HMGB Proteins Research Articles

Specificity of the Stimulatory Interaction between Chromosomal HMGB Proteins and the Transcription Factor Dof2 and Its Negative Regulation by Protein Kinase CK2-mediated Phosphorylation

The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that can contribute to transcriptional control by interaction with certain transcription factors. Using the transcription factor Dof2 and five different maize HMGB proteins, we have examined the specificity of the HMGB-transcription factor interaction. The HMG-box DNA binding domain of HMGB1 is sufficient for the interaction with Dof2. Although all tested HMGB proteins can interact with Dof2, the various HMGB proteins stimulate the binding of Dof2 to its DNA target site with different efficiencies. The HMGB5 protein is clearly the most potent facilitator of Dof2 DNA binding. Maximal stimulation of the DNA binding by the HMGB proteins requires association of HMGB and Dof2 prior to DNA binding. HMGB5 and Dof2 form a ternary complex with the DNA, but within the protein-DNA complex the interaction of HMGB5 and Dof2 is different from that in solution, as in contrast to the proteins in solution, they cannot be cross-linked with glutaraldehyde when bound to DNA. Phosphorylation of HMGB1 by protein kinase CK2 abolishes the interaction with Dof2 and the stimulation of Dof2 DNA binding. These findings indicate that transcription factors may recruit certain members of the HMGB family as assistant factors.

Differential chromatin association and nucleosome binding of the maize HMGA, HMGB, and SSRP1 proteins.

In plants, chromosomal high mobility group (HMG) proteins have been identified in the HMGA family, containing A/T-hook DNA binding motifs, and in the HMGB family, containing an HMG-box DNA binding domain, that are considered architectural factors in chromatin. We have characterized the association of the HMGA protein, five different HMGB proteins, and the structure-specific recognition protein 1 (SSRP1) with maize chromatin by extraction experiments using NaCl, ethidium bromide, spermine, and distamycin A. The difference in the release of the proteins from chromatin by these reagents indicates that they are differentially associated with chromatin. This was confirmed by treatment of chromatin with micrococcal nuclease, demonstrating that the HMGA, HMGB2/3, and SSRP1 proteins are enriched in the highly nuclease-sensitive fraction of chromatin, which is likely to be transcriptionally competent. As examined by electrophoretic mobility shift analyses, the HMGA protein and the proteins containing an HMG domain (HMGB proteins and SSRP1) bind specifically to purified maize mononucleosomes that contain a histone octamer and approximately 165 bp of DNA. The mode of interaction with the nucleosomes differs for HMGA and HMGB proteins. In the case of the HMGB1 protein, the full-length protein is required for specific nucleosome binding, as the individual HMG-box DNA binding domain (which is sufficient for DNA interactions) interacts nonspecifically with the nucleosomes. Collectively, these findings indicate that HMGA, the various HMGB proteins, and SSPR1 are differentially associated with plant chromatin and may act as architectural factors in different nucleoprotein structures.

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2.

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.

Stability of the maize chromosomal high-mobility-group proteins, HMGa and HMGb,in vivo

Chromosomal non-histone high-mobility-group (HMG) proteins represent essential components of eukaryotic chromatin and have also been isolated from a variety of plants. In maize, studies on structure and function of the two larger of the four major HMG proteins have recently been performed and are now extended by analysis of their in vivo stability using pulse-chase experiments in a cell suspension culture. The half-life of the analyzed HMGa and HMGb proteins was found to be 65 h or more than 78 h, respectively.