Nuclear Localization of the Saccharomyces cerevisiae HMG Protein NHP6A Occurs by a Ran-Independent Nonclassical Pathway

The Saccharomyces cerevisiae non-histone protein 6-A (NHP6A) is a member of the high-mobility group 1/2 protein family that bind and bend DNA of mixed sequence. NHP6A has only one high-mobility group 1/2 DNA binding domain and also requires a 16-amino-acid basic tail at its N-terminus for DNA binding. We show in this report that nuclear accumulation of NHP6A is strictly correlated with its DNA binding properties since only nonhistone protein 6 A–green fluorescent protein chimeras that were competent for DNA binding were localized to the nucleus. Despite the requirement for basic residues within the N-terminal segment for DNA binding and nuclear accumulation, this region does not appear to contain a nuclear localization signal. Moreover, NHP6A does not bind to the yeast nuclear localization signal receptor SRP1 and nuclear targeting of NHP6A does not require the function of the 14 different importins. Unlike histone H2B1 which contains a classical nuclear localization signal, entry of NHP6A into the nucleus was found to be independent of Ran as judged by coexpression of Ran GTPase mutants and was shown to occur at 0 °C after a 15-min induction. These unusual properties lead us to suggest that NHP6A entry into the nucleus proceeds by a nonclassical Ran-independent pathway.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.