Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of SNF5 binding in human pluripotent embryonic carcinoma NCCIT and SNF5 overexpressed NCCIT cells. We generated genome-wide cSNF5 maps of NCCIT and SNF5 overexpressed NCCIT cells from chromatin immunoprecipitated DNA. SNF5 and OCT4 seem not to share their binding in OCT4 centered binding plot in control, while SNF5 overexpression directs SNF5 to OCT4 target genes. Examination of the relationship between SNF5 and OCT4 binding in control and SNF5 overexpressed cells.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of SNF5 binding in human pluripotent embryonic carcinoma NCCIT and SNF5 overexpressed NCCIT cells. We generated genome-wide cSNF5 maps of NCCIT and SNF5 overexpressed NCCIT cells from chromatin immunoprecipitated DNA. SNF5 and OCT4 seem not to share their binding in OCT4 centered binding plot in control, while SNF5 overexpression directs SNF5 to OCT4 target genes.
Project description:Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionallyin vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHB's anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demonstrate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.
Project description:Analysis of global gene expression by SNF5 level change in human pluripotent and differentiated cells. The core subunit of BAF complex SNF5 is at the nexus of the link between chromatin remodeling and tumor suppression. We demonstrated a role for the remodeler SNF5 as a key executor in regulating pluripotency gene networks during differentiation by using loss and gain of function experiments followed by gene-expression arrays. Total RNA obtained from pluripotent state, differentiated state, absense of SNF5 and overexpression of SNF5
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes