Harmonics of Circadian Gene Transcription in Mammals

The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse

Telomerase Transduction in Cord Blood CD34+ Cells Interferes with Cellular Differentiation and Does Not Increase Their Proliferative Capacity.

Loss of telomeric repeats has been causally linked to replicative senescence and ageing in human cells. In contrast to normal somatic cells, which are telomerase-negative, hematopoietic stem cells have low levels of telomerase, which can be transiently up-regulated upon cytokine stimulation. Despite telomerase actions, proliferation of progenitor cells is accompanied by telomere shortening possibly leading to a reduction of their replicative potential. To test whether ectopic expression of telomerase can overcome telomere erosion in hematopoietic progenitor cells, we transduced CD34+ cord blood (CB) cells (n=7) with a retroviral vector including hTERT, the catalytic component of telomerase. CD34+ cells were cultured for 4 weeks in serum-free medium in the presence of stem cell factor (SCF). Flt-3 ligand (Flt-3 L), interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF). The hTERT transduced CD34+ CB cells exhibited significantly elevated telomerase activity (10.4-fold) compared to vector-only-transduced CD34+ cells. A peak telomere elongation of 500 bp was found in hTERT transduced CB cells at day 14 of the culture, but telomere shortening was not completely prevented. Surprisingly, the overall expansion potential was found to be significantly reduced in hTERT transduced cells (5.6-fold) relative to control cells which was due to an accelerated loss of CD34 expression and an increased cellular differentiation. These results indicate that telomeres can be elongated in hematopoietic progenitor cells by ectopic expression of hTERT although telomere shortening is not prevented. In addition, our data suggest that hTERT transduction in CD34+ CB cells does not increase their proliferative capacity, but appears to interfere with their differentiation potential

The Cytokine-inducible Scr Homology Domain-containing Protein Negatively Regulates Signaling by Promoting Apoptosis in Erythroid Progenitor Cells

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines, but its role in proliferation, differentiation, and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells, we generated green fluorescent protein (GFP)-tagged versions of wild type CIS, a mutant harboring an inactivated SH2 domain (CIS R107K), and a mutant with a deletion of the SOCS Box (CISΔBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence, both are essential for the growth inhibitory effect of CIS, whereas the CIS SOCS box is dispensable. Accordingly, the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly, apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus, CIS negatively regulates signaling at two levels, apoptosis and proliferation, and thereby sets a threshold for signal transduction

Distinct roles of Mdm2 and Mdm4 in red cell production

info:eu-repo/semantics/publishe

Identification of nucleocytoplasmic cycling as a remote sensor in cellular signaling by databased modeling

Considerable progress has been made in identifying the molecular composition of complex signaling networks controlling cell proliferation, differentiation, and survival. However, to discover general building principles and predict the dynamic behavior of signaling networks, it is necessary to develop quantitative models based on experimental observations. Here we report a mathematical model of the core module of the Janus family of kinases (JAK)–signal transducer and activator of transcription (STAT) signaling pathway based on time-resolved measurements of receptor and STAT5 phosphorylation. Applying the fitted model, we can determine the quantitative behavior of STAT5 populations not accessible to experimental measurement. By in silico investigations, we identify the parameters of nuclear shuttling as the most sensitive to perturbations and verify experimentally the model prediction that inhibition of nuclear export results in a reduced transcriptional yield. The model reveals that STAT5 undergoes rapid nucleocytoplasmic cycles, continuously coupling receptor activation and target gene transcription, thereby forming a remote sensor between nucleus and receptor. Thus, dynamic modeling of signaling pathways can promote functional understanding at the systems level