Fig. 5
NM1 controls the levels of SNF2h at class II promoters but it is not required for chromatin remodeling. NM1 C-terminus and intact motor function are required for the association of Pol II with promoters of class II genes. a Venn diagrams displaying overlap of occupancy between NM1 and SNF2h across genic regions. b, c ChIP and qPCR analysis on chromatin isolated from NM1 knockdown cells (NM1 RNAi) and control cells (scrRNAi), using antibodies against WSTF, SNF2h and NM1. The qPCR analysis was performed with primers amplifying the gene promoters of (b) the mouse checkpoint clamp complex protein Rad9a gene (Rad9a), (c) the mouse ribosomal protein gene Rpl19. In all cases, the values are presented as the percentage of the input signal for each primer pair. All ChIP experiments were performed at least three time (n = 3). Error bars represent standard deviations. Significances (p-values) were obtained by Student’s t-test, two-sample equal variance. Panel B, pWSTF = 0.042 (*), pSNF2h = 0.0036 (**); panel C, pWSTF = 0.001 (**), pSNF2h = 0.017 (*). ns = non-significant. d Chromatin profile from HEK293T cells subjected to control (scrRNAi) or to NM1 gene silencing (NM1 RNAi) shown as 2ΔCt of undigested and MNase digested cross-linked chromatin. Similar to panel H, the position of each primer pair within the human RPL19 gene is indicated below the graph. The location +100 bp corresponds to the coding region of the gene; the location +1 bp corresponds to the transcription start site; the location −70 bp corresponds to a sequence upstream the transcription start site within the gene promoter. The results were successfully reproduced in four separate experiments (n = 4). Error bars represent standard deviations of four separate experiments. e Schematic illustration of the V5-tagged NM1 constructs expressed in HEK293T cells (top panel) and their expressions as monitored on immunoblots using anti-V5 epitope antibodies as well as expressions of actin, SNF2h and WSTF. f 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 within the human RPL19 gene is indicated below the graph. The location +100 bp corresponds to the coding region of the gene; the location +1 bp corresponds to the transcription start site; the location −70 bp corresponds to a sequence upstream the transcription start site within the gene promoter. The results were successfully reproduced in four separate experiments (n = 4). Error bars represent standard deviations of four separate experiments. An effect is detected upon expression of V5-RK605AA NM1 at −70, p = 0.01 (*), and at −140 p = 0.008 (**). g ChIP assays and qPCR analysis on chromatin isolated from HEK293T cells stably expressing V5-wtNM1 or V5-RK605AA NM1 using antibodies against V5, WSTF, SNF2h, actin and non-specific IgGs. qPCR analysis was performed with primers amplifying around the −70 bp region upstream the transcription start site within the human RPL19 gene promoter. The values are presented as the percentage of the input signal for each pair. The experiment was successfully reproduced two times (n = 2). h Pol II transcription analysis performed in HEK293T cells stably expressing V5-wtNM1, V5-RK605AA or V5-ΔC NM1 mutants. For the analysis, relative mRNA levels from the human genes RAD9A and RPL19 were monitored from polyA mRNA preparations by RT-qPCR using GAPDH mRNA as internal control. Error bars represent the standard deviation of three independent experiments (n = 3). RAD9 mRNA levels, pRK605AA NM1 = 5.552E-06 (****), pΔC NM1 = 0.000186 (***); RPL19 mRNA levels, pRK605AA NM1 = 0.05543 (*), pΔC NM1 = 0.000142 (***). i ChIP assays performed on crosslinked chromatin isolated from HEK293T cells constitutively expressing V5-wt NM1, V5-RK605AA NM1 or V5-ΔC NM1 using an antibody against the active Pol II (4H8) and non-specific IgGs. The precipitated DNA was analyzed by qPCR with primers amplifying the human RAD9A and RPL19 promoters. The bar diagrams show the relative amounts of DNA precipitated with the indicated antibodies. The values are presented as the percentage of the input signal for each pair. Error bars represent standard deviations of three independent experiments (n = 3). Left panel (RAD9 promoter occupancy), pRK605AA NM1 = 0.000118 (***), pΔC NM1 = 0.00032 (***); right panel (RPL19 promoter occupancy), pRK605AA NM1 = 5.826E-06 (****), pΔC NM1 = 5.288E-06 (****) bp base pair, ChIP-Seq chromatin immunoprecipitation and deep sequencing, NM1 nuclear myosin 1c, qPCR quantitative polymerase chain reaction