|
|
|
|
|
|
|||||||
|
 |
|||||||
|
Contents
*To download a copy |
Chromatin Epigenetics: Nucleosome-binding Proteins Modulate the Levels of Histone Posttranslational Modifications in Chromatin
We explored the possibility that chromatin-binding structural proteins, such as HMGNs, which are devoid of enzymatic activity, are part of the mechanism that regulates the levels of chromatin modifications. HMGNs are a family of proteins that bind to nucleosomes without specificity for the underlying DNA sequence and induce structural and functional changes in chromatin. We reasoned that the binding of these proteins to nucleosomes may induce local or global changes in chromatin and shift the equilibrium between the activities of histone-modifying and -demodifying enzymes. To test this hypothesis, we used specific antibodies to examine the levels of several modifications in the tail of histone H3, isolated from either wild-type, or from Hmgn1–/– mouse embryonic fibroblasts (MEFs). We found that loss of HMGN1 protein elevated the levels of phosphorylation at serine 10 (H3S10p) and serine 28 (H3S28p) but decreased the levels of acetylation at lysine 14 (H3K14ac). Reexpression of the HMGN1 in the Hmgn1–/– cells, by induction of stably integrated vectors, elevated the levels of H3K14ac and reduced the levels of H3S10p, proof that the levels of these modifications are indeed regulated by HMGN1. Significantly, reexpression of a mutant HMGN1 that does not bind to chromatin did not alter the levels of these modifications, an indication that HMGN1 modulates histone modifications by binding to chromatin. Further in vitro studies and analysis of cells treated with HDAC inhibitors revealed that HMGN1 elevates the levels of H3K14ac by enhancing the activity of a specific HAT, rather than by reducing the demodifying activity of an HDAC. Similar studies on the mechanisms whereby HMGN1 reduces the levels of H3S10p indicate that the binding of the protein to nucleosomes hinders the ability of specific kinases to access and modify their targets. The in vivo studies are fully supported by in vitro analyses indicating that HMGN1 affects these H3 modifications only in the context of chromatin. The modification of purified H3 protein is not affected by HMGN1. Thus, HMGN1 can either enhance or reduce the level of a specific modification in chromatin. Interestingly, a close homolog of HMGN1, named HMGN2, enhanced the acetylation of H3K14 more efficiently than HMGN1 but did not inhibit the phosphorylation of H3S10, suggesting HMGN variant–specific effects on histone modification. Current analysis of a series of mutants in which distinct domains of HMGN1 were swapped with domains of HMGN2 indicate that distinct domains of the proteins are involved in enhancement of acetylation and reduction of phosphorylation, further proof for HMGN-specific effects on histone modifications. We have previously demonstrated that the linker histone H1 inhibits the acetylation of H3K14 (Herrera JE et al. Mol Cell Biol 20: 523–9, 2000). Taken together, these studies establish that chromatin-binding structural proteins modulate the levels of chromatin modifications, most likely by altering the ability of chromatin modifiers to access and modify their targets. HMGNs and H1 may affect the accessibility of sites in chromatin either by inducing global changes in the “compaction” of the chromatin fiber, or by inducing steric changes at the local levels of the nucleosome. Our studies demonstrate that structural proteins alter the equilibrium generated by the activities of the enzymes that determine the levels of chromatin modifications, and point to an additional mechanism that regulates these epigenetic markers (Figure 1). Figure 1. Model illustrating the effects of the structural chromatin-binding proteins H1, HMGN1, and HMGN2 on the levels of specific histone modifications. These proteins affect the levels of histone modifications by altering the dynamic equilibrium between the enzymes that continuously add or remove chemical tags, such as acetyl or phosphate groups, to histone tails. N2, HMGN2; S10, serine 10; K14, lysine 14; S10p, phosphorylated S10; H1, histone 1; K14ac, acetylated K14; HAT, histone acetylase; HDAC, histone deacetylase.
|
|||||||
|
 |
osttranslational
modifications in the tails of the nucleosomal histones are important epigenetic
markers and serve as potential targets for cancer therapy. In living cells,
the levels of histone modifications are not fixed. They are in a continuous
state of flux and reflect the equilibrium reached between the activities of
enzymes that continuously modify, and those that continuously demodify specific
histone residues. For example, the equilibrium between the opposing activities
of histone acetylases (HATs) and deacetylases (HDACs) or kinases and phosphatases
determines the levels of acetylation and phosphorylation in chromatin. Recruitment
of histone-modifying or -demodifying enzyme complexes to specific sites is part
of the mechanism that regulates gene expression. 