HMGB2

Abstract: Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development1. In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development2. Here we show that the Dorsal switch protein 1 (DSP1), a Drosophila HMGB2 homologue, binds to a sequence present within Ab-Fab and in other characterized PREs. Addition of this motif to an artificial sequence containing Pleiohomeotic and GAGA factor consensus sites is sufficient for PcG protein recruitment in vivo. Mutations that abolish DSP1 binding to Ab-Fab and to a PRE from the engrailed locus lead to loss of PcG protein binding, loss of silencing, and switching of these PREs into constitutive TREs. The binding of DSP1 to PREs is therefore important for the recruitment of PcG proteins.

Abstract: Cellular senescence is a stable cell growth arrest that is characterized by the silencing of proliferation-promoting genes through compaction of chromosomes into senescence-associated heterochromatin foci (SAHF). Paradoxically, senescence is also accompanied by increased transcription of certain genes encoding for secreted factors such as cytokines and chemokines, known as the senescence-associated secretory phenotype (SASP). How SASP genes are excluded from SAHF-mediated global gene silencing remains unclear. In this study, we report that high mobility group box 2 (HMGB2) orchestrates the chromatin landscape of SASP gene loci. HMGB2 preferentially localizes to SASP gene loci during senescence. Loss of HMGB2 during senescence blunts SASP gene expression by allowing for spreading of repressive heterochromatin into SASP gene loci. This correlates with incorporation of SASP gene loci into SAHF. Our results establish HMGB2 as a novel master regulator that orchestrates SASP through prevention of heterochromatin spreading to allow for exclusion of SASP gene loci from a global heterochromatin environment during senescence.

Abstract: Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Abstract: The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 μmol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no γ-H2AX accumulated after 24–72 hours of incubation with 1 μmol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.[Mol Cancer Ther 2009;8(4):864–72]