The RING Finger Protein SNURF Is a Bifunctional Protein Possessing DNA Binding Activity

Abstract

The small nuclear C 3 HC 4 finger protein (SNURF), RNF4, acts as transcriptional coactivator for both steroiddependent and -independent promoters such as those driven by androgen response elements and GC boxes, respectively. However, SNURF does not possess intrinsic transcription activation function, and the precise molecular mechanism of its action is unknown. We have studied herein the interaction of SNURF with DNA in vitro. SNURF binds to linear double-stranded DNA with no apparent sequence specificity in a cooperative fashion that is highly dependent on the length of the DNA fragment used. SNURF interacts efficiently with both supercoiled circular and four-way junction DNA, and importantly, it also recognizes nucleosomes. An intact RING structure of SNURF is not mandatory for DNA binding, whereas mutations of specific positively charged residues in the N terminus (amino acids 8 -11) abolish DNA binding. Interestingly, the ability of SNURF to interact with DNA is associated with its capability to enhance transcription from promoters containing GC box elements. Because SNURF can interact with both DNA and protein (transcription) factors, it may promote assembly of nucleoprotein structures. RING1 (really interesting new gene) finger is a motif of conserved cysteines and histidines that coordinate two zinc atoms in a "cross-brace" system, a ligation scheme distinct from those of the classical zinc fingers (1, 2). The RING motifs can be classified into two subgroups according to the presence of a cysteine or histidine in the fifth position: C 3 HC 4 (RING-HC) and C 3 H 2 C 3 (RING-H2) fingers. Otherwise their composition and length can vary substantially. The RING finger has been found in a variety of eukaryotic proteins of diverse evolutionary origin that are involved in various cellular processes such as oncogenesis, development, signal transduction, and apoptosis (1-3). RING fingers have been shown to mediate protein-protein interactions and formation of multi-protein complexes. The RING motif of promyelocytic leukemia gene product is important in the assembly of protein complexes linked to SUMO-1 (a small ubiquitin-like modifier protein) modifications (4). RING finger has also been suggested to act as a DNAbinding motif (5). The function of many RING-containing proteins can be mediated through DNA binding or chromatin association. RAG1 is involved in V(D)J recombination complex, and RAD-16 participates in DNA repair (6). RING finger-containing polycomb group proteins Psc, Su(z)2, Bmi-1, and RING1 are involved in the maintenance of the transcriptionally repressed state of genes by regulating chromatin structure (7-9), and Mel-18 is shown to act as a transcriptional repressor via binding to specific DNA sequence (10). Nuclear receptor mediator TIF␣ is tightly associated with euchromatin (11), whereas BRCA1 appears to be associated with the RNA polymerase II holoenzyme (12). However, the RING structures of these latter proteins have not been implicated in mediating their binding to chromatin or DNA.Recent intrig...

Fission Yeast Rnf4 Homologs Are Required for DNA Repair

“…Given that these phenotypes require the deletion of both genes, we cannot formally determine whether Rnf4 is functioning as Rfp1, Rfp2, or a combination of the two. Rnf4 contains both DNA binding and E3 ubiquitin ligase activities (21,25) and has been shown to interact with several transcription factors that are not conserved in S. pombe, including the androgen (24) and estrogen receptors (44). It is not known whether Rnf4 is required for DNA repair in human cells.…”

“…These proteins function redundantly in DNA repair; a double mutant is hypersensitive to different forms of DNA damage, although checkpoint signaling through to Chk1 activation is normal. We show that human Rnf4, an E3 ubiquitin ligase that has been implicated as a cofactor for several transcription factors (21)(22)(23)(24)(25), is a close sequence relative of Rfp1 and Rfp2 and can indeed substitute functionally for these proteins. We show that the control over protein SUMOylation is defective in cells lacking Rfp1 and Rfp2 and that indeed this is the cause of the DNA repair defect.…”

Interplay of Posttranslational Modifications in Sp1 Mediates Sp1 Stability during Cell Cycle Progression

“…30,32,36,38,[40][41][42] In vitro ubiquitination assays have demonstrated that RNF4 functions as a ubiquitin E3 ligase and that its E3 ligase activity is closely linked to its transcription regulatory functions. 37,43 The same group also demonstrated that SUMO-1 promotes the association between RNF4 and PML nuclear bodies. 44 Moreover, RNF4 recruitment to sumoylated proteins was mediated through tandem SIMs within the N-terminus of RNF4.…”

“…36 RNF4 was primarily identified as a bridging factor that regulated steroid-receptordependent transcription. 37,38 However, RNF4 may participate in Sp1-dependent transcription and cooperate with androgen receptor and Sp1. 39,40 RNF4 was able to stimulate the rat luteinizing hormone-β promoter by interacting with Sp1 and steroidogenic factor-1 and then protect it from androgen suppression.…”

Cooperative Coactivation of Estrogen Receptor α in ZR-75 Human Breast Cancer Cells by SNURF and TATA-binding Protein

“…1-4) support a novel mechanism for SNURF-mediated coactivation of ER␣. We recently showed that the N-terminal region of SNURF (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] is required for nonspecific DNA binding (95), and deletion of this domain also resulted in loss of ER␣ coactivation (Fig. 4A).…”