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

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

rRNA gene occupancy of NM1, SNF2h, and actin is cell cycle-dependent.

<p>(A) Schematic representation of the human rDNA transcription unit. (B) ChIP and qPCR on growing HeLa cells with antibodies as indicated, detected all along the human rDNA transcription unit. The values are presented as the percentages of the input signal for each primer pair. Error bars represent standard deviations. (C) Cell cycle profiles monitored by FACS analysis on growing HeLa cells, on nocodazole-blocked HeLa cells and on HeLa cells 2 h after release from the nocodazole block. (D) ChIP and qPCR on HeLa cells in early G1 at the rRNA gene promoter, 18S rDNA, 28S rDNA and IGS with antibodies as indicated below the bars. The values are presented as the percentage of the input signal for each primer pair. (E) ChIP and qPCR on mitotic HeLa cells blocked in prometaphase at the rRNA gene promoter, 18S and 28S rDNA as well as IGS with antibodies indicated below the bars. The values are presented as the percentage of the input signal for each primer pair.</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

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