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Biomedical subjects

Katherine L West

Publications and source records attributed to Katherine L West.

8 recordsLinked to original sources
PubMed2005

Chromosomal protein HMGN1 enhances the acetylation of lysine 14 in histone H3.

The acetylation levels of lysine residues in nucleosomes, which are determined by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin-related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1-/- cells are lower than in Hmgn1+/+ cells. Induced expression of wild-type HMGN1, but not of a mutant that does not bind to chromatin, in Hmgn1-/- cells elevates the levels of H3K14ac. In vivo, HMGN1 elevates the levels of H3K14ac by enhancing the action of HAT. In vitro, HMGN1 enhances the ability of PCAF to acetylate nucleosomal, but not free, H3. Thus, HMGN1 modulates the levels of H3K14ac by binding to chromatin. We suggest that HMGN1, and perhaps similar architectural proteins, modulates the levels of acetylation in chromatin by altering the equilibrium generated by the opposing enzymatic activities that continuously modify and de-modify the histone tails in nucleosomes.

Acetylation↗
PubMed2005

Increased tumorigenicity and sensitivity to ionizing radiation upon loss of chromosomal protein HMGN1.

We report that loss of HMGN1, a nucleosome-binding protein that alters the compaction of the chromatin fiber, increases the cellular sensitivity to ionizing radiation and the tumor burden of mice. The mortality and tumor burden of ionizing radiation-treated Hmgn1-/- mice is higher than that of their Hmgn1+/+ littermates. Hmgn1-/- fibroblasts have an altered G2-M checkpoint activation and are hypersensitive to ionizing radiation. The ionizing radiation hypersensitivity and the aberrant G2-M checkpoint activation of Hmgn1-/- fibroblasts can be reverted by transfections with plasmids expressing wild-type HMGN1, but not with plasmids expressing mutant HMGN proteins that do not bind to chromatin. Transformed Hmgn1-/- fibroblasts grow in soft agar and produce tumors in nude mice with a significantly higher efficiency than Hmgn1+/+ fibroblasts, suggesting that loss of HMGN1 protein disrupts cellular events controlling proliferation and growth. Hmgn1-/- mice have a higher incidence of multiple malignant tumors and metastases than their Hmgn1+/+ littermates. We suggest that HMGN1 optimizes the cellular response to ionizing radiation and to other tumorigenic events; therefore, loss of this protein increases the tumor burden in mice.

Animals↗
PubMed2005

Chromosomal protein HMGN1 modulates the expression of N-cadherin.

HMGN1 is a nuclear protein that binds to nucleosomes and alters the accessibility of regulatory factors to their chromatin targets. To elucidate its biological function and identify specific HMGN1 target genes, we generated Hmgn1-/- mice. DNA microarray analysis of Hmgn1+/+ and Hmgn1-/- embryonic fibroblasts identified N-cadherin as a potential HMGN1 gene target. RT-PCR and western blot analysis confirmed a linkage between HMGN1 expression and N-cadherin levels. In both transformed and primary mouse embryonic fibroblasts (MEFs), HMGN1 acted as negative regulator of N-cadherin expression. Likewise, the N-cadherin levels in early embryos of Hmgn1-/- mice were higher than those of their Hmgn1+/+ littermates. Loss of HMGN1 increased the adhesiveness, motility and aggregation potential of Hmgn1-/- MEFs, a phenotype consistent with increased levels of N-cadherin protein. Re-expression of wild-type HMGN1, but not of the mutant HMGN1 protein that does not bind to chromatin, in Hmgn1-/- MEFs, decreased the levels of N-cadherin and restored the Hmgn1+/+ phenotype. These studies demonstrate a role for HMGN1 in the regulation of specific gene expression. We suggest that in MEFs, and during early mouse development, the interaction of HMGN1 with chromatin down-regulates the expression of N-cadherin.

Animals↗
PubMed2004

Chromosomal protein HMGN1 modulates histone H3 phosphorylation.

Here we demonstrate that HMGN1, a nuclear protein that binds to nucleosomes and reduces the compaction of the chromatin fiber, modulates histone posttranslational modifications. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of phospho-S10-H3 and enhances the rate of stress-induced phosphorylation of S10-H3. In vitro, HMGN1 reduces the rate of phospho-S10-H3 by hindering the ability of kinases to modify nucleosomal, but not free, H3. During anisomycin treatment, the phosphorylation of HMGN1 precedes that of H3 and leads to a transient weakening of the binding of HMGN1 to chromatin. We propose that the reduced binding of HMGN1 to nucleosomes, or the absence of the protein, improves access of anisomysin-induced kinases to H3. Thus, the levels of posttranslational modifications in chromatin are modulated by nucleosome binding proteins that alter the ability of enzymatic complexes to access and modify their nucleosomal targets.

Animals↗
PubMed2004

Chromosomal proteins HMGN3a and HMGN3b regulate the expression of glycine transporter 1.

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.

Amino Acid Transport Systems, Neutral↗
PubMed2003

Chromosomal protein HMGN1 enhances the rate of DNA repair in chromatin.

We report that HMGN1, a nucleosome binding protein that destabilizes the higher-order chromatin structure, modulates the repair rate of ultraviolet light (UV)-induced DNA lesions in chromatin. Hmgn1(-/-) mouse embryonic fibroblasts (MEFs) are hypersensitive to UV, and the removal rate of photoproducts from the chromatin of Hmgn1(-/-) MEFs is decreased as compared with the chromatin of Hmgn1(+/+) MEFs; yet, host cell reactivation assays and DNA array analysis indicate that the nucleotide excision repair (NER) pathway in the Hmgn1(-/-) MEFs remains intact. The UV hypersensitivity of Hmgn1(-/-) MEFs could be rescued by transfection with plasmids expressing wild-type HMGN1 protein, but not with plasmids expressing HMGN1 mutants that do not bind to nucleosomes or do not unfold chromatin. Transcriptionally active genes, the main target of the NER pathways in mice, contain HMGN1 protein, and loss of HMGN1 protein reduces the accessibility of transcribed genes to nucleases. By reducing the compaction of the higher-order chromatin structure, HMGN1 facilitates access to UV-damaged DNA sites and enhances the rate of DNA repair in chromatin.

Animals↗
PubMed2002

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability.

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIbeta. We show that topo IIbeta is a DNA-dependent ATPase that appears to fit Michaelis-Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k(cat) for ATP hydrolysis by human DNA topo IIbeta in the presence of DNA is 2.25 s(-1). We have characterised a topo IIbeta derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s(-1), while not significantly altering the apparent K(m). The specificity constant for the interaction between ATP and topo IIbeta (kcat/K(mapp)) showed a 90% reduction for betaS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIbeta. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.

Adenosine Triphosphatases↗