Age of Huntington's disease (HD) motoric onset is strongly related to the number of CAG trinucleotide repeats in the huntingtin gene, suggesting that biological tissue age plays an important role in disease etiology. Recently, a DNA methylation based biomarker of tissue age has been advanced as an epigenetic aging clock. We sought to inquire if HD is associated with an accelerated epigenetic age. DNA methylation data was generated for 475 brain samples from various brain regions of 26 HD cases and 39 controls. Overall, brain regions from HD cases exhibit a significant epigenetic age acceleration effect (p=0.0012). A multivariate model analysis suggests that HD status increases biological age by 3.2 years. Accelerated epigenetic age can be observed in specific brain regions (frontal lobe, parietal lobe, and cingulate gyrus). After excluding controls, we observe a negative correlation (r=−0.41, p=5.5×10−8) between HD gene CAG repeat length and the epigenetic age of HD brain samples. Using correlation network analysis, we identify 11 co-methylation modules with a significant association with HD status across 3 broad cortical regions. In conclusion, HD is associated with an accelerated epigenetic age of specific brain regions and more broadly with substantial changes in brain methylation levels.
Specific detection of mRNA cleavage by 5′RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5′-RNA-linker-mediated RACE (5′-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5′-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. With its sensitivity and specificity, this variation on the 5′RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo.
Summary A murine recombinant retrovirus containing the cDNA encoding the human p55 interleukin-2 (IL2)-binding protein was used to insert this gene into a murine interleukin-3 (IL3)-dependentcell line, FD.C/1. Virus-infected cells, maintained in medium supplemented with IL3, expressedhumanp55on the cell surface and readily adapted to growth using human IL2. In the presence of human IL2. the synthesis ofthe endogenous murine p55 binding protein was induced in FD.C/1 cells, making it difficult to determine whether the human p55 protein was actively involved in the process of growth signal transduction. A cloned cell line, FD.huIL2R-2, was identified which grew in the presence of human IL2 but which had lost the ability to synthesize murine p55 protein. Growth of this clone was inhibited by the monoclonal antibody 2A3 which specifically blocked binding of human 1L2 to the human p55 binding protein. Analysis of restriction enzyme digests of FD.huIL2R-2 cell DNA revealed that a rearrangement of a murine p55 gene had occurred, implying that virus infection had resulted in the integration of retroviral DNA at a site close to or within a murine p55 gene. 1 f IL2 signal transduction involves binding to a surface heterodimeric receptor for IL2, it is argued that FD.huIL2R-2 cells contain an IL2 receptor complex of murine p70 and human p55 IL2-binding proteins. Alternatively, it is possible that integration of human p55 DNA into a site close to a murine p55 gene may lead to a hybrid p55 IL2-binding protein. If FD.huIL2R-2 cells express murine p70 IL2-binding protein as part ofthe receptor complex, the inability of cells to grow in murine IL2 implies that IL2 binding to p70 protein alone is insufficient for a growth signal in these cells. FD.huIL2R-2 cells grow at rates similar in IL3-or human IL2-dependent states. It is likely therefore that the biochemical pathways that control each of these lymphokine-dependent growth states are ver>' similar.
The cloned murine interleukin 3 (IL 3)-dependent cell lines FD.C/1, 32Dc1-23, and KP3 can each be switched to interleukin 2 (IL 2)-dependent growth states. Replication-defective retroviral vectors have been used to introduce the v-src oncogene into each of these cell lines maintained in either an IL 3- or an IL 2-dependent growth state. These cell lines maintained in an IL 3-dependent growth state were converted to lymphokine-independent growth after infection with v-src. These same cells maintained in an IL 2-dependent growth state and infected with v-src maintained strict lymphokine dependence for growth. Another cloned murine IL 3-dependent cell line, GM, can be switched to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent growth state. GM cells maintained as IL 3- or GM-CSF-dependent cells readily converted to a lymphokine-independent growth state when infected with v-src. These experiments indicate that either there exist differences in the biochemical mechanisms of signal transduction through the IL 3- and IL 2-specific receptors, or developmental processes associated with the switching of cells to an IL 2-dependent growth state influence expression of the v-src gene product. These cell lines offer new ways not only for analyzing biochemical pathways that regulate cell growth, but also for analyzing the control of oncogene expression.
Three cloned murine interleukin 3 (IL-3)-dependent cell lines have been converted to interleukin 2 (IL-2) or granulocyte-macrophage colony-stimulating factor (GM-CSF) growth-dependent states. FD.C/1 32Dcl-23 and GM cells grown and maintained as IL-3-dependent cell lines, and cells grown with GM-CSF have been infected with a murine recombinant retrovirus containing the v-src oncogene, and grown as lymphokine-independent cell lines. There is a significant increase in tyrosine kinase activity in cells which become lymphokine-independent. FD.C/1 and 32Dcl-23 cells maintained as IL-2-dependent cells lines and infected with the same virus did not grow as IL-2-independent cells. The lymphokine-independent cells FD.C/1src, 32Dsrc, and GMsrc all expressed high levels of tyrosine kinase activity, ranging from 5- to 20-fold more than levels measured in virus-infected cell lines maintained as IL-2-dependent cells. The exposure of FD.C/1src and 32Dsrc cells to IL-3, and GMsrc cells to IL-3 or GM-CSF, resulted in significant decreases in tyrosine kinase activity. These changes were rapidly reversed by removal of IL-3 or GM-CSF from these cells. However, the synthesis of v-src-specific RNA was not affected by the presence of IL-3 or GM-CSF in these cell lines. The biochemical pathways activated by IL-3 or GM-CSF inhibit the activity of the tyrosine kinase encoded by the v-src oncogene without altering gene transcription.
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