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  • 1.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Non-protein-coding RNA: Transcription and regulation of ribosomal RNA2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized.

    In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network.

    Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.

  • 2.
    Eberle, Andrea B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Böhm, Stefanie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Farrants, Ann-Kristin Östlund
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation2012In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 426, no 2, p. 147-152Article in journal (Refereed)
    Abstract [en]

    Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate the interactions between proteins and DNA in vivo. ChIP is often used as a quantitative tool, and proper quantification relies on the use of adequate references for data normalization. However, many ChIP experiments involve analyses of samples that have been submitted to experimental treatments with unknown effects, and this precludes the choice of suitable internal references. We have developed a normalization method based on the use of a synthetic DNA-antibody complex that can be used as an external reference instead. A fixed amount of this synthetic DNA-antibody complex is spiked into the chromatin extract at the beginning of the ChIP experiment. The DNA-antibody complex is isolated together with the sample of interest, and the amounts of synthetic DNA recovered in each tube are measured at the end of the process. The yield of synthetic DNA recovery in each sample is then used to normalize the results obtained with the antibodies of interest. Using this approach, we could compensate for losses of material, reduce the variability between ChIP replicates, and increase the accuracy and statistical resolution of the data.

  • 3.
    Hobein, Matthias
    et al.
    Stockholm University, Faculty of Science, Department of Physics.
    Orban, Istvan
    Stockholm University, Faculty of Science, Department of Physics.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, Department of Physics.
    Solders, Andreas
    Stockholm University, Faculty of Science, Department of Physics.
    Suhonen, Markus
    Stockholm University, Faculty of Science, Department of Physics.
    Fritioff, T.
    Stockholm University, Faculty of Science, Department of Physics.
    Tashenov, Stanislav
    Stockholm University, Faculty of Science, Department of Physics.
    Schuch, Reinhold
    Stockholm University, Faculty of Science, Department of Physics.
    Optimization of the Stockholm R-EBIT for the production and extraction of highly charged ions2010In: Journal of Instrumentation, ISSN 1748-0221, Vol. 5, no C11003Article in journal (Refereed)
    Abstract [en]

    We describe a refrigerated EBIT (R-EBIT) commissioned at the AlbaNova Research Center at Stockholm University. As an innovative solution, the superconducting magnet and the trapping drift tubes of the R-EBIT are cooled to a temperature of 4 K by a set of two cooling heads connected to helium compressors. This dry, i.e. liquid helium and liquid nitrogen free, system is easily operated and creates highly charged ions at a fraction of the cost of traditional liquid-cooled systems. A pulsed and continuous gas injection system was developed to feed neutral particles into the electron beam in the trap region. This improves significantly the highly charged ion production and R-EBIT performance. Fast extraction of ions from the R-EBIT yields very short ( < 100 ns), charge-separated ion bunches which can be either analysed using a straight time-of-flight section or sent to experimental beam lines following selection in a bending magnet. An emittance meter was used to measure the emittance of the ions extracted from the R-EBIT. The extracted ions were also re-trapped in a cylindrical Penning trap and properties of the re-trapped ions have been measured using the emittance meter. Results of these measurements are reported in this publication.

  • 4.
    Ryme, Jessica
    et al.
    Stockholm University.
    Asp, Patrik
    Stockholm University.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Cavellan, Erica
    Stockholm University.
    Farrants, Ann-Kristin Östlund
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Variations in the Composition of Mammalian SWI/SNF Chromatin Remodelling Complexes2009In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 108, no 3, p. 565-576Article in journal (Refereed)
    Abstract [en]

    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 BAF 180. 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.

  • 5. Sadeghif, Fatemeh
    et al.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Vintermist, Anna
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Östlund Farrant, Ann-Kristin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The B-WICH chromatin-remodelling complex initiates the regulation of RNA polymerase III by c-MycManuscript (preprint) (Other academic)
    Abstract [en]

    Transcription by RNA polymerase III in eukaryotic cells is closely associated with cell growth and proliferation, and regulated by several proliferative signals. In addition, the chromatin-remodelling complex B-WICH, comprised of William syndrome transcription factor, the ATPase SNF2h and nuclear myosin, binds to the 5S rRNA and 7SL genes and activates transcription, but the mechanism behind is poorly understood. Here, we have used high‑resolution MN walking to show that the role of B-WICH in RNA polymerase III transcription is to induce local alterations of the chromatin structure in the vicinity of the 5S rRNA and 7SL RNA genes. In the 5S rDNA, the remodelled region harbours an E-box, to which c-Myc, together with Max, binds in a B-WICH dependent way.  Both B-WICH and c-Myc are required for the subsequent histone acetylation of histone H3. Our results present two ways for c-Myc to alter 5S rRNA transcription; to bind to the RNA polymerase III machinery at the promoter and to an E-box in the intergenic spacer. We propose a model in which the B-WICH complex is required to maintain an open chromatin structure at these RNA polymerase III genes, which is a prerequisite for other regulatory factors to bind at the gene.

  • 6.
    Sadeghifar, Fatemeh
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Vintermist, Anna
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Östlund Farrants, Ann-Kristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    The B-WICH chromatin-remodelling complex facilitates the binding of c-Myc and histone acetyl transferases and regulates RNA pol III transcriptionManuscript (preprint) (Other academic)
    Abstract [en]

    Transcription of the 5S rRNA genes and 7SL genes by RNA polymerase III is necessary for cell growth and proliferation. The chromatin-remodelling complex B-WICH is associated with these genes, and siRNA-silencing of one component, the WSTF protein, reduces the level of transcription. However, the molecular mechanism is unclear. We show here that the role of B-WICH is to promote the binding of RNA polymerase III and RNA polymerase III factors, TFIIIA, TFIIIB and TFIIIC. WSTF knock down by siRNA resulted in a decreased recruitment of these initiation factors and, consequently, RNA polymerase III, to promoters. In addition, B-WICH induced a local alteration of the chromatin structure around the 5S rRNA and 7SL RNA genes, leading to a reduced acetylation of histone H3, in particular H3K9-Ac. A reduction in the level of WSTF also caused a loss of c-myc binding to the genes. We propose a model in which B-WICH complex is required to maintain an open chromatin structure around these RNA polymerase III genes, a prerequisite for other factors to associate at the gene.

  • 7.
    Sadeghifar, Fatemeh
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Vintermist, Anna
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Östlund Farrants, Ann-Kristin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The B-WICH chromatin-remodelling complex regulates RNA polymerase III transcription by promoting Max-dependent c-Myc binding2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 9, p. 4477-4490Article in journal (Refereed)
    Abstract [en]

    The chromatin-remodelling complex B-WICH, comprised of William syndrome transcription factor, the ATPase SNF2h and nuclear myosin, specifically activates RNA polymerase III transcription of the 5S rRNA and 7SL genes. However, the underlying mechanism is unknown. Using high-resolution MN walking we demonstrate here that B-WICH changes the chromatin structure in the vicinity of the 5S rRNA and 7SL RNA genes during RNA polymerase III transcription. The action of B-WICH is required for the binding of the RNA polymerase machinery and the regulatory factors c-Myc at the 5S rRNA and 7SL RNA genes. In addition to the c-Myc binding site at the 5S genes, we have revealed a novel c-Myc and Max binding site in the intergenic spacer of the 5S rDNA. This region also contains a region remodelled by B-WICH. We demonstrate that c-Myc binds to both sites in a Max-dependent way, and thereby activate transcription by acetylating histone H3. The novel binding patterns of c-Myc and Max link transcription of 5S rRNA to the Myc/Max/Mxd network. Since B-WICH acts prior to c-Myc and other factors, we propose a model in which the B-WICH complex is required to maintain an open chromatin structure at these RNA polymerase III genes. This is a prerequisite for the binding of additional regulatory factors.

  • 8. Sarshad, Aishe
    et al.
    Sadeghifar, Fatemeh
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Louvet, Emilie
    Mori, Raffaele
    Böhm, Stefanie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Al-Muzzaini, Bader
    Vintermist, Anna
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Fomproix, Nathalie
    Östlund, Ann-Kristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Percipalle, Piergiorgio
    Nuclear Myosin 1c Facilitates the Chromatin Modifications Required to Activate rRNA Gene Transcription and Cell Cycle Progression2013In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 9, no 3, article id e1003397Article in journal (Refereed)
    Abstract [en]

    Actin and nuclear myosin 1c (NM1) cooperate in RNA polymerase I (pol I) transcription. NM1 is also part of a multiprotein assembly, B-WICH, which is involved in transcription. This assembly contains the chromatin remodeling complex WICH with its subunits WSTF and SNF2h. We report here that NM1 binds SNF2h with enhanced affinity upon impairment of the actin-binding function. ChIP analysis revealed that NM1, SNF2h, and actin gene occupancies are cell cycle-dependent and require intact motor function. At the onset of cell division, when transcription is temporarily blocked, B-WICH is disassembled due to WSTF phosphorylation, to be reassembled on the active gene at exit from mitosis. NM1 gene knockdown and motor function inhibition, or stable expression of NM1 mutants that do not interact with actin or chromatin, overall repressed rRNA synthesis by stalling pol I at the gene promoter, led to chromatin alterations by changing the state of H3K9 acetylation at gene promoter, and delayed cell cycle progression. These results suggest a unique structural role for NM1 in which the interaction with SNF2h stabilizes B-WICH at the gene promoter and facilitates recruitment of the HAT PCAF. This leads to a permissive chromatin structure required for transcription activation.

  • 9.
    Stefanie, Böhm
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Judith, Domingo Prim
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Anna, Vintermist
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ann-Kristin, Östlund Farrants
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Non-coding RNAs from the rDNA intergenic repeat are transcribed by RNA polymerase I and II and have different functionsManuscript (preprint) (Other academic)
    Abstract [en]

    Long intergenic non-coding RNA, linc RNA, are often produced from intergenic sequences and have been ascribed diverse functions, such as regulating mRNA levels and being involved in the formation of heterochromatin. We show here that the intergenic spacer region (IGS) of the ribosomal DNA gene repeat in human cells is transcribed. Three ncRNAs, the IGS19asRNA, the IGS32asRNA and the IGS38RNA, of 500, 800 and 1300 bases, respectively, were isolated and investigated. Two of them, the IGS19asRNA and the IGS32asRNA, were transcribed in the antisense direction with respect to the rRNA and in the sense direction for the IGS38RNA. We also showed that the ncRNAs were transcribed by different RNA polymerases; the IGS19asRNA and the IGS38RNA were transcribed by RNA polymerase II and the IGS32asRNA were transcribed by RNA polymerase I. The three ncRNAs were also differentially regulated; IGS19asRNA induced upon heat shock and the level of the IGS32asRNA increased upon glucose feeding, similar to the 45S rRNA. In addition, the ncRNAs IGS19asRNA and IGS32asRNA were found at different locations in the nucleus, with IGS19asRNA located in a speckled pattern in the nucleus and IGS32asRNA associated with chromatin bound to heterochromatin protein 1. This suggests that the IGS32asRNA has a role in heterochromatin formation.

  • 10.
    Vintermist, Anna
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Sadeghifar, Fatemeh
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Louvet, Emilie
    Mansén, Anethe
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Percipalle, Pergiorgio
    Östlund Farrants, Ann-Kristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 4, article id e19184Article in journal (Refereed)
    Abstract [en]

    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.

  • 11.
    Yu, Simei
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Waldholm, Johan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Brahma regulates a specific trans-splicing event at the mod(mdg4) locus of Drosophila melanogaster2014In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 11, no 2, p. 134-145Article in journal (Refereed)
    Abstract [en]

    The mod(mdg4) locus of Drosophila melanogaster contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. We have studied the transcripts derived from one of the anti-sense regions within the mod(mdg4) locus in order to shed light on the expression of this complex locus. We have characterized the expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped their transcription start sites and cleavage sites, identified and quantified alternatively spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. In a previous study, we had shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. Here we show, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity.

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