The underlying mechanisms by which cell identity is achieved in a cell type-specific manner during development are unknown. In this project, we examine the mechanisms through which genomic architecture is regulated by different protein factors and how these proteins in turn regulate gene expression in the cardiomyocyte. We search for cardiac chromatin structural factors that are important for the establishment of genomic architecture during differentiation. We hypothesized that these candidates would also be implicated in pathological gene expression upon the onset of heart failure. Instead, we found that the expression changes of chromatin structural genes across a panel of different mouse strains were not universal, nor did they correlate with cardiac phenotype after pathological stress. Most of our current knowledge of signaling mechanisms in the heart has stemmed from genetic manipulations in a single mouse strain. Here, we examined well-characterized regulators of cardiac phenotype and showed that the relationships between gene expression and cardiac phenotype are lost when expanding across multiple genetic backgrounds. More importantly, these data demonstrate that there is no single signature gene that drives heart disease (nor is there a single gene whose expression is a biomarker of the condition), highlighting the role of genetic variability to differentially sculpt the transcriptome in the development and progression of complex diseases. In addition, our findings demonstrate that regulation of gene expression by genetics occurs in a tissue-dependent manner. We previously identified High Mobility Group B2 as an important chromatin structural protein in the heart and showed its involvement in pathological gene expression. These studies suggested this regulation occurs by remodeling global transcriptional activity. To characterize structural organization of cellular transcription, we show that transcriptional activity is compartmentalized into stable factories in the heart that undergo functional changes in vivo in response to disease stimuli. We provide evidence of direct reorganization of genomic structure by showing that nuclear positioning of cardiac genes with respect to chromatin environments and transcription factories correlates with changes in their expression. In summary, this project explores the mechanisms of cardiac gene regulation and illustrates multiple levels of regulation, with influences from genetics and chromatin architecture.

When faced with chronic stress, the heart enters a compensatory hypertrophic stage; without intervention it eventually succumbs to decompensation marked by a dilated left ventricular chamber and decreased ejection fraction. While the morphological cardiac remodeling that occurs during the progression of heart failure is well characterized, the exact molecular cause for this gradual switch to failure is not known. In addition to the numerous alterations in signaling pathways, a conserved switch in the transcriptome, known as the fetal gene program, occurs during hypertrophy as a protective effort to sustain contractility by reverting to fetal isoforms of metabolic, contractile and calcium handling genes. We hypothesize that the reproducible, coordinated reprogramming of gene expression is orchestrated by a change in chromatin structure that enables pathologic gene expression. To determine the proteins involved in repackaging chromatin during cardiac pathology, we performed quantitative proteomic analyses of nuclear proteins in a mouse model of pressure overload hypertrophy and failure. Among the hundreds of proteins we measured on chromatin, my subsequent analyses have focused on two candidates that had the potential to alter gene expression by directly affecting chromatin packing. The first was Nucleolin, a major component of the nucleolus where it mediates ribosomal biogenesis. Using isolated myocytes and the developing zebrafish embryo, we uncovered a role for Nucleolin to regulate cardiac looping, with its effect on hypertrophy context dependent, such that in isolated myoctyes knockdown can promote pathologic gene expression, but loss of Nucleolin during development does not alter myocyte size, instead affecting differentiation along the cardiac lineage. The second protein I functionally validated was High mobility group protein B2 (HMGB2), a non-histone chromatin structural protein that increases 3-fold in our proteomic analyses. We show that HMGB2 is necessary for ribosomal RNA transcription and is enriched in the nucleolus in hypertrophy; however, overexpression of HMGB2 shuts down transcription globally by compacting DNA. Furthermore, we find HMGB2 knockdown alters the chromatin environment of individual gene promoters in the same manner as hypertrophic agonist signaling in isolated myocytes. Finally, we find that the effect of HMGB2 abundance on the expression of individual genes can be partially explained by the chromatin context, and specifically identify a novel relationship between HMGB2 and CTCF. These studies add to the growing body of work characterizing chromatin remodeling in hypertrophy, and demonstrate that this remodeling extends outside of gene bodies and promoters. Finally, this work begins to uncover what features of chromatin are responsible for tailoring the effects of ubiquitous chromatin proteins toward a cell-type specific outcome.

Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.

With the invitation to edit this volume, I wanted to take the opportunity to assemble reviews on different aspects of circadian clocks and rhythms. Although most c- tributions in this volume focus on mammalian circadian clocks, the historical int- duction and comparative clocks section illustrate the importance of various other organisms in deciphering the mechanisms and principles of circadian biology. Circadian rhythms have been studied for centuries, but only recently, a mole- lar understanding of this process has emerged. This has taken research on circadian clocks from mystic phenomenology to a mechanistic level; chains of molecular events can describe phenomena with remarkable accuracy. Nevertheless, current models of the functioning of circadian clocks are still rudimentary. This is not due to the faultiness of discovered mechanisms, but due to the lack of undiscovered processes involved in contributing to circadian rhythmicity. We know for example, that the general circadian mechanism is not regulated equally in all tissues of m- mals. Hence, a lot still needs to be discovered to get a full understanding of cir- dian rhythms at the systems level. In this respect, technology has advanced at high speed in the last years and provided us with data illustrating the sheer complexity of regulation of physiological processes in organisms. To handle this information, computer aided integration of the results is of utmost importance in order to d- cover novel concepts that ultimately need to be tested experimentally.

Understanding mechanisms of gene regulation that are independent of the DNA sequence itself - epigenetics - has the potential to overthrow long-held views on central topics in biology, such as the biology of disease or the evolution of species. High throughput technologies reveal epigenetic mechanisms at a genome-wide level, giving rise to epigenomics as a new discipline with a distinct set of research questions and methods. Leading experts from academia, the biotechnology and pharmaceutical industries explain the role of epigenomics in a wide range of contexts, covering basic chromatin biology, imprinting at a genome-wide level, and epigenomics in disease biology and epidemiology. Details on assays and sequencing technology serve as an up-to-date overview of the available technological tool kit. A reliable guide for newcomers to the field as well as experienced scientists, this is a unique resource for anyone interested in applying the power of twenty-first-century genomics to epigenetic studies.