Vishal R. Patel

Coordination of the transcriptome and metabolome by the circadian clock

The circadian clock governs a large array of physiological functions through the transcriptional control of a significant fraction of the genome. Disruption of the clock leads to metabolic disorders, including obesity and diabetes. As food is a potent zeitgeber (ZT) for peripheral clocks, metabolites are implicated as cellular transducers of circadian time for tissues such as the liver. From a comprehensive dataset of over 500 metabolites identified by mass spectrometry, we reveal the coordinate clock-controlled oscillation of many metabolites, including those within the amino acid and carbohydrate metabolic pathways as well as the lipid, nucleotide, and xenobiotic metabolic pathways. Using computational modeling, we present evidence of synergistic nodes between the circadian transcriptome and specific metabolic pathways. Validation of these nodes reveals that diverse metabolic pathways, including the uracil salvage pathway, oscillate in a circadian fashion and in a CLOCK-dependent manner. This integrated map illustrates the coherence within the circadian metabolome, transcriptome, and proteome and how these are connected through specific nodes that operate in concert to achieve metabolic homeostasis.

Reprogramming of the Circadian Clock by Nutritional Challenge

Circadian rhythms and cellular metabolism are intimately linked. Here, we reveal that a high-fat diet (HFD) generates a profound reorganization of specific metabolic pathways, leading to widespread remodeling of the liver clock. Strikingly, in addition to disrupting the normal circadian cycle, HFD causes an unexpectedly large-scale genesis of de novo oscillating transcripts, resulting in reorganization of the coordinated oscillations between coherent transcripts and metabolites. The mechanisms underlying this reprogramming involve both the impairment of CLOCK:BMAL1 chromatin recruitment and a pronounced cyclic activation of surrogate pathways through the transcriptional regulator PPARγ. Finally, we demonstrate that it is specifically the nutritional challenge, and not the development of obesity, that causes the reprogramming of the clock and that the effects of the diet on the clock are reversible.

Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock

Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS3 analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS3) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

Circadian acetylome reveals regulation of mitochondrial metabolic pathways

The circadian clock is constituted by a complex molecular network that integrates a number of regulatory cues needed to maintain organismal homeostasis. To this effect, posttranslational modifications of clock proteins modulate circadian rhythms and are thought to convert physiological signals into changes in protein regulatory function. To explore reversible lysine acetylation that is dependent on the clock, we have characterized the circadian acetylome in WT and Clock-deficient (Clock−/−) mouse liver by quantitative mass spectrometry. Our analysis revealed that a number of mitochondrial proteins involved in metabolic pathways are heavily influenced by clock-driven acetylation. Pathways such as glycolysis/gluconeogenesis, citric acid cycle, amino acid metabolism, and fatty acid metabolism were found to be highly enriched hits. The significant number of metabolic pathways whose protein acetylation profile is altered in Clock−/− mice prompted us to link the acetylome to the circadian metabolome previously characterized in our laboratory. Changes in enzyme acetylation over the circadian cycle and the link to metabolite levels are discussed, revealing biological implications connecting the circadian clock to cellular metabolic state.

MotifMap: integrative genome-wide maps of regulatory motif sites for model species

BackgroundA central challenge of biology is to map and understand gene regulation on a genome-wide scale. For any given genome, only a small fraction of the regulatory elements embedded in the DNA sequence have been characterized, and there is great interest in developing computational methods to systematically map all these elements and understand their relationships. Such computational efforts, however, are significantly hindered by the overwhelming size of non-coding regions and the statistical variability and complex spatial organizations of regulatory elements and interactions. Genome-wide catalogs of regulatory elements for all model species simply do not yet exist.ResultsThe MotifMap system uses databases of transcription factor binding motifs, refined genome alignments, and a comparative genomic statistical approach to provide comprehensive maps of candidate regulatory elements encoded in the genomes of model species. The system is used to derive new genome-wide maps for yeast, fly, worm, mouse, and human. The human map contains 519,108 sites for 570 matrices with a False Discovery Rate of 0.1 or less. The new maps are assessed in several ways, for instance using high-throughput experimental ChIP-seq data and AUC statistics, providing strong evidence for their accuracy and coverage. The maps can be usefully integrated with many other kinds of omic data and are available at http://motifmap.igb.uci.edu/.ConclusionsMotifMap and its integration with other data provide a foundation for analyzing gene regulation on a genome-wide scale, and for automatically generating regulatory pathways and hypotheses. The power of this approach is demonstrated and discussed using the P53 apoptotic pathway and the Gli hedgehog pathways as examples.

Leptin engages a hypothalamic neurocircuitry to permit survival in the absence of insulin

The dogma that life without insulin is incompatible has recently been challenged by results showing the viability of insulin-deficient rodents undergoing leptin monotherapy. Yet, the mechanisms underlying these actions of leptin are unknown. Here, the metabolic outcomes of intracerebroventricular (i.c.v.) administration of leptin in mice devoid of insulin and lacking or re-expressing leptin receptors (LEPRs) only in selected neuronal groups were assessed. Our results demonstrate that concomitant re-expression of LEPRs only in hypothalamic γ-aminobutyric acid (GABA) and pro-opiomelanocortin (POMC) neurons is sufficient to fully mediate the lifesaving and antidiabetic actions of leptin in insulin deficiency. Our analyses indicate that enhanced glucose uptake by brown adipose tissue and soleus muscle, as well as improved hepatic metabolism, underlies these effects of leptin. Collectively, our data elucidate a hypothalamic-dependent pathway enabling life without insulin and hence pave the way for developing better treatments for diseases of insulin deficiency.

CircadiOmics: integrating circadian genomics, transcriptomics, proteomics and metabolomics

jModelTest 2 is written in Java, and it can run on Windows, Macintosh and Linux platforms. Source code and binaries are freely available from https://code.google.com/p/jmodeltest2/. The package includes detailed documentation and examples, and a discussion group is available at https://groups.google.com/forum/#!forum/jmodeltest/. We evaluated the accuracy of jModelTest 2 using 10,000 data sets simulated under a large variety of conditions (Supplementary Note 3). Using the Bayesian information criterion4 for model selection, jModelTest 2 identified the generating model 89% of the time (Supplementary Table 2); in the remaining cases, jModelTest 2 selected a model similar to the generating one. Accordingly, model-averaged estimates of model parameters were highly precise (Supplementary Table 3). In these simulations, the two selection heuristics that we developed were accurate and efficient. Using the hierarchical clustering heuristic, we found the same best-fit model as the full search 95% of the time. With the similarity filtering approach, we reduced the number of models evaluated by 60% on average and found the global best-fit model 99% of the time (Fig. 1 and Supplementary Note 2). jModelTest 2 can be executed in high-performance computing environments as (i) a desktop version with a user-friendly interface for multicore processors, (ii) a cluster version that distributes the computational load among nodes, and (iii) as a hybrid version that can take advantage of a cluster of multicore nodes. An experimental study with real and simulated data sets showed remarkable computational speedups compared to previous versions (Supplementary Note 4). For example, the hybrid approach executed on the Amazon EC2 cloud with 256 processes was 182–211 times faster. For relatively large alignments (138 sequences and 10,693 sites), this could be equivalent to a reduction of the running time from nearly 8 days to around 1 hour.

Cycles in spatial and temporal chromosomal organization driven by the circadian clock

Dynamic transitions in the epigenome have been associated with regulated patterns of nuclear organization. The accumulating evidence that chromatin remodeling is implicated in circadian function prompted us to explore whether the clock may control nuclear architecture. We applied the chromosome conformation capture on chip technology in mouse embryonic fibroblasts (MEFs) to demonstrate the presence of circadian long-range interactions using the clock-controlled Dbp gene as bait. The circadian genomic interactions with Dbp were highly specific and were absent in MEFs whose clock was disrupted by ablation of the Bmal1 gene (also called Arntl). We establish that the Dbp circadian interactome contains a wide variety of genes and clock-related DNA elements. These findings reveal a previously unappreciated circadian and clock-dependent shaping of the nuclear landscape.

Mapping the Structural Topology of the Yeast 19S Proteasomal Regulatory Particle Using Chemical Cross-linking and Probabilistic Modeling

Structural characterization of proteasome complexes is an essential step toward understanding the ubiquitin-proteasome system. Currently, high resolution structures are not available for the 26S proteasome holocomplex as well as its subcomplex, the 19S regulatory particle (RP). Here we have employed a novel integrated strategy combining chemical cross-linking with multistage tandem mass spectrometry to define the proximity of subunits within the yeast 19S RP to elucidate its topology. This has resulted in the identification of 174 cross-linked peptides of the yeast 19S RP, representing 43 unique lysine-lysine linkages within 24 nonredundant pair-wise subunit interactions. To map the spatial organization of the 19S RP, we have developed and utilized a rigorous probabilistic framework to derive maximum likelihood (ML) topologies based on cross-linked peptides determined from our analysis. Probabilistic modeling of the yeast 19S AAA-ATPase ring (i.e., Rpt1–6) has produced an ML topology that is in excellent agreement with known topologies of its orthologs. In addition, similar analysis was carried out on the 19S lid subcomplex, whose predicted ML topology corroborates recently reported electron microscopy studies. Together, we have demonstrated the effectiveness and potential of probabilistic modeling for unraveling topologies of protein complexes using cross-linking data. This report describes the first study of the 19S RP topology using a new integrated strategy combining chemical cross-linking, mass spectrometry, and probabilistic modeling. Our results have provided a solid foundation to advance our understanding of the 19S RP architecture at peptide level resolution. Furthermore, our methodology developed here is a valuable proteomic tool that can be generalized for elucidating the structures of protein complexes.