Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

Cdc6, which alters chromatin ultrastructure to allow DNA replication in muscle stem cells transitioning out of quiescence, is identified as a target of the MyoD transcription factor

The dynamics of E1A in regulating networks and canonical pathways in quiescent cells

<p>Abstract</p> <p>Background</p> <p>Adenoviruses force quiescent cells to re-enter the cell cycle to replicate their DNA, and for the most part, this is accomplished after they express the E1A protein immediately after infection. In this context, E1A is believed to inactivate cellular proteins (e.g., p130) that are known to be involved in the silencing of E2F-dependent genes that are required for cell cycle entry. However, the potential perturbation of these types of genes by E1A relative to their functions in regulatory networks and canonical pathways remains poorly understood.</p> <p>Findings</p> <p>We have used DNA microarrays analyzed with Bayesian ANOVA for microarray (BAM) to assess changes in gene expression after E1A alone was introduced into quiescent cells from a regulated promoter. Approximately 2,401 genes were significantly modulated by E1A, and of these, 385 and 1033 met the criteria for generating networks and functional and canonical pathway analysis respectively, as determined by using Ingenuity Pathway Analysis software. After focusing on the highest-ranking cellular processes and regulatory networks that were responsive to E1A in quiescent cells, we observed that many of the up-regulated genes were associated with DNA replication, the cell cycle and cellular compromise. We also identified a cadre of up regulated genes with no previous connection to E1A; including genes that encode components of global DNA repair systems and DNA damage checkpoints. Among the down-regulated genes, we found that many were involved in cell signalling, cell movement, and cellular proliferation. Remarkably, a subset of these was also associated with p53-independent apoptosis, and the putative suppression of this pathway may be necessary in the viral life cycle until sufficient progeny have been produced.</p> <p>Conclusions</p> <p>These studies have identified for the first time a large number of genes that are relevant to E1A's activities in promoting quiescent cells to re-enter the cell cycle in order to create an optimum environment for adenoviral replication.</p

MOLECULAR LUMINESCENCE SPECTRA OF COUMARINS AND FUROCOUMARINS AT 77∘K77^{\circ} K

Author Institution: Department of Chemistry, Texas Tech UniversityThe lowest excited singlet and triplet states of coumarins and furocoumarins (psoralens) have been assigned to the $(\pi,\pi^{*})$ type on the basis of luminescence and photoselection measurements in ethanol at $77^{\circ} K$, Surprisingly, coumarins and furoumarins without substituent at positions 3 and 4 of the pyrone moiety showed their 0-0 phosphorescence bands at essentially the same location regardless of size and substituent of these molecules. For example, the 0-0 bands are 457 (coumarin), 456, 450 (isopsoralen), 457 (8-methoxy-psoralen), and ca. 452 nm (4,5,8-trimethly psoralen). Polarizations of the 0-0 bands are negative or minimum, and out-of-plane vibration (possibly C-H) with maximum polarization is also present. 5-Methoxypsoralen shows its 0-0 band at 473 nm. Phosphorescence lifetimes of these compounds are found to be 0.5-1 sec. The above results are interpreted in terms of highly localized triplet states in the region of the pyrone double bond, and SCF molecular orbital data will be presented in support of the localized nature of the triplet states. Relative directions of the two transition moments $(\pi, \pi^{*})$ have also been determined from the lowest polarized fluorescence excitation spectra

E1A Interacts with Two Opposing Transcriptional Pathways To Induce Quiescent Cells into S Phase ▿

Despite data suggesting that the adenovirus E1A protein of 243 amino acids creates an S-phase environment in quiescent cells by overcoming the nucleosomal repression of E2F-regulated genes, the precise mechanisms underlying E1A's ability in this process have not yet been defined at the biochemical level. In this study, we show by kinetic analysis that E1A, as opposed to an E1A mutant failing to bind p130, can temporally eliminate corepressor complexes consisting of p130-E2F4 and HDAC1/2-mSin3B from the promoters of E2F-regulated genes in quiescent cells. Once the complexes are removed, the di-methylation of H3K9 at these promoters becomes dramatically diminished, and this in turn allows for the acetylation of H3K9/14 and the recruitment of activating E2F family members, which is then followed by the transcriptional activity of the E2F-regulated genes. Remarkably, although an E1A mutant that can no longer bind to a histone acetyltransferase (PCAF) is as capable as wild-type E1A in eliminating corepressor complexes and methyl groups from the promoters of these genes, it cannot mediate the acetylation of H3K9/14 or induce their transcription. These findings suggest that corepressors as well as coactivators are acted upon by E1A to derepress E2F-regulated genes in quiescent cells. Thus, our results highlight for the first time a functional relationship between E1A and two transcriptional pathways of differing functions for transitioning cells out of quiescence and into S phase

A Purified Adenovirus 289-Amino-Acid ElA Protein Activates RNA Polymerase III Transcription In Vitro and Alters Transcription Factor TFIIIC

RESEARCH PAPERWe have previously demonstrated that a purified bacterially synthesized ElA 289-amino-acid protein is capable of stimulating transcription from the promoters of genes transcribed by RNA polymerase II in vitro (R. Spangler, M. Bruner, B. Dalie, and M. L. Harter, Science 237:1044-1046, 1987). In this study, we show that this protein is also capable of transactivating in vitro the adenovirus virus-associated (VA1) RNA gene transcribed by RNA polymerase UI. Pertinent to the transcription of this gene is the rate-limiting component, TFIIIC, which appears to be of two distinct forms in uninfected HeLa cells. The addition of an oligonucleotide containing a TFIIIC binding site to HeLa whole-cell extracts inhibits VAT transcription by sequestering TFIIIC. However, the addition of purified ETA to extracts previously challenged with the TFIIIC oligonucleotide restores the level of VAT transcription. When included in the same reaction, an ElA-specific monoclonal antibody reverses the restoration. Incubation of purified ETA with either HeLa cell nuclear or whole-cell extracts alters the DNA-binding properties of TFIIIC as detected by gel shift assays. This alteration does not occur if ElA-specific antibody and ETA protein are added simultaneously to the extract. In contrast, the addition of this antibody to extracts at a later time does not reverse the alteration observed in the TFIIIC binding activities. Never at any time did we note the formation of novel TFIIIC-promoter complexes after the addition of ETA to nuclear extracts. These results clearly establish that ETA mediates its effect on VAT transcription through TFIIIC in a very rapid yet indirect manner. The results also establish that a bacterially produced ETA protein can directly participate in RNA polymerase II transcription without the requirement of celiular protein synthesis or other viral proteins.RUTGERS UNIVERSITY SCHOOL OF MEDICINE (UMDNJ) and THE CLEVELAND CLINIC FOUNDATION (CCF

A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program

The molecular mechanism(s) that are responsible for suppressing MyoD’s transcriptional activities in undifferentiated skeletal muscle cells have not yet been determined. We now show that MyoD associates with a histone deacetylase-1 (HDAC1) in these cells and that this interaction is responsible for silencing MyoD-dependent transcription of endogenous p21 as well as muscle-specific genes. Specifically, we present evidence that HDAC1 can bind directly to MyoD and use an acetylated MyoD as a substrate in vitro, whereas a mutant version of HDAC1 (H141A) can not. Further more, this mutant also fails to repress MyoD-mediated transcription in vivo, and unlike wild-type HDAC1 it can not inhibit myogenic conversion, as judged by confocal microscopy. Finally, we show that an endogenous MyoD can be acetylated upon its conversion to a hypophosphorylated state and only when the cells have been induced to differentiate. These results provide for a model which postulates that MyoD may be co-dependent on HDAC1 and P/CAF for temporally controlling its transcriptional activity before and after the differentiation of muscle cells

p21 and retinoblastoma protein control the absence of DNA replication in terminally differentiated muscle cells

Abstract. During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E–cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state. Key words: C2C12 cells • E1A • cell cycle • p21 • DNA replicatio

The Response of microRNAs to Solar UVR in Skin-Resident Melanocytes Differs between Melanoma Patients and Healthy Persons

<div><p>The conversion of melanocytes into cutaneous melanoma is largely dictated by the effects of solar ultraviolet radiation (UVR). Yet to be described, however, is exactly how these cells are affected by intense solar UVR while residing in their natural microenvironment, and whether their response differs in persons with a history of melanoma when compared to that of healthy individuals. By using laser capture microdissection (LCM) to isolate a pure population of melanocytes from a small area of skin that had been intermittingly exposed or un-exposed to physiological doses of solar UVR, we can now report for the first time that the majority of UV-responsive microRNAs (miRNAs) in the melanocytes of a group of women with a history of melanoma are down-regulated when compared to those in the melanocytes of healthy controls. Among the miRNAs that were commonly and significantly down-regulated in each of these women were miR-193b (<i>P</i><0.003), miR-342-3p (<i>P</i><0.003), miR186 (<i>P</i><0.007), miR-130a (<i>P</i><0.007), and miR-146a (<i>P</i><0.007). To identify genes potentially released from inhibition by these repressed UV-miRNAs, we analyzed databases (e.g., DIANA-TarBase) containing experimentally validated microRNA-gene interactions. In the end, this enabled us to construct UV-miRNA-gene regulatory networks consisting of individual genes with a probable gain-of-function being intersected not by one, but by several down-regulated UV-miRNAs. Most striking, however, was that these networks typified well-known regulatory modules involved in controlling the epithelial-to-mesenchymal transition and processes associated with the regulation of immune-evasion. We speculate that these pathways become activated by UVR resulting in miRNA down regulation only in melanocytes susceptible to melanoma, and that these changes could be partially responsible for empowering these cells toward tumor progression.</p></div

MicroRNAs differentially expressed in melanocytes of skin after exposure to ssUVR.

<p><b>(a and b)</b> A color-coded heat map representations of UV-miRNAs in melanocytes commonly expressed in melanoma patients or healthy individuals. The map in <b>(a)</b> ranks each miRNA by their fold-change (RQ), beginning with those having the most negative RQ value (top) to the highest negative RQ value (bottom), whereas the map in <b>(b)</b> ranks each from the most negative to the most positive RQ value. The color bars at the top of <b>(a)</b> and <b>(b)</b> represent the normalized intensity of down-regulated or up-regulated UV-miRNAs with the shaded blue corresponding to a decrease in expression and that of shaded red to an increase in expression. The left margin shows the representative miRNAs and the bottom of the columns show each individual sample. MicroRNAs in the heat map of <b>(a)</b> and which match those previously identified in melanoma tissue are marked by a star. <b>(c)</b> An identical set of UV-miRNAs in the melanocytes of patients or healthy persons is effected differently by ssUVR. The <i>y</i>-axis indicates relative gene expression, and the whisker bars indicate the standard error of the mean. MicroRNAs that match those previously identified in melanoma tissue are marked by a star.</p