Regulation of the actin-activated MgATPase activity of Acanthamoeba myosin II by phosphorylation of serine 639 in motor domain loop 2.

It had been proposed previously that only filamentous forms of Acanthamoeba myosin II have actin-activated MgATPase activity and that this activity is inhibited by phosphorylation of up to four serine residues in a repeating sequence in the C-terminal nonhelical tailpiece of the two heavy chains. We have reinvestigated these issues using recombinant WT and mutant myosins. Contrary to the earlier proposal, we show that two nonfilamentous forms of Acanthamoeba myosin II, heavy meromyosin and myosin subfragment 1, have actin-activated MgATPase that is down-regulated by phosphorylation. By mass spectroscopy, we identified five serines in the heavy chains that can be phosphorylated by a partially purified kinase preparation in vitro and also are phosphorylated in endogenous myosin isolated from the amoebae: four serines in the nonhelical tailpiece and Ser639 in loop 2 of the motor domain. S639A mutants of both subfragment 1 and full-length myosin had actin-activated MgATPase that was not inhibited by phosphorylation of the serines in the nonhelical tailpiece or their mutation to glutamic acid or aspartic acid. Conversely, S639D mutants of both subfragment 1 and full-length myosin were inactive, irrespective of the phosphorylation state of the serines in the nonhelical tailpiece. To our knowledge, this is the first example of regulation of the actin-activated MgATPase activity of any myosin by modification of surface loop 2.

SATB1-mediated functional packaging of chromatin into loops.

Mammalian genomes are organized into multiple layers of higher-order chromatin structure, and in this organization chromatin looping is a striking and crucial feature that brings together distal genomic loci into close spatial proximity. Such three-dimensional organization of chromatin has been suggested to be functionally important in gene regulation. Many important questions need to be addressed, such as what types of nuclear proteins are responsible for folding chromatin into loops, whether there are any genomic marks that serve as the core sites of chromatin folding events, how distal genomic sites are brought together, and what are the biological consequences for interactions between distal genomic loci. In order to address these fundamental questions, it is essential to devise and employ methods that can capture higher-order structures formed by specific nuclear proteins at high resolution. In this article, in order to describe methods of analyzing protein-mediated chromatin interactions, we will use as an example a global genome-organizer protein, SATB1, which mediates chromatin looping.

SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.

SATB1 (special AT-rich sequence binding protein 1) organizes cell type-specific nuclear architecture by anchoring specialized DNA sequences and recruiting chromatin remodeling factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4 and Il13, located in the 200-kb T-helper 2 (T(H)2) cytokine locus on mouse chromosome 11. We show that on T(H)2 cell activation, SATB1 expression is rapidly induced to form a unique transcriptionally active chromatin structure at the cytokine locus. In this structure, chromatin is folded into numerous small loops, all anchored to SATB1 at their base. In addition, histone H3 is acetylated at Lys9 and Lys14, and the T(H)2-specific factors GATA3, STAT6 and c-Maf, the chromatin-remodeling enzyme Brg1 and RNA polymerase II are all bound across the 200-kb region. Before activation, the T(H)2 cytokine locus is already associated with GATA3 and STAT6, showing some looping, but these are insufficient to induce cytokine gene expression. Using RNA interference, we show that on cell activation, SATB1 is required not only for compacting chromatin into dense loops at the 200-kb cytokine locus but also for inducing Il4, Il5, Il13 and c-Maf expression. Thus, SATB1 is a necessary determinant for the hitherto unidentified higher-order, transcriptionally active chromatin structure that forms on T(H)2 cell activation.

Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome.

Mutations in MECP2 are associated with Rett syndrome, an X-linked neurodevelopmental disorder. To identify genes targeted by Mecp2, we sequenced 100 in vivo Mecp2-binding sites in mouse brain. Several sequences mapped to an imprinted gene cluster on chromosome 6, including Dlx5 and Dlx6, whose transcription was roughly two times greater in brains of Mecp2-null mice compared with those of wild-type mice. The maternally expressed gene DLX5 showed a loss of imprinting in lymphoblastoid cells from individuals with Rett syndrome. Because Dlx5 regulates production of enzymes that synthesize gamma-aminobutyric acid (GABA), loss of imprinting of Dlx5 may alter GABAergic neuron activity in individuals with Rett syndrome. In mouse brain, Dlx5 imprinting was relaxed, yet Mecp2-mediated silent-chromatin structure existed at the Dlx5-Dlx6 locus in brains of wild-type, but not Mecp2-null, mice. Mecp2 targeted histone deacetylase 1 to a sharply defined, approximately 1-kb region at the Dlx5-Dlx6 locus and promoted repressive histone methylation at Lys9 at this site. Chromatin immunoprecipitation-combined loop assays showed that Mecp2 mediated the silent chromatin-derived 11-kb chromatin loop at the Dlx5-Dlx6 locus. This loop was absent in chromatin of brains of Mecp2-null mice, and Dlx5-Dlx6 interacted with far distant sequences, forming distinct active chromatin-associated loops. These results show that formation of a silent-chromatin loop is a new mechanism underlying gene regulation by Mecp2.

Tissue-specific nuclear architecture and gene expression regulated by SATB1.

Eukaryotic chromosomes are packaged in nuclei by many orders of folding. Little is known about how higher-order chromatin packaging might affect gene expression. SATB1 is a cell-type specific nuclear protein that recruits chromatin-remodeling factors and regulates numerous genes during thymocyte differentiation. Here we show that in thymocyte nuclei, SATB1 has a cage-like 'network' distribution circumscribing heterochromatin and selectively tethers specialized DNA sequences onto its network. This was shown by fluorescence in situ hybridization on wild-type and Satb1-null thymocytes using in vivo SATB1-bound sequences as probes. Many gene loci, including that of Myc and a brain-specific gene, are anchored by the SATB1 network at specific genomic sites, and this phenomenon is precisely correlated with proper regulation of distant genes. Histone-modification analyses across a gene-enriched genomic region of 70 kb showed that acetylation of histone H3 at Lys9 and Lys14 peaks at the SATB1-binding site and extends over a region of roughly 10 kb covering genes regulated by SATB1. By contrast, in Satb1-null thymocytes, this site is marked by methylation at H3 Lys9. We propose SATB1 as a new type of gene regulator with a novel nuclear architecture, providing sites for tissue-specific organization of DNA sequences and regulating region-specific histone modification.

SATB1 targets chromatin remodelling to regulate genes over long distances.

Eukaryotic chromosomes are organized inside the nucleus in such a way that only a subset of the genome is expressed in any given cell type, but the details of this organization are largely unknown. SATB1 ('special AT-rich sequence binding 1'), a protein found predominantly in thymocytes, regulates genes by folding chromatin into loop domains, tethering specialized DNA elements to an SATB1 network structure. Ablation of SATB1 by gene targeting results in temporal and spatial mis-expression of numerous genes and arrested T-cell development, suggesting that SATB1 is a cell-type specific global gene regulator. Here we show that SATB1 targets chromatin remodelling to the IL-2Ralpha ('interleukin-2 receptor alpha') gene, which is ectopically transcribed in SATB1 null thymocytes. SATB1 recruits the histone deacetylase contained in the NURD chromatin remodelling complex to a SATB1-bound site in the IL-2Ralpha locus, and mediates the specific deacetylation of histones in a large domain within the locus. SATB1 also targets ACF1 and ISWI, subunits of CHRAC and ACF nucleosome mobilizing complexes, to this specific site and regulates nucleosome positioning over seven kilobases. SATB1 defines a class of transcriptional regulators that function as a 'landing platform' for several chromatin remodelling enzymes and hence regulate large chromatin domains.