Project description:Though it is well established that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression and immune responses of CD4+ T cells purified from blood of healthy subjects at different time points throughout the day. Circadian clock function as well as immune function was further analyzed in cultivated T cells and circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, of IFN-g production and CD40L expression in both freshly isolated and in cultured CD4+ T cells. Moreover, circadian luciferase reporter activities in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed a rhythmic regulation of the NF-kB pathway as a candidate mechanism regulating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic adaptive immune responses in vitro and in vivo. The study is designed with 3 biological replicates from three different time points.
Project description:We have completed the high quality reference genome for domestic sheep (Oar v3.1). Early-stage Illumina GA sequence platform sequenced less reads in high GC content regions than in other regions. To read through higher GC content regions, we generated 2 Gb MeDIP-seq data for filling gaps in sheep reference genome assembly.
Project description:Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes. As genome-wide transcription is organized under the high-order chromosome structure, it is unclear how circadian gene expression is influenced by chromosome structure. In this study, we focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. We analyzed the interactome of a Bmal1-bound enhancer upstream of a clock gene, Nr1d1, by 4C-seq and observed that cohesin binding sites are enriched in the interactome. Integrating circadian transcriptome data and cistrome data, we found that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites facilitate the interaction between circadian enhancer and promoter. A coarse-grained model integrating the long-range effect of cohesin and CTCF markedly improved our mechanistic understanding of circadian gene expression. This model is subsequently supported by our RNA-seq data from cohesin knockout cells. Cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. Taken together, our study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure. Bmal1 ChIP-Seq in WT mouse embryonic fibroblast cells
Project description:We have completed the high quality reference genome for domestic sheep (Oar v3.1) and performed a detailed survey of gene expression across different tissues. RNA-seq data of 7 tissue types from the reference female Texel and skin tissue from a Gansu alpine fine wool sheep were sequenced.
Project description:The mammalian circadian clock involves a transcriptional feedback loop in which CLOCK and BMAL1 activate the Period and Cryptochrome genes, which then feedback and repress their own transcription. We have interrogated the transcriptional architecture of the circadian transcriptional regulatory loop on a genome scale in mouse liver and find a stereotyped, time-dependent pattern of transcription factor binding, RNA polymerase II (RNAPII) recruitment, RNA expression and chromatin states. We find that the circadian transcriptional cycle of the clock consists of three distinct phases - a poised state, a coordinated de novo transcriptional activation state, and a repressed state. Interestingly only 22% of mRNA cycling genes are driven by de novo transcription, suggesting that both transcriptional and post-transcriptional mechanisms underlie the mammalian circadian clock. We also find that circadian modulation of RNAPII recruitment and chromatin remodeling occurs on a genome-wide scale far greater than that seen previously by gene expression profiling. Examination of 9 transcriptional regulators, 2 RNAPII and 6 histone modifications every 4hr during the circadian cycle in mouse liver
Project description:Though it is well established that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression and immune responses of CD4+ T cells purified from blood of healthy subjects at different time points throughout the day. Circadian clock function as well as immune function was further analyzed in cultivated T cells and circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, of IFN-g production and CD40L expression in both freshly isolated and in cultured CD4+ T cells. Moreover, circadian luciferase reporter activities in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed a rhythmic regulation of the NF-kB pathway as a candidate mechanism regulating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic adaptive immune responses in vitro and in vivo.
Project description:Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockM-NM-^T19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockM-NM-^T19 mutation to a M-bM-^HM-<900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKM-NM-^T19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals. DOI:http://dx.doi.org/10.7554/eLife.00426.001. Examination of 3 transcriptional regulators in mouse liver at ZT8