Haifang Wang

Analysis of Gene Regulatory Networks in the Mammalian Circadian Rhythm

Circadian rhythm is fundamental in regulating a wide range of cellular, metabolic, physiological, and behavioral activities in mammals. Although a small number of key circadian genes have been identified through extensive molecular and genetic studies in the past, the existence of other key circadian genes and how they drive the genomewide circadian oscillation of gene expression in different tissues still remains unknown. Here we try to address these questions by integrating all available circadian microarray data in mammals. We identified 41 common circadian genes that showed circadian oscillation in a wide range of mouse tissues with a remarkable consistency of circadian phases across tissues. Comparisons across mouse, rat, rhesus macaque, and human showed that the circadian phases of known key circadian genes were delayed for 4–5 hours in rat compared to mouse and 8–12 hours in macaque and human compared to mouse. A systematic gene regulatory network for the mouse circadian rhythm was constructed after incorporating promoter analysis and transcription factor knockout or mutant microarray data. We observed the significant association of cis-regulatory elements: EBOX, DBOX, RRE, and HSE with the different phases of circadian oscillating genes. The analysis of the network structure revealed the paths through which light, food, and heat can entrain the circadian clock and identified that NR3C1 and FKBP/HSP90 complexes are central to the control of circadian genes through diverse environmental signals. Our study improves our understanding of the structure, design principle, and evolution of gene regulatory networks involved in the mammalian circadian rhythm.

Shotgun Proteomics Analysis of Hibernating Arctic Ground Squirrels

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1–2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.

Modulation of gene expression in heart and liver of hibernating black bears (Ursus americanus)

BackgroundHibernation is an adaptive strategy to survive in highly seasonal or unpredictable environments. The molecular and genetic basis of hibernation physiology in mammals has only recently been studied using large scale genomic approaches. We analyzed gene expression in the American black bear, Ursus americanus, using a custom 12,800 cDNA probe microarray to detect differences in expression that occur in heart and liver during winter hibernation in comparison to summer active animals.ResultsWe identified 245 genes in heart and 319 genes in liver that were differentially expressed between winter and summer. The expression of 24 genes was significantly elevated during hibernation in both heart and liver. These genes are mostly involved in lipid catabolism and protein biosynthesis and include RNA binding protein motif 3 (Rbm3), which enhances protein synthesis at mildly hypothermic temperatures. Elevated expression of protein biosynthesis genes suggests induction of translation that may be related to adaptive mechanisms reducing cardiac and muscle atrophies over extended periods of low metabolism and immobility during hibernation in bears. Coordinated reduction of transcription of genes involved in amino acid catabolism suggests redirection of amino acids from catabolic pathways to protein biosynthesis. We identify common for black bears and small mammalian hibernators transcriptional changes in the liver that include induction of genes responsible for fatty acid β oxidation and carbohydrate synthesis and depression of genes involved in lipid biosynthesis, carbohydrate catabolism, cellular respiration and detoxification pathways.ConclusionsOur findings show that modulation of gene expression during winter hibernation represents molecular mechanism of adaptation to extreme environments.

Computational analysis of gene regulation in animal sleep deprivation

Sleep is an animal behavior shared by a wide range of species, suggesting that it must serve fundamental functions. However, the functions and molecular mechanisms underlying sleep are largely unknown. Through a meta-analysis of all available short-term sleep deprivation (SD) microarray data in mouse brain, we identified 91 key mouse SD-affected genes and two RBM3 isoforms showing opposite changes of expression during SD. Although most of the key SD-affected genes showed consistent changes of expression during SD across brain subregions despite their heterogeneous basal expression levels, we also identified the genes whose SD responses strongly depend upon the brain subregion. A gene regulatory network was also constructed for these genes showing that cAMP-responsive element (CRE) is one of the key cis-regulatory elements controlling SD-affected genes. We observed that SD during an animals normal sleeping time could significantly disturb the circadian oscillation of clock genes. Surprisingly, synaptogenesis markers were significantly underexpressed in SD mice, differing from the previous findings in rat and fly. Comparing SD microarray data in mouse, rat, sparrow, and fly, we identified Egr and Nr4a gene families as conserved SD-affected genes, thus shedding new light on the origin of sleep in animals.

Genomic Analysis of miRNAs in an Extreme Mammalian Hibernator, the Arctic Ground Squirrel.

MicroRNAs (miRNAs) are 19- to 25-nucleotide-long small and noncoding RNAs now well-known for their regulatory roles in gene expression through posttranscriptional and translational controls. Mammalian hibernation is a physiological process involving profound changes in set-points for food consumption, body mass and growth, body temperature, and metabolic rate in which miRNAs may play important regulatory roles. In an initial study, we analyzed miRNAs in the liver of an extreme hibernating species, the Arctic ground squirrel (Spermophilus parryii), using massively parallel Illumina sequencing technology. We identified >200 ground squirrel miRNAs, including 18 novel miRNAs specific to ground squirrel and mir-506 that is fast evolving in the ground squirrel lineage. Comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 mo after hibernation had ended (nonhibernating), we identified differentially expressed miRNAs during hibernation, which are also compared with the results from two other miRNA profiling methods: Agilent miRNA microarray and real-time PCR. Among the most significant miRNAs, miR-320 and miR-378 were significantly underexpressed during both stages of hibernation compared with nonhibernating animals, whereas miR-486 and miR-451 were overexpressed in late torpor but returned in early arousal to the levels similar to those in nonhibernating animals. Analyses of their putative target genes suggest that these miRNAs could play an important role in suppressing tumor progression and cell growth during hibernation. High-throughput sequencing data and microarray data have been submitted to GEO database with accession: GSE19808.

Analysis of a Gene Regulatory Cascade Mediating Circadian Rhythm in Zebrafish

In the study of circadian rhythms, it has been a puzzle how a limited number of circadian clock genes can control diverse aspects of physiology. Here we investigate circadian gene expression genome-wide using larval zebrafish as a model system. We made use of a spatial gene expression atlas to investigate the expression of circadian genes in various tissues and cell types. Comparison of genome-wide circadian gene expression data between zebrafish and mouse revealed a nearly anti-phase relationship and allowed us to detect novel evolutionarily conserved circadian genes in vertebrates. We identified three groups of zebrafish genes with distinct responses to light entrainment: fast light-induced genes, slow light-induced genes, and dark-induced genes. Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade. Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafishs adaptation to daily light cycling. Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.

Preservation of bone mass and structure in hibernating black bears (Ursus americanus) through elevated expression of anabolic genes

Physical inactivity reduces mechanical load on the skeleton, which leads to losses of bone mass and strength in non-hibernating mammalian species. Although bears are largely inactive during hibernation, they show no loss in bone mass and strength. To obtain insight into molecular mechanisms preventing disuse bone loss, we conducted a large-scale screen of transcriptional changes in trabecular bone comparing winter hibernating and summer non-hibernating black bears using a custom 12,800 probe cDNA microarray. A total of 241 genes were differentially expressed (P < 0.01 and fold change >1.4) in the ilium bone of bears between winter and summer. The Gene Ontology and Gene Set Enrichment Analysis showed an elevated proportion in hibernating bears of overexpressed genes in six functional sets of genes involved in anabolic processes of tissue morphogenesis and development including skeletal development, cartilage development, and bone biosynthesis. Apoptosis genes demonstrated a tendency for downregulation during hibernation. No coordinated directional changes were detected for genes involved in bone resorption, although some genes responsible for osteoclast formation and differentiation (Ostf1, Rab9a, and c-Fos) were significantly underexpressed in bone of hibernating bears. Elevated expression of multiple anabolic genes without induction of bone resorption genes, and the down regulation of apoptosis-related genes, likely contribute to the adaptive mechanism that preserves bone mass and structure through prolonged periods of immobility during hibernation.

Comparative functional genomics of adaptation to muscular disuse in hibernating mammals

Hibernation is an energy‐saving adaptation that involves a profound suppression of physical activity that can continue for 6–8 months in highly seasonal environments. While immobility and disuse generate muscle loss in most mammalian species, in contrast, hibernating bears and ground squirrels demonstrate limited muscle atrophy over the prolonged periods of physical inactivity during winter, suggesting that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional programmes that underlie molecular mechanisms preventing muscle loss, we conducted a large‐scale gene expression screen in hind limb muscles comparing hibernating and summer‐active black bears and arctic ground squirrels using custom 9600 probe cDNA microarrays. A molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during torpor of hibernation that suggests induction of translation at different hibernation states. The induction of protein biosynthesis probably contributes to attenuation of disuse muscle atrophy through the prolonged periods of immobility of hibernation. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction in multiple genes involved in oxidation–reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation.

Oscillating primary transcripts harbor miRNAs with circadian functions.

The roles of miRNAs as important post-transcriptional regulators in the circadian clock have been suggested in several studies. But the search for circadian miRNAs has led to disparate results. Here we demonstrated that at least 57 miRNA primary transcripts are rhythmically transcribed in mouse liver. Most of these transcripts are under the regulation of circadian transcription factors such as BMAL1/CLOCK and REV-ERBα/β. However, the mature miRNAs derived from these transcripts are either not oscillating or oscillating at low amplitudes, which could explain the inconsistency of different circadian miRNA studies. In order to show that these circadian primary transcripts can give rise to miRNAs with circadian functions, we over-expressed one of them, miR-378, in mouse by adenovirus injection. We found a significant over-representation of circadian oscillating genes under-expressed by miR-378 over-expression in liver. In particular, we observed that miR-378 modulates the oscillation amplitudes of Cdkn1a in the control of cell cycle and Por in the regulation of oxidation reduction by forming partnership with different circadian transcription factors. Our study suggests that circadian transcription of miRNA at primary transcript level can be a good indicator for circadian miRNA functions.

A class of circadian long non-coding RNAs mark enhancers modulating long-range circadian gene regulation

Abstract Circadian rhythm exerts its influence on animal physiology and behavior by regulating gene expression at various levels. Here we systematically explored circadian long non-coding RNAs (lncRNAs) in mouse liver and examined their circadian regulation. We found that a significant proportion of circadian lncRNAs are expressed at enhancer regions, mostly bound by two key circadian transcription factors, BMAL1 and REV-ERBα. These circadian lncRNAs showed similar circadian phases with their nearby genes. The extent of their nuclear localization is higher than protein coding genes but less than enhancer RNAs. The association between enhancer and circadian lncRNAs is also observed in tissues other than liver. Comparative analysis between mouse and rat circadian liver transcriptomes showed that circadian transcription at lncRNA loci tends to be conserved despite of low sequence conservation of lncRNAs. One such circadian lncRNA termed lnc-Crot led us to identify a super-enhancer region interacting with a cluster of genes involved in circadian regulation of metabolism through long-range interactions. Further experiments showed that lnc-Crot locus has enhancer function independent of lnc-Crots transcription. Our results suggest that the enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs.