HMGN proteins promote chromatin unfolding, enhance access to nucleosomes, and modulate transcription from chromatin templates. It is not known whether they act indiscriminately as general modulators of transcription or whether they regulate specific gene expression. Here, we investigated the role of HMGN3, a recently discovered HMGN family member, in transcription in vivo. We created cell lines overexpressing HMGN3a or its splice variant, HMGN3b, and analyzed their gene expression profiles using microarrays and reverse transcriptase PCR. We found that ectopic expression of HMGN3a alters the expression of approximately 0.8% of genes. Both HMGN3a and HMGN3b upregulate the expression of the glycine transporter 1 gene (Glyt1). Glyt1 encodes a membrane transporter that regulates the glycine concentration in synaptic junctions. Both GLYT1 and HMGN3 are highly expressed in glia cells and the eye, and we show that both proteins are coexpressed in the retina. Chromatin immunoprecipitation assays showed that HMGN3 protein is recruited to a region of the Glyt1 gene encompassing the Glyt1a transcriptional start site. These results suggest that HMGN3 regulates Glyt1 expression and demonstrate that members of the HMGN family can regulate the transcription of specific genes.In eukaryotes, all of the DNA is complexed with histone proteins and packaged into a highly folded, well-ordered, and dynamic structure called chromatin. This packaging modulates the ability of regulatory factors to access their DNA targets and plays a major role in regulating various nuclear activities, including transcription (18,48). Chromatin folding is modulated by numerous nuclear factors including nucleosome remodeling complexes, histone-modifying enzymes, and architectural proteins such as linker histones and HMG proteins. Members of the HMG superfamily interact with chromatin and DNA and affect a wide range of DNA-dependent activities such as transcription, replication, and recombination (9).One of the HMG families, the HMGN family, is comprised of small, basic proteins that bind specifically to nucleosomes (8). HMGN proteins are highly conserved and found only in vertebrates. The two founding members of the family, HMGN1 and HMGN2 (formerly named HMG-14 and HMG-17) (8), have been studied extensively. They contain a highly conserved nucleosome binding domain, a bipartite nuclear localization signal, and a C-terminal chromatin-unfolding domain (12, 47). When incorporated into minichromosomes, HMGN proteins confer a more open chromatin structure that is more sensitive to nucleases and that is transcribed and replicated more efficiently (13,14,35,46,50). Their ability to unfold chromatin also enhances the rate of DNA repair, as recently demonstrated in mice lacking HMGN1 (5).Although HMGN proteins display little or no DNA sequence specificity when binding to nucleosomes (45), several lines of evidence indicate that HMGN binding within the nucleus is nonrandom. Immunofluorescence studies have shown that HMGN proteins are localized in many foci within t...