NBP-45, a Novel Nucleosomal Binding Protein with a Tissue-specific and Developmentally Regulated Expression

Abstract

Here we characterize a novel murine nuclear protein, which we named NBP-45, that is related to the ubiquitous nuclear proteins HMG-14/-17, binds specifically to nucleosome core particles, and can function as a transcriptional activator. NBP-45 mRNA is expressed at low levels and in variable amounts in all mouse tissues tested but is especially abundant in RNA extracted from 7-day-old mouse embryos, suggesting that it functions in early embryonic development. NBP-45 is composed of 406 amino acids and is encoded by a single size transcript. The region spanning the N-terminal 85 amino acids contains three segments that are highly homologous to functionally important domains in the HMG-14/-17 protein family: the nuclear localization signal, the nucleosome binding domain, and the chromatin unfolding domain. The protein region spanning the C-terminal 321 amino acids has a 42% content of negatively charged residues. The first 23 amino acids contain a region necessary for nuclear entry of the protein, the region spanning residues 12-40 is the main nucleosomal binding domain of the protein, and the negatively charged, Cterminal domain is necessary for transcription activation. The functional domains of NBP-45 are indicative of a nuclear protein that binds to nucleosomes, thereby creating a chromatin region of high local negative charge. Our studies establish the nucleosomal binding domain as a protein motif that is present in other than just the ubiquitous HMG-14/-17 proteins. We suggest that the nucleosomal binding domain motif is a protein module that facilitates binding to nucleosomes in chromatin.In the cell nucleus, the orderly progression of many DNArelated activities, such as transcription, replication, recombination, and repair, are associated with changes in the higher order structure of the chromatin fiber and with a temporal disassembly of the nucleosome. These structural changes are facilitated by multiple types of reversible posttranslational modifications of the histones and by the activities of various multiprotein complexes that disrupt the histone-DNA interactions in nucleosomes (1-9). In addition, structural proteins that lack known enzymatic activity, such as the high mobility group (HMG) 1 proteins, are also known to modify the structure of their DNA binding site and induce an architecture that facilitates and enhances various DNA-related activities (10, 11). The HMG protein family is subdivided into three subfamilies: the HMG-1/-2 subfamily, the HMG-I/Y subfamily, and the HMG-14/-17 subfamily. Each of these subfamilies has a unique protein signature and a distinct functional motif. The HMG-1 domain is the functional domain of the HMG-1/-2 subfamily, the AT-hook is the functional domain of the HMG-I/Y family, and the nucleosomal binding domain is the functional motif of the HMG-14/-17 proteins. Through these domains the HMG proteins bind to their DNA or chromatin target, with little if any specificity for the underlying DNA sequence (10, 11).Two of the HMG functional domains, the HMG-1, and the AT-hoo...

Distinct Properties of Human HMGN5 Reveal a Rapidly Evolving but Functionally Conserved Nucleosome Binding Protein

“…HMGN5 was cloned into pGEX4T2 at BamHI and EcoRI sites, and Hmgn5 was cloned as previously described (32). All proteins were expressed in bacteria as previously described (34). Histone H5 was purified from chicken erythrocytes by acid extraction and Mono S ion-exchange chromatography.…”

“…The HMGN1, HMGN2, HMGN3, and HMGN4 proteins contain fewer than 100 amino acids, and each has a distinct sequence that is highly conserved throughout the animal kingdom (5,27). HMGN5, which is the most recently discovered HMGN variant (32,34), differs from the other HMGN members in both size and sequence conservation. For example, mouse HMGN5 (mHMGN5) contains 406 amino acids, including a highly acidic, 300-amino-acid C terminus, and is 4 times larger than the other HMGN variants (35).…”

Knockdown of HMGN5 Expression by RNA Interference Induces Cell Cycle Arrest in Human Lung Cancer Cells

“…HMGNs have been studied extensively for their ability to modulate transcription (Furusawa et al, 2006;Zhu and Hansen, 2007;Ueda et al, 2009). HMGN5/NSBP1 is a typical member of the HMGN family which is localized to the nucleus and contains a functional NBD and a conserved motif in the C-terminus domain (Shirakawa et al, 2000). However, unlike the other HMGNs, HMGN5 contains a longer acidic C-terminal domain which affects cellular localization and architectural properties of the protein (Rochman et al, 2010).…”

Differential expression of the HMGN family of chromatin proteins during ocular development

“…Notably, HMGN1 inhibits the phosphorylation of histone H3 by MSK1 while HMGN2 does not, showing that different family members probably have distinct biological functions (Ueda et al, 2006). Currently, the HMGN protein family (as defined by the presence of the NBD) consists of five members, the extensively studied HMGN1 and 2 (Bustin, 2001) as well as HMGN3 (also reported as the thyroid hormone receptor binding protein Trip7) (Amano et al, 2002;), HMGN4 ) and NBP45/NSBP1 (Shirakawa et al, 2000). HMGN1 is highly expressed in early mouse embryos (Mohamed et al, 2001), with levels generally downregulating, except in self renewing cell populations, as development proceeds .…”