with the Gal4-ZID, Gal4-BCL6, and Gal4-ZF5 fusion proteins. The Gal4 DNA binding domain alone did not alter

Discussion

We previously demonstrated the interaction between a brain POZ/BTB protein, NAC1 and other POZ/BTB proteins, and we hypothesized that NAC1 is a corepressor functioning by recruiting other POZ/BTB transcription regulators and known corepressors. Mammalian two hybrids demonstrated cellular interactions between NAC1 with other POZ/BTB proteins and with the known corepressor BCoR. GST pulldowns further supported these observations, with either lNAC1 or sNAC1 interacting directly with the proteins (

Acknowledgements

This work was supported by the Veterans Administration (DVA-MR-393), NIH (DA 11809-03) and the University of Pennsylvania Research Foundation and the Joseph Alexander Foundation. The authors appreciate the editorial efforts of Dana R. Williams and Ryan Thomas-Degnan. Plasmids were generously provided by Dr. Vivian Bardwell.

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