Ann-Kristin Östlund Farrants

The chromatin remodelling complex WSTF–SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription

Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein–protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference‐mediated knockdown of NM1 and WSTF reduced pre‐rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre‐assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.

The WSTF-SNF2h Chromatin Remodeling Complex Interacts with Several Nuclear Proteins in Transcription

The WSTF (Williams syndrome transcription factor) protein is involved in vitamin D-mediated transcription and replication as a component of two distinct ATP-dependent chromatin remodeling complexes, WINAC and WICH, respectively. We show here that the WICH complex (WSTF-SNF2h) interacts with several nuclear proteins as follows: Sf3b155/SAP155, RNA helicase II/Guα, Myb-binding protein 1a, CSB, the proto-oncogene Dek, and nuclear myosin 1 in a large 3-MDa assembly, B-WICH, during active transcription. B-WICH also contains RNAs, 45 S rRNA, 5 S rRNA, 7SL RNA, and traces of the U2 small nuclear RNA. The core proteins, WSTF, SNF2h, and nuclear myosin 1, are associated with the RNA polymerase III genes 5 S rRNA genes and 7SL, and post-transcriptional silencing of WSTF reduces the levels of these transcripts. Our results show that a WSTF-SNF2h assembly is involved in RNA polymerase III transcription, and we suggest that WSTF-SNF2h-NM1 forms a platform in transcription while providing chromatin remodeling.

The histone acetyltransferase PCAF associates with actin and hnRNP U for RNA polymerase II transcription.

ABSTRACT Actin is a key regulator of RNA polymerase (pol) II transcription. In complex with specific hnRNPs, it has been proposed that actin functions to recruit pol II coactivators during the elongation of nascent transcripts. Here, we show by affinity chromatography, protein-protein interaction assays, and biochemical fractionation of nuclear extracts that the histone acetyltransferase (HAT) PCAF associates with actin and hnRNP U. PCAF and the nuclear actin-associated HAT activity detected in the DNase I-bound protein fraction could be released by disruption of the actin-hnRNP U complex. In addition, actin, hnRNP U, and PCAF were found to be associated with the Ser2/5- and Ser2-phosphorylated pol II carboxy-terminal domain construct. Chromatin and RNA immunoprecipitation assays demonstrated that actin, hnRNP U, and PCAF are present at the promoters and coding regions of constitutively expressed pol II genes and that they are associated with ribonucleoprotein complexes. Finally, disruption of the actin-hnRNP U interaction repressed bromouridine triphosphate incorporation in living cells, suggesting that actin and hnRNP U cooperate with PCAF in the regulation of pol II transcription elongation.

Chromatin remodelling and actin organisation.

Chromatin remodelling is a prerequisite for nuclear processes, and cells have several different ways of remodelling the chromatin structure. The ATP‐dependent chromatin remodelling complexes are large multiprotein complexes that use ATP to change DNA–histone contacts. These complexes are classified into 4 sub‐families depending on the central ATPase. The switch mating type/sucrose non‐fermenting (SWI/SNF) complexes are mainly involved in transcriptional regulation, and this means that they are involved in many processes, such as the formation of actin filaments in the cytoplasm. SWI/SNF complexes are involved in the regulation of genes expressing cell adhesion proteins and extracellular matrix proteins. Actin is also present in the nucleus, affecting transcription, RNA processing and export. In addition, actin and actin‐related proteins are subunits of SWI/SNF complexes and the INO80‐containing complexes, another subfamily of ATP‐dependent chromatin remodelling complexes. Not all functions of the actin and actin‐related proteins in the complexes are yet clear: it is known that they play important roles in maintaining the stability of the proteins, possibly by bridging subunits and recruiting the complexes to chromatin.

Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy

The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.

The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes

The chromatin remodelling complex B-WICH, which comprises the William syndrome transcription factor (WSTF), SNF2h, and nuclear myosin 1 (NM1), is involved in regulating rDNA transcription, and SiRNA silencing of WSTF leads to a reduced level of 45S pre-rRNA. The mechanism behind the action of B-WICH is unclear. Here, we show that the B-WICH complex affects the chromatin structure and that silencing of the WSTF protein results in a compaction of the chromatin structure over a 200 basepair region at the rRNA promoter. WSTF knock down does not show an effect on the binding of the rRNA-specific enhancer and chromatin protein UBF, which contributes to the chromatin structure at active genes. Instead, WSTF knock down results in a reduced level of acetylated H3-Ac, in particular H3K9-Ac, at the promoter and along the gene. The association of the histone acetyl-transferases PCAF, p300 and GCN5 with the promoter is reduced in WSTF knock down cells, whereas the association of the histone acetyl-transferase MOF is retained. A low level of H3-Ac was also found in growing cells, but here histone acetyl-transferases were present at the rDNA promoter. We propose that the B-WICH complex remodels the chromatin structure at actively transcribed rRNA genes, and this allows for the association of specific histone acetyl-transferases.

Variations in the Composition of Mammalian SWI/SNF Chromatin Remodelling Complexes

The ATP‐dependent chromatin remodelling complexes SWI/SNF alter the chromatin structure in transcriptional regulation. Several classes of mammalian SWI/SNF complex have been isolated biochemically, distinguished by a few specific subunits, such as the BAF‐specific BAF250A, BAF250B and BRM, and the PBAF‐specific BAF180. We have determined the complex compositions using low stringency immunoprecipitation (IP) and shown that the pattern of subunit interactions was more diverse than previously defined classes had predicted. The subunit association at five gene promoters that depend on the SWI/SNF activity varied and the sequential chromatin immunoprecipitations revealed that different class‐specific subunits occupied the promoters at the same time. The low‐stringency IP showed that the BAF‐specific BAF250A and BAF250B and the PBAF‐specific BAF180 co‐exist in a subset of SWI/SNF complexes, and fractionation of nuclear extract on size‐exclusion chromatography demonstrated that sub‐complexes with unorthodox subunit compositions were present in the cell. We propose a model in which the constellations of SWI/SNF complexes are “tailored” for each specific chromatin target and depend on the local chromatin environment to which complexes and sub‐complexes are recruited. J. Cell. Biochem. 108: 565–576, 2009.

SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster

BackgroundThe SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA.ResultsWe have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo.ConclusionsWe have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.

Nuclear myosin 1 contributes to a chromatin landscape compatible with RNA polymerase II transcription activation

BackgroundNuclear myosin 1c (NM1) is emerging as a regulator of transcription and chromatin organization.ResultsUsing chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in combination with molecular analyses, we investigated the global association of NM1 with the mammalian genome. Analysis of the ChIP-Seq data demonstrates that NM1 binds across the entire mammalian genome with occupancy peaks correlating with distributions of RNA Polymerase II (Pol II) and active epigenetic marks at class II gene promoters. In mouse embryonic fibroblasts subjected to RNAi mediated NM1 gene silencing, we show that NM1 synergizes with polymerase-associated actin to maintain active Pol II at the promoter. NM1 also co-localizes with the nucleosome remodeler SNF2h at class II promoters where they assemble together with WSTF as part of the B-WICH complex. A high resolution micrococcal nuclease (MNase) assay and quantitative real time PCR shows that this mechanism is required for local chromatin remodeling. Following B-WICH assembly, NM1 mediates physical recruitment of the histone acetyl transferase PCAF and the histone methyl transferase Set1/Ash2 to maintain and preserve H3K9acetylation and H3K4trimethylation for active transcription.ConclusionsWe propose a novel genome-wide mechanism where myosin synergizes with Pol II-associated actin to link the polymerase machinery with permissive chromatin for transcription activation.

The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal

Hrp65, an evolutionary conserved RNA-binding protein from the midge Chironomus tentans, has a conserved DBHS (Drosophila behavior, human splicing) domain that is also present in several mammalian proteins. In a yeast two-hybrid screening we found that Hrp65 can interact with itself. Here we confirm the Hrp65 self-interaction by in vitro pull-down experiments and map the sequences responsible for the interaction to a region that we refer to as the protein-binding domain located within the DBHS domain. We also show that the protein-binding domains of Drosophila NonA and human PSF, two other proteins with conserved DBHS domains, bind to Hrp65 in the yeast two-hybrid system. These observations indicate that the protein-binding domain can mediate homodimerization of Hrp65 as well as heterodimerization between different DBHS-containing proteins. Moreover, analyses of recombinant Hrp65 by gel-filtration chromatography show that Hrp65 can not only dimerize but also oligomerize into complexes of at least three to six molecules. Furthermore, we have analyzed the functional significance of the Hrp65 self-interaction in cotransfection assays, and our results suggest that the interaction between different Hrp65 isoforms is crucial for their intracellular localization.