Project description:RNAseq transcriptional profiling of Drosophila brains from wildtype, and period loss-of-function animals with time points taken over two days. 2 days of brain collection, time points at ZT0, ZT6, ZT12, and ZT18; wildtype and per0 flies. 10-12 brains per time point.
Project description:The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. We perform in vivo antibody-independent ChIP-sequencing of endogenously-expressed REVERBα, in order to map its cistrome and so define its repressive targets. We find that REVERBα binding sites harbour consensus RORE or RevDR2 motifs, and overlap with corepressor complex binding, without over-representation of putative tethering factors. When Reverbα is deleted in a hepatocyte-selective fashion, only modest physiological and transcriptional effects are seen, with de-repressed target genes tightly associating with REVERBα binding sites. Thus, contrary to previous reports, REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional programme is revealed only by perturbation (the metabolically abnormal global Reverbα-/- mouse, or acutely mistimed feeding), supporting instead a role for this core clock protein in buffering against metabolic challenge.
Project description:The glucocorticoid receptor (GR) is a nuclear hormone receptor critical to the regulation of energy metabolism and the inflammatory response. The actions of GR have been shown to be highly dependent on context. Here, we performed GR ChIP-seq in mouse liver to demonstrate the necessity for liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining tissue-specificity of GR action. In normal liver, the HNF4 motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites found within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodelled, with both loss and gain of GR recruitment evident. Lost sites are characterised by HNF4 motifs and weak GRE motifs. Gained sites are characterised by strong GRE motifs, and typically show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is further demonstrated by evidence of an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.
Project description:We identified a new type of histone mark-lysine Ã-hydroxybutyrylation (Kbhb). This ketone body derived histone mark (Kbhb) was dramatically induced in livers during starvation. To charactize histopne Kbhb: 1) We mapped genomic distributions of histone Kbhb marks (H3K9bhb, H3K4bhb and H4K8bhb) by ChIP-seq in mouse liver. 2) We examined the response of histone Kbhb mark to starvation by carrying out ChIP-seq experiments for H3K9bhb in both "starved" and "fed" mouse liver. 3) We also examined differentially-expressed genes during starvation by carrying out RNA-seq experiments in both "starved" and "fed" mouse liver. By integrating analyses of ChIP-seq and RNA-seq data, we tried to get a correlation between H3K9bhb mark and gene expression in response to starvation. Sequencing was performed on the HiSeq2000 (Illumina). ChIP-seq for histone Kbhb marks in both "starved (ST)" and "fed (AL)" mouse liver cells The anti-H3K4bhb, -H3K9bhb, and -H4K8bhb antibodies were generated from PTM biolabs. The process for generating antibodies were described similarly in Cell, 2011. 146: p. 1016-1028, Mol Cell, 2015. 58(2): p. 203-15, Nat Chem Biol, 2014. 10(5): p. 365-70, except for using different immunogens.
Project description:The circadian clock component REVERBα is considered a dominant regulator of lipid metabolism, with global Reverbα deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion (ReverbαFlox2-6AdipoCre), and transcriptional profiling demonstrates that, under basal conditions, direct targets of REVERBα regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, ReverbαFlox2-6AdipoCre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT REVERBα cistrome with differential gene expression reveals broad control of metabolic processes by REVERBα which is unmasked in the obese state.
Project description:ChIP-Sequencing on Shox2-HA E12.5 and E13.5 Limb and Palate, as well as Pbx on E12.5 limb . Abstract: Vertebrate appendage patterning is programmed by Hox-TALE factors-bound regulatory elements. However, it remains enigmatic which cell lineages are commissioned by Hox-TALE factors to generate regional specific pattern and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis, and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis. Shox2/TALE For ChIP-Seq, the list of libraries below, including controls, were generated [listed in the format of (antibody)-target-tissue-stage]: (α-HA)-Shox2-Limb-E12.5, (α-HA)-Shox2-Limb-E13.5, (α-HA)-Shox2-Palate-E12.5, (α-HA)-Shox2-Limb/Palate-E12.5, (α-Pbx)-Pbx-Limb-E12.5, Input (control), (α-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control). *The attached signal tracks(*.bigwig) were generated by âbdgcmp (MACS2) to filter out background signal(by filtering against the signal track obtained from (α-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control)) and subsequently convert to bigwig for analysis and visualization.
Project description:Memory CD8+ T cells have the ability to provide lifelong immunity against pathogens. Although memory features generally arise after challenge with a foreign antigen, naïve CD8 single positive (SP) thymocytes may acquire phenotypic and functional characteristics of memory cells in response to cytokines such as interleukin-4. This process is associated with the induction of the T-box transcription factor Eomesodermin (EOMES). However, the underlying molecular mechanisms remain ill-defined. Using epigenomic profiling, we show that these innate memory CD8SP cells acquire only a portion of the active enhancer repertoire of conventional memory cells. This reprograming is secondary to EOMES recruitment, mostly to RUNX3-bound enhancers. Furthermore, EOMES is found within chromatin-associated complexes containing BRG1 and promotes the recruitment of this chromatin remodelling factor. Also, the in vivo acquisition of EOMES-dependent program is BRG1-dependent. In conclusion, our results support a strong epigenetic basis for the EOMES-driven establishment of CD8+ T cell innate memory program.
Project description:Glucocorticoids are critical regulators of energy metabolism and immunity and major drug targets in inflammatory disease. It is also known that the circadian clock regulates both metabolism and immunity. This experiment is part of wider study to understand the coupling between circadian clock components and Glucocorticoid receptor in multiple mouse tissues. Here, RNA-seq analysis was performed in vehicle and dexamethasone (a synthetic Glucocorticoid) treated WT mouse liver and lung tissues at two circadian times, to understand time of the day variation in Glucocorticoid regulated genes. Mice were treated at ZT6 (6 hours after lights on, 1:30pm) or at ZT18 (6 hours after lights off, 1:30am) with dexamethasone (1 mg/kg intraperitoneal) or vehicle (methylcyclodextrin 1mg/kg intraperitoneal) for 2 hours before sacrifice by cervical dislocation. Lung and liver tissue were lysed and total RNA prepared using SV Total RNA Isolation System (Promega). Quality and integrity of total RNA samples were assessed by 2100 Bioanalyzer or a 2200 TapeStation (Agilent Technologies) according to the manufacturer’s instructions. RNA sequencing (RNA-seq) libraries were generated using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer’s protocol. Then paired-end sequenced (101 + 101 cycles, plus indices) on an Illumina HiSeq2500 instrument.