Selective transcriptional regulation by Myc: Experimental design and computational analysis of high-throughput sequencing data

AbstractThe gene expression programs regulated by the Myc transcription factor were evaluated by integrated genome-wide profiling of Myc binding sites, chromatin marks and RNA expression in several biological models. Our results indicate that Myc directly drives selective transcriptional regulation, which in certain physiological conditions may indirectly lead to RNA amplification. Here, we illustrate in detail the experimental design concerning the high-throughput sequencing data associated with our study (Sabò et al., Nature. (2014) 511:488–492) and the R scripts used for their computational analysis

A model of N-terminal Cyclin T1 based on FRET experiments

Human Cyclin T1 is the cyclin partner of kinase CDK9 in the positive transcription elongation factor b (P-TEFb). P-TEFb is recruited by Tat, the transactivator of the human immunodeficiency virus type 1 (HIV-1), to the viral promoter by direct interactions between Tat, Cyclin T1 and thecis-acting transactivation-responsive region (TAR) present at the 5′-end of each viral mRNA. At present, no structural data for Cyclin T1 are available. Here, we build a structural model of an N-terminus portion of Cyclin T1 (aa 27–263) based on the X-ray structure of Cyclin H. The model is compared with site directed mutagenesis data from the literature and validated by fluorescence resonance energy transfer (FRET) using Tat as a probe in living cells. This model provides a first step towards the structural characterization of the CDK9–CycT1–Tat-TAR complex, which is crucial for HIV-1 replication and may constitute a promising target for pharmaceutical intervention

Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity

Abstract: It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies

Acetylation of Conserved Lysines in the Catalytic Core of Cyclin-Dependent Kinase 9 Inhibits Kinase Activity and Regulates Transcription▿ †

Promoter clearance and transcriptional processivity in eukaryotic cells are fundamentally regulated by the phosphorylation of the carboxy-terminal domain of RNA polymerase II (RNAPII). One of the kinases that essentially performs this function is P-TEFb (positive transcription elongation factor b), which is composed of cyclin-dependent kinase 9 (CDK9) associated with members of the cyclin T family. Here we show that cellular GCN5 and P/CAF, members of the GCN5-related N-acetyltransferase family of histone acetyltransferases, regulate CDK9 function by specifically acetylating the catalytic core of the enzyme and, in particular, a lysine that is essential for ATP coordination and the phosphotransfer reaction. Acetylation markedly reduces both the kinase function and transcriptional activity of P-TEFb. In contrast to unmodified CDK9, the acetylated fraction of the enzyme is specifically found in the insoluble nuclear matrix compartment. Acetylated CDK9 associates with the transcriptionally silent human immunodeficiency virus type 1 provirus; upon transcriptional activation, it is replaced by the unmodified form, which is involved in the elongating phase of transcription marked by Ser2-phosphorylated RNAPII. Given the conservation of the CDK9 acetylated residues in the catalytic task of virtually all CDK proteins, we anticipate that this mechanism of regulation might play a broader role in controlling the function of other members of this kinase family

N-Myc is SUMOylated at lysine 349.

<p>(<b>A</b>) Mouse N-Myc protein sequence (from aa 301 to 450) with consensus SUMO acceptor sites (as predicted by SUMOplot™) highlighted in red. (<b>B</b>) 293T cells were transfected with plasmids expressing the indicated proteins. Lysates were analyzed by immunoprecipitation and immunoblotting as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091072#pone-0091072-g001" target="_blank">Fig. 1</a>. (<b>C</b>) The Myc region corresponding to the SUMO acceptor site in mouse N-Myc is aligned with N-Myc sequences from other species and also c-Myc and L-Myc corresponding regions. Mouse N-Myc lysine 349 is highlighted in red.</p

SUMOylation of Myc proteins.

<p>(<b>A</b>) Immunoblot analysis in denaturing conditions of 293T cells transfected with plasmids expressing the indicated proteins. (<b>B</b>) Lysates from (<b>A</b>) were immunoprecipitated (IP) in denaturing conditions with anti-Flag beads, and the precipitates subsequently analyzed by immunoblotting (IB) with the indicated antibodies. (<b>C</b>) as in (<b>A</b>) and (<b>B</b>) but for HeLa and U2OS cells.</p

Protein stresses induce N-Myc SUMOylation.

<p>(<b>A</b>) HeLa cells were transfected with plasmids expressing the indicated proteins and either mock treated or treated for 30 min with 0.7 M NaCl, 3.7% EtOH or heat-shock at 43°C (HS). Lysates were analyzed by immunoprecipitation and immunoblotting in denaturing conditions with the indicated antibodies. The histogram at the bottom represents the quantification of the ratio of monoSUMOylated to total wild-type N-Myc, normalized to the mock-treated cells: values represent the mean and s.d. from five independent experiments. (<b>B</b>) Lan-1 cells were either mock treated, treated with 10 µM MG132 for 6 h or heat-shocked at 43°C for 1 h (HS). Lysates were analyzed as in <b>A</b>. CTRL IP: IP with N-Myc antibody without cell lysate.</p

SUMOylation of N-Myc in neuroblastoma cells.

<p>(<b>A</b>) Immunoblot analysis in denaturing conditions of different neuroblastoma cell lines after 6 h treatment with 10 µM MG132. (*): non-specific band. (<b>B</b>) Immunoprecipitation (IP) with anti-N-Myc antibodies in denaturing conditions and analysis by immunoblotting with the indicated antibodies. (<b>C</b>) SHSY5Y cells were infected with pQCXIP retroviral vectors expressing Flag-HA tagged WT or K349R N-Myc proteins and expression of selected mRNAs was measured by RT-PCR (after normalization to the housekeeper RPPO). The histogram represents the mean and s.d. of three independent experiments. The total levels of the indicated proteins were assessed by immunoblot. (<b>D-F</b>) SK-N-BE(2) cells were infected with pQCXIN empty vector, Flag-HA-N-Myc WT or K349R (mouse cDNA) and superinfected with pGIPZ-PURO shN-Myc or control shRNA. (<b>D</b>) Immunoblot analysis of the level of endogenous and Flag-HA tagged exogenous N-Myc protein in mock treated cells and cells treated with MG132 10 µM. (<b>E</b>) FACS analysis of BrdU incorporation (% of positive cells); the histogram represents the mean and s.d. of three independent experiments. The numbering of the samples corresponds to that in (<b>D</b>). In neither of the quantitative assays used (panels C, E) was a statistically significant difference observed between the WT and K349R forms of N-Myc. (<b>F</b>) Colony Assay. Cells were plated in 6-well plates, incubated for 7 days and stained with Crystal violet. Numbers in each plate indicate relative cell densities, as assessed by absorbance at 595 nm following solubilization of the dye with acetic acid.</p

SUMOylation defective N-Myc mutant does not reveal critical differences respect to the wild type counterpart.

<p>(<b>A</b>) U2OS cells transfected with Flag tagged N-Myc WT or K349R mutant were treated with CHX 50 µg/ml for the indicated times. Cells were then lysed and the levels of the Flag-N-Myc protein were measured by quantitative immunoblotting. The graph represents the means of three independent experiments. Immunoblot of one representative experiment is shown. It is noteworthy that the half-life of exogenous N-Myc measured here (ca. 110 min) is in range with that seen in analogous experiments for either N-Myc (157 min <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091072#pone.0091072-Otto1" target="_blank">[27]</a>) or c-Myc (97-100 min <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091072#pone.0091072-Faiola1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091072#pone.0091072-Popov1" target="_blank">[65]</a>). (<b>B</b>) 293T cells were transfected with plasmids expressing the indicated proteins. Lysates were analyzed by immunoprecipitation and immunoblotting as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091072#pone-0091072-g001" target="_blank">Fig. 1</a>, but in non-denaturing conditions (co-IP lysis buffer: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091072#s2" target="_blank">methods</a>). (<b>C</b>) HeLa cells were transfected with the reporter plasmids pNuc-Luc (left panel) or p15-Luc (right panel) together with pRL-TK (as a normalizer) and expression plasmids for Flag-c-Myc, Flag-N-Myc WT or Flag-N-Myc K349R, as indicated. The transcriptional activity was measured with a Dual Luciferase Assay kit (Promega). The histograms represent the mean and s.d. of three independent experiments. The total levels of the indicated proteins were assessed by immunoblot. (<b>D-H</b>) Primary MEFs were infected with retroviral vectors coding for N-MycER™ WT or K349R and treated or not with 4-hydroxy-tamoxifen (OHT). (<b>D</b>) RT-PCR measurement of target mRNAs in OHT treated (48 h) versus control cells after normalization to the housekeeper RPPO. The histogram represents the mean and s.d. of three independent experiments. (<b>E</b>) Growth curves showing cumulative cell numbers for N-MycER™ WT or K349R MEFs treated or not with OHT for up to 11 days. The curves represent the mean and s.d. of three independent cell counts. (<b>F</b>) FACS analysis of BrdU incorporation (% of positive cells) after 24 h of OHT or control treatment. The histogram represents the mean and s.d. of three independent experiments. (<b>G</b>) Luminescence-based measurement of Caspase activity (Caspase-Glo 3/7, Promega) after 24 h of OHT or control treatment, the bars represent the mean and s.d. of three independent experiments. In neither of the quantitative assays used (panels A, C, D, E, F, G) was a statistically significant difference observed between the WT and K349R forms of N-Myc. (<b>H</b>) Immunoblot analysis of p53, ARF and γH2AX after 48 of OHT or control treatment.</p

SUMOylation of c-Myc at lysines 323 and 326.

<p>(<b>A</b>) 293T cells were transfected with plasmids expressing the indicated proteins. Lysates were analyzed by immunoprecipitation and immunoblotting as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091072#pone-0091072-g001" target="_blank">Fig. 1</a>. Cells were treated with MG132 10 µM or DMSO for 6 h, as indicated. (<b>B</b>) as in (<b>A</b>) but with wild type or K323, 326R (2KR) mutant c-Myc proteins.</p