Transcriptional regulatory logic of the diurnal cycle in the mouse liver.

Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1-/- livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1-/- compared to WT livers, the amplitudes in Bmal1-/- were generally lower. The residual rhythms in Bmal1-/- reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK:BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver.

Integrative genomics identifies the corepressor SMRT as a gatekeeper of adipogenesis through the transcription factors C/EBPß and KAISO.

The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPß-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.

Longitudinal expression changes are weak correlates of disease progression in Huntington's disease.

Huntington's disease is a severe but slowly progressive hereditary illness for which only symptomatic treatments are presently available. Clinical measures of disease progression are somewhat subjective and may require years to detect significant change. There is a clear need to identify more sensitive, objective and consistent measures to detect disease progression in Huntington's disease clinical trials. Whereas Huntington's disease demonstrates a robust and consistent gene expression signature in the brain, previous studies of blood cell RNAs have lacked concordance with clinical disease stage. Here we utilized longitudinally collected samples from a well-characterized cohort of control, Huntington's disease-at-risk and Huntington's disease subjects to evaluate the possible correlation of gene expression and disease status within individuals. We interrogated these data in both cross-sectional and longitudinal analyses. A number of changes in gene expression showed consistency within this study and as compared to previous reports in the literature. The magnitude of the mean disease effect over 2 years' time was small, however, and did not track closely with motor symptom progression over the same time period. We therefore conclude that while blood-derived gene expression indicators can be of value in understanding Huntington's disease pathogenesis, they are insufficiently sensitive to be of use as state biomarkers.