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

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

A functional NM1 is required for the activation of pol I transcription.

<p>(A) RNAi-mediated NM1 gene knockdown in HeLa cells analyzed by double immunostaining and confocal microscopy with antibodies to NM1 and fibrillarin, after transfection of NM1-specific siRNA or control oligos (scrRNAi). Scale bars, 5 µm. (B) Steady state expression levels for NM1, WSTF, SNF2h and actin monitored on immunoblots of total lysates prepared from HeLa cells subjected to NM1 gene knockdown (NM1 RNAi) or from cells subjected to scrRNAi. (C) Semiquantitative densitometric quantification of NM1 steady state protein expression relative to actin. (D) qRT-PCR analysis of 45S pre-rRNA performed on total RNA prepared from HeLa cells subjected to NM1 gene knockdown (NM1 RNAi) or from cells subjected to control siRNA oligonucleotides (scrRNAi). The 45S pre-rRNA levels are relative to GAPDH mRNA. (E–F) FUrd incorporation assays on living HeLa cells subjected to (E) NM1 gene knockdown by RNAi, or (F) treated with BDM. Transcription was monitored by short FUrd pulses to follow incorporation into nascent nucleolar transcripts. Quantification of the FUrd foci after immunostaining and confocal microscopy was performed by measurements on randomly selected nucleolar regions in the images. The signal was quantified using ImageJ software. The average of the mean grey values in control cells was determined, and defined as hundred percent of signal. The average of the mean grey values measured after treatment was expressed proportionally. n = number of cells in each experiment. Error bars represent standard deviations. (G) rRNA synthesis in HEK293T cells stably expressing V5-wtNM1, V5-RK605AA, V5-ΔC or V5-ΔIQ NM1 mutants. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT–qPCR using GAPDH mRNA as internal control. Error bars represent the standard deviation of three independent experiments. Significances [<i>p</i>_{RK605AA NM1} = 0.019 (*), <i>p</i>_{ΔC NM1} = 0.0006 (***), <i>p</i>_{ΔIQ NM1} = 0.05 (*)] were obtained by Student's T-test, two-sample, equal variance.</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

During cell division, WSTF phosphorylation events govern the assembly and disassembly of B-WICH.

<p>(A) NM1, SNF2h and WSTF are co-precipitated from protein extracts prepared from growing HeLa cells. Bound proteins were detected on immunoblots. 10% of the input is shown in Lane 1. IP, immunoprecipitation. (B) Co-precipitations of NM1, SNF2h and WSTF are impaired from protein extracts prepared from mitotic HeLa cells at t = 0 min from the nocodazole block. Bound proteins were detected on immunoblots. 10% of the input is loaded in Lane 1. IP, immunoprecipitation. (C) WSTF becomes phosphorylated at the onset of mitosis. Lysates were prepared from growing HeLa cells (Lane 1), mitotic HeLa cells at t = 0 min from the nocodazole block (Lanes 2 and 3) or HeLa cells after t = 120 min release from the block (Lanes 4 and 5). Where indicated extracts were subjected to phosphatase treatment (Lanes 3 and 5). In all cases lysates were separated by 7% phospho-affinity SDS-PAGE and analyzed on immunoblots for WSTF and actin. (D) Co-precipitations of WSTF, SNF2h and NM1 are dependent on WSTF phosphorylation. Mitotic extracts from HeLa cells at t = 0 min from the nocodazole block, untreated (Lanes 1–3) or treated with phosphatase (Lanes 4–6), were subjected to immunoprecipitations with anti-WSTF antibodies or non-specific IgGs. Bound proteins were separated by SDS PAGE and analyzed on immunoblots for WSTF, SNF2h and NM1. 5% of the input is shown in Lane 1. IP, immunoprecipitation. (E–F) WSTF steady state expression levels on immunoblots of lysates prepared from control (scrRNAi) and WSTF-silenced HeLa cells. Right panel, densitometric quantification of WSTF steady state protein expression relative to actin. (G) In growing HeLa cells, co-precipitations of NM1 and SNF2h are impaired as a consequence of WSTF gene knockdown by siRNA (WSTF RNAi) but not when cells are subjected to control experiments with scrambled RNAi oligonucleotides (scrRNAi). In Lanes 1 and 4, 20% of the input material was loaded.</p

NM1 C-terminus and intact motor function are required for the association of NM1, SNF2h, and actin with rRNA genes.

<p>(A) ChIP assays performed on HEK293T cells or HEK293T cells constitutively expressing V5-tagged wt and mutated NM1 constructs (as indicated) were analyzed by qPCR. The bars diagrams show the relative amounts of rDNA promoter, 18S rDNA and 28S rDNA precipitated with V5 antibodies. The values are presented as the percentage of the input signal for each pair. Error bars represent standard deviations. (B) Steady state expression levels of endogenous WSTF and SNF2h analyzed on immunoblots of total cell lysates from HEK293T cells expressing V5-wtNM1, V5-RK605AA NM1 and V5-ΔC NM1 mutants. WSTF and SNF2h steady state expression levels were normalized against endogenous actin levels. Expressions of V5-wtNM1, V5-RK605AA NM1 and V5-ΔC NM1 mutants were monitored with anti-V5 antibodies. (C–E) ChIP assays on chromatin from HEK293T cells stably expressing V5-wtNM1, V5-RK605AA and V5-ΔC NM1 mutants with antibodies against V5, WSTF, SNF2h and actin. qPCR analysis was performed with primers amplifying promoter and 18S rDNA. The values are presented as the percentage of the input signal for each pair. Error bars represent standard deviations. (F–G) ChIP assays on chromatin from HeLa cells untreated (F) or treated with BDM (G) using the indicated antibodies (see below the bars). qPCR analysis was performed with primers amplifying rRNA gene promoter, 18S and 28S rDNA as well as IGS. The values are presented as the percentage of the input signal for each primer pair. Error bars represent standard deviations.</p

NM1 interacts with SNF2h and the HAT PCAF.

<p>(A) Schematic representation of V5-tagged wt and mutated NM1 constructs stably expressed in HEK293T cell lines. (B) Steady state expressions of V5-tagged wt and mutated NM1 constructs were monitored on immunoblots for V5 and actin antibodies as loading control. (C) Co-precipitations of SNF2h and PCAF from total lysates obtained from HEK293T cells stably expressing wt and mutated V5-tagged NM1 constructs as indicated. 10% of the input is shown in Lane 1. IP, immunoprecipitation. (D) Semiquantitative densitometric analysis of endogenous SNF2h levels co-precipitated with V5-tagged NM1 constructs relative to input.</p

A speculative two-step model in which NM1 bridges the pol I machinery and chromatin <i>via</i> an interaction with SNF2h that competes with actin.

<p>(I) NM1 interacts with polymeric actin and with the rDNA <i>via</i> its C-terminus, generating local force that pulls the polymerase along active gene. (II) Upon NM1 dissociation from actin, NM1 interacts with SNF2h in a WSTF-dependent manner, a mechanism that provides a way to stabilize the WICH complex on the rDNA, to recruit PCAF, and to maintain the levels of H3K9 acetylation required for transcription activation.</p