Sp2 Localizes to Subnuclear Foci Associated with the Nuclear Matrix

Abstract

We have reported that extracts prepared from many human and mouse cell lines show little or no Sp2 DNA-binding activity and that Sp2 has little or no capacity to stimulate transcription of promoters that are activated by Sp1, Sp3, and Sp4. Using an array of chimeric Sp1/Sp2 proteins we showed further that Sp2 DNA-binding activity and trans-activation are each negatively regulated in mammalian cells. As part of an ongoing effort to study Sp2 function and regulation we characterized its subcellular localization in comparison with other Sp-family members in fixed and live cells. We report that 1) Sp2 localizes largely within subnuclear foci associated with the nuclear matrix, and 2) these foci are distinct from promyelocytic oncogenic domains and appear to be stable during an 18-h time course of observation. Deletion analyses identified a 37 amino acid sequence spanning the first zinc-"finger" that is sufficient to direct nuclear matrix association, and this region also encodes a bipartite nuclear localization sequence. A second nuclear matrix targeting sequence is encoded within the Sp2 trans-activation domain. We conclude that Sp2 preferentially associates with the nuclear matrix and speculate that this subcellular localization plays an important role in the regulation of Sp2 function.

C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix

“…This is similar to the behaviour of Sp2, for which two independent regions are required to specify correct matrix-associated patterning (Moorefield et al, 2006). Thus, it seems likely that the concept of a simple NMTS will have to give way to a more complex model in which higher order structural determinants influence patterning.…”

“…DNA is stained with Hoechst 33258 (blue) and imaged to reflect the DNA content after each treatment. Zaidi et al, 2001;Zeng et al, 1997), Nxp2 (Kimura et al, 2002), Hairless (Djabali and Christiano, 2004) and Sp2 (Moorefield et al, 2006) the interaction is driven by what has been termed a 'nuclear matrix targeting signal' or NMTS. Whereas some NMTSs have been narrowed down to less than 50 amino acids, little sequence homology has been identified between them, and we can find no striking similarity with sequences contained within Ciz1 708-830.…”

Sp2 Is a Maternally Inherited Transcription Factor Required for Embryonic Development

“…Transient Transfections, Western Blotting, trans-Activation and Subcellular Localization Assays, and Fluorescence Microscopy-Transient transfection assays were performed as described (32,43) except that zebrafish Zf4 cells (a fibroblastlike cell line derived from embryos 24 h post-fertilization (hpf)) were included as recipients. Extracts to be utilized for Western blotting were harvested 48 h after transfection and analyzed as described (32).…”

“…Extracts to be utilized for Western blotting were harvested 48 h after transfection and analyzed as described (32). Transient trans-activation assays were performed via co-transfection of pCMV4-ZfSp2/flu, pCMV4-hSp1/flu (42), or pCMV4-hSp3/flu (44) with a DHFR-firefly luciferase reporter plasmid (DHFR-Lux (31)) and a Renilla luciferase reporter plasmid (phRL-⌬53MLP (43)) as a control for transfection efficiency.…”

“…To identify domains of Sp2 that limit its capacity to stimulate transcription and/or bind DNA, we prepared and characterized the activities of chimeric molecules in which portions of Sp1 and Sp2 were exchanged. Studies using Sp1/Sp2 chimeras revealed that the Sp2 trans-activation and DNA binding domains are each negatively regulated in vitro, and further analyzes have shown that each of these domains carries amino acid sequences that independently target Sp2 to the nuclear matrix (32). It remains uncertain whether the tethering of Sp2 to the nuclear matrix accounts for the limited capacities of Sp2 to bind DNA and stimulate transcription.…”