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1.  The STAT3-DNMT1 connection 
JAK-STAT  2012;1(4):257-260.
Gene activity is regulated by transcriptional and epigenetic mechanisms. A paper in 2005 by Zhang et al.1 showed that STAT3 binds to the DNA methyl transferase, DNMT1 and their data indicated that STAT3 may cause epigenetic gene silencing by targeting DNMT1 to the PTPN6 promoter. Now, a paper by Lee et al.2 has fleshed out the mechanism. They provide evidence that acetylation of STAT3 regulates the binding of DNMT1, CpG DNA methylation and regulation of several genes, including that encoding the estrogen receptor α (ESR1) in breast cancer cells.
doi:10.4161/jkst.22436
PMCID: PMC3670282  PMID: 24058781
STAT3; DNMT1; acetylation; DNA methylation; epigenetics; transcription; cancer
2.  Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry 
Proteomic analysis of T cells emerging from quiescence identifies dynamic network-level changes in key cellular processes. Disruption of two such processes, ribosome biogenesis and RNA splicing, reveals that the programs controlling cell growth and cell-cycle entry are separable.
The authors conduct a proteomic and protein interaction network analysis of human T lymphocytes during entry into the first cell cycle.Inhibiting the induction of eIF6 (60S ribosome biogenesis) causes T cells to enter the cell cycle without growing in size.Inhibiting the induction of SF3B2/SF3B4 (U2/U12-dependent RNA splicing) allows an increase in cell size without entering the cell cycle.These results provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells.
Regulating the transition of cells such as T lymphocytes from quiescence (G0) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G0. We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G0 into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.
doi:10.1038/msb.2012.5
PMCID: PMC3321526  PMID: 22415777
cell cycle; cell size; mass spectrometry; proteomics; T cells
3.  A functional assay for microRNA target identification and validation 
Nucleic Acids Research  2012;40(10):e75.
MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3′-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.
doi:10.1093/nar/gks145
PMCID: PMC3378903  PMID: 22323518
4.  p27Kip1 and p130 Cooperate To Regulate Hematopoietic Cell Proliferation In Vivo†  
Molecular and Cellular Biology  2006;26(16):6170-6184.
To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1−/−; p130−/− mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1−/− counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1−/−; p130−/− animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1−/− splenocytes. The finding that the p27Kip1−/−; p130−/− splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.
doi:10.1128/MCB.02182-05
PMCID: PMC1592787  PMID: 16880527
5.  Commitment Point during G0→G1 That Controls Entry into the Cell Cycle 
Molecular and Cellular Biology  2003;23(7):2351-2361.
Initiation of T-lymphocyte-mediated immune responses involves two cellular processes: entry into the cell cycle (G0→G1) for clonal proliferation and coordinated changes in surface and secreted molecules that mediate effector functions. However, a point during G0→G1 beyond which T cells are committed to enter the cell cycle has not been defined. We define here a G0→G1 commitment point that occurs 3 to 5 h after CD3 and CD28 stimulation of human CD4 or CD8 T cells. Transition through this point requires cdk6/4-cyclin D, since inhibition with TAT-p16INK4A during the first 3 to 5 h prevents cell cycle entry and maintains both naive and memory T cells in G0. Transition through the G0→G1 commitment point is also necessary for T cells to increase in size, i.e., to enter the cellular growth cycle. However, transition through this point is not required for the induction of effector functions. These can be initiated while cells are maintained in G0 with TAT-p16INK4A. We have termed this quiescent, activated state G0(A). Our data provide proof of the principle that entry of T cells into the cell cycle and cellular growth cycles are coupled at the G0→G1 commitment point but that these processes can be uncoupled from the early expression of molecules of effector functions.
doi:10.1128/MCB.23.7.2351-2361.2003
PMCID: PMC150729  PMID: 12640120
6.  FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal 
The Journal of Cell Biology  2002;156(3):531-542.
Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim−/− mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling.
These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB–mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.
doi:10.1083/jcb.200108084
PMCID: PMC2173339  PMID: 11815629
apoptosis; cytokine; mitochondria; PI3K; forkhead
7.  Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27KIP1 
Molecular and Cellular Biology  2000;20(24):9138-9148.
Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1 transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.
PMCID: PMC102172  PMID: 11094066
8.  Polyribosome binding of rabbit globin messenger RNA and messenger ribonucleoprotein labelled with bacteriophage-T4 RNA ligase and 5′-[32P] phosphocytidine 3′-phosphate 
Nucleic Acids Research  1983;11(1):1-10.
Rabbit polyribosomal globin messenger RNA (mRNA) and messenger ribonucleoprotein (mRNP) were labelled at the 3′ poly(A) tail to high specific activity with T4 RNA ligase and [5′-32P]pCp without consequent loss of functional activity. Labelled message was translated in both micrococcal nuclease treated and untreated rabbit reticulocyte lysates, as shown by the formation of labelled polyribosomes. The utilisation of labelled messenger was abolished by T2 toxin or sodium fluoride which are known to inhibit protein synthesis.
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PMCID: PMC325686  PMID: 6866762

Results 1-8 (8)