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

Chromatin remodelling of ribosomal genes - be bewitched by B-WICH

Transcription of the ribosomal genes accounts for the majority of transcription in the cell due to the constant high demand for ribosomes. The number of proteins synthesized correlates with an effective ribosomal biogenesis, which is regulated by cell growth and proliferation. In the work presented in this thesis, we have investigated the ribosomal RNA genes 45S and 5S rRNA, which are transcribed by RNA Pol I and RNA Pol III, respectively. The focus of this work is the chromatin remodelling complex B-WICH, which is composed of WSTF, the ATPase SNF2h and NM1. We have studied in particular its role in ribosomal gene transcription. We showed in Study I that B-WICH is required to set the stage at rRNA gene promoters by remodelling the chromatin into an open, transcriptionally active configuration. This results in the binding of histone acetyl transferases to the genes and subsequent histone acetylation, which is needed for ribosomal gene activation. Study II investigated the role of B-WICH in transcription mediated by RNA polymerase III. We showed that B-WICH is essential to create an accessible chromatin atmosphere at 5S rRNA genes, which is compatible with the results obtained in Study 1. In this case, however, B-WICH operates as a licensing factor for c-Myc and the Myc/Max/Mxd network. Study III confirmed the importance and the function of the B-WICH complex as an activator of ribosomal genes. We demonstrated that B-WICH is important for the remodelling of the rDNA chromatin into an active, competent state in response to extracellular stimuli, and that the association of the B-WICH complex to the rRNA gene promoter is regulated by proliferative and metabolic changes in cells. The work presented in this thesis has confirmed that the B-WICH complex is an important regulator and activator of Pol I and Pol III transcription. We conclude that B-WICH is essential for remodelling the rDNA chromatin into a transcriptionally active state, as required for efficient ribosomal gene transcription.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. </p

Chromatin remodelling of ribosomal genes - be bewitched by B-WICH

Transcription of the ribosomal genes accounts for the majority of transcription in the cell due to the constant high demand for ribosomes. The number of proteins synthesized correlates with an effective ribosomal biogenesis, which is regulated by cell growth and proliferation. In the work presented in this thesis, we have investigated the ribosomal RNA genes 45S and 5S rRNA, which are transcribed by RNA Pol I and RNA Pol III, respectively. The focus of this work is the chromatin remodelling complex B-WICH, which is composed of WSTF, the ATPase SNF2h and NM1. We have studied in particular its role in ribosomal gene transcription. We showed in Study I that B-WICH is required to set the stage at rRNA gene promoters by remodelling the chromatin into an open, transcriptionally active configuration. This results in the binding of histone acetyl transferases to the genes and subsequent histone acetylation, which is needed for ribosomal gene activation. Study II investigated the role of B-WICH in transcription mediated by RNA polymerase III. We showed that B-WICH is essential to create an accessible chromatin atmosphere at 5S rRNA genes, which is compatible with the results obtained in Study 1. In this case, however, B-WICH operates as a licensing factor for c-Myc and the Myc/Max/Mxd network. Study III confirmed the importance and the function of the B-WICH complex as an activator of ribosomal genes. We demonstrated that B-WICH is important for the remodelling of the rDNA chromatin into an active, competent state in response to extracellular stimuli, and that the association of the B-WICH complex to the rRNA gene promoter is regulated by proliferative and metabolic changes in cells. The work presented in this thesis has confirmed that the B-WICH complex is an important regulator and activator of Pol I and Pol III transcription. We conclude that B-WICH is essential for remodelling the rDNA chromatin into a transcriptionally active state, as required for efficient ribosomal gene transcription.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. </p

The B-WICH chromatin-remodelling complex initiates the regulation of RNA polymerase III by c-Myc

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.</p

Non-coding RNAs from the rDNA intergenic repeat are transcribed by RNA polymerase I and II and have different functions

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.</p

Nuclear Myosin 1c Facilitates the Chromatin Modifications Required to Activate rRNA Gene Transcription and Cell Cycle Progression

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.AuthorCount:10;</p

NM1 controls the levels of H3K9 acetylation for the activation of pol I transcription.

<p>(A) Chromatin profile from NM1 knockdown cells (NM1 RNAi, red line) and control cells (Control scrRNAi, blue line) shown as 2ΔCt of undigested and MNase digested cross-linked chromatin. The position of each primer pair used is given below the graph; 2c (coding) denotes Position 2 in the coding region. Error bars represent standard deviations of three separate experiments. (B) Chromatin profile from HEK293T cells stably expressing V5-wtNM1, V5-RK605AA NM1 or V5-ΔC NM1 shown as 2ΔCt of undigested and MNase digested cross-linked chromatin. The position of each primer pair is indicated below the graph; 2c (coding), Position 2 in the coding region. Error bars represent standard deviations of three separate experiments. (C–E) ChIP and qPCR analysis on chromatin isolated from NM1 knockdown cells (NM1 RNAi) and control cells (scrRNAi), (C) using antibody against WSTF, SNF2h, NM1 and actin, (D) antibodies to pol I, UBF or PCAF, and (E) antibodies against histone H3 acetylated on K9 (H3K9Ac) or histone H3 acetylated on K14 (H3K14Ac). In all cases, qPCR analysis was performed with primers amplifying rRNA gene promoters. The values are presented as the percentage of the input signal for each primer pair. Error bars represent standard deviations. Significances [(*), <i>p</i> = 0.05 and (**), <i>p</i> = 0.02] were obtained by Student's T-test, two-sample equal variance.</p

NM1 gene silencing by RNAi leads to a delay in cell cycle progression.

<p>(A) Cell cycle profile measured by flow cytometry on propidium iodide-stained HeLa cells subjected to RNAi-mediated NM1 gene knockdown or control non-specific RNAi (scrRNAi). Standard ratios of G1, S and G2/M phases are shown in relation to scrRNAi. Where indicated, significances (<i>p</i>-values) were calculated by Student's T-test relative to control RNAi. S-phase, <i>p</i>_RK605AA = 0.0395 (*). (B) Analysis of cell cycle profile on HeLa cells subjected to BDM treatment. The control unsynchronized population of HeLa cells shows standard ratios of G1 and G2/M phases. Increase of the G2/M cell population is observed after a nocodazole block. Prometaphase cells were harvested by mechanical shock and released from the nocodazole block (0 h), in which case the cell cycle profile exhibits only a mitotic population. Two hours after release from the nocodazole block, appearance of the G1 peak indicates that cells have exited mitosis. However, the presence of 20 mM BDM added immediately after release of the nocodazole block led to delayed cell cycle progression with an increased number of cells in mitosis. (C) Cell cycle profiles for HEK293T cells expressing V5-wtNM1, V5-RK605AA NM1, V5-ΔIQ NM1 and V5-ΔC NM1. DNA staining was done by propidium iodide. Where indicated, significances (<i>p</i>-values) were calculated by Student's T-test relative to HEK293T cells not expressing any of the V5-tagged NM1 constructs. G1 phase, <i>p</i>_{ΔC NM1} = 0.02 (**), <i>p</i>_{ΔIQ NM1} = 0.006 (***); S-phase, <i>p</i>_RK605AA = 0.0008 (***), <i>p</i>_{ΔC NM1} = 0.016 (*); G2/M phase, <i>p</i>_{ΔC NM1} = 0.007 (***). (D–E) Cell cycle profiles obtained by EdU incorporation for HEK293T cells expressing V5-wtNM1, V5-RK605AA NM1,V5-ΔIQ NM1 and V5-ΔC NM1, following synchronization in G1/S phase with aphidicolin. In Panel (E), the numbers of cells in S phase were quantified 2 h after release from the aphidicolin block.</p