1. The local organization of transcription factor chromatin interaction sites is highly cell specific, and strongly correlated with the transcriptional response. Factor sites linked to promoters that are non-responsive in a given cell type are refractory to factor directed remodeling, and resistant to factor binding. This leads to the hypothesis that local chromatin structural organization in a given cell type is a major determinant of tissue specific factor action. Factor binding to these sites is also highly dynamic, with residence times on the template in the range of 5-20 seconds. Integration of these concepts led to the development of a new paradigm for transcription factor interaction with chromatin, termed assisted loading. 2. Genomic footprinting has emerged as an unbiased discovery method for transcription factor (TF) occupancy at cognate DNA in vivo. A basic premise of footprinting is that sequence-specific TF-DNA interactions are associated with localized resistance to nucleases, leaving observable signatures of cleavage within accessible chromatin. This phenomenon is interpreted to imply protection of the critical nucleotides by the stably bound protein factor. However, this model conflicts with previous reports of many TFs exchanging with specific binding sites in living cells on a timescale of seconds. We show that TFs with short DNA residence times have no footprints at bound motif elements. Moreover, the nuclease cleavage profile within a footprint originates from the DNA sequence in the factor-binding site, rather than from the protein occupying specific nucleotides. These findings suggest a revised understanding of TF footprinting and reveal limitations in comprehensive reconstruction of the TF regulatory network using this approach. 3. During fasting the liver supplies the organism's energy demands by producing glucose and ketones. Fuel production during fasting is temporally organized whereby glucose serves as the major fuel produced in short-term fasting while ketones are produced in longer fasts as gluconeogenic precursors deplete. However, the regulatory process dictating this temporally-organized metabolic output is unexplored. We showed that a cascade of transcription factors (TFs) sequentially activated by hormonal signals, TF gene expression and assisted loading onto specific 'fasting enhancers' generate a framework for the temporal organization of fuel production during fasting. Fasting leads to massive chromatin re-organization, exposing enhancers in which a complex set of regulatory signals converge to regulate transcription. Glucagon secreted in early fasting activates cAMP responsive element binding protein (CREB) leading to increased expression of many TF genes, including CCAAT enhancer binding protein beta (C/EBPbeta) which relaxes chromatin, thereby potentiating glucocorticoid receptor (GR) binding to fasting enhancers upon its activation by corticosterone in mid-term fasting. Then, GR augments glucagon-dependent gluconeogenesis but also supports a peroxisome proliferator receptor a (PPARa)-dependent ketogenic gene program to sustain ketogenesis during prolonged fasting. Our results bridge over a long-lasting gap between the characterized temporal organization of fuel output during fasting and the undefined role of transcription and chromatin regulation in generating this output. Because a de-regulated response to fasting is a hallmark of diabetes progression, these findings may promote our understanding of this complex disease. 4. Cancer discovery has been focused primarily on the identification of critical mutated pathways, and the development of drugs to target the gene products of these so-called driver mutants. Critical driver mutants are not commonly identified, and therapies targeting these pathways are frequently followed by relapse. In fact, the regulatory networks that control global gene expression are massively transformed during cancer initiation and progression. The failure to normalize gene regulation and return cells to normal growth control is likely a major factor in treatment failure. To treat cancer effectively, a key issue is to identify the regulatory elements that create this abnormal expression pattern, rather than simply describing the gene expression pattern in tumor cells. Our primary goal is to analyze the status of enhancer networks in cancer cells, and identify enhancer signatures characteristic of progression and metastasis. Enhancer signatures predictive of cancer progression can then inform threat level for a specific tumor, appropriate patient therapy, and treatment progress for favorable response in enhancer status.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005450-32
Application #
9153476
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
32
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Goldstein, Ido; Hager, Gordon L (2018) The Three Ds of Transcription Activation by Glucagon: Direct, Delayed, and Dynamic. Endocrinology 159:206-216
Saloura, Vassiliki; Vougiouklakis, Theodore; Sievers, Cem et al. (2018) The role of protein methyltransferases as potential novel therapeutic targets in squamous cell carcinoma of the head and neck. Oral Oncol 81:100-108
Swinstead, Erin E; Paakinaho, Ville; Hager, Gordon L (2018) Chromatin reprogramming in breast cancer. Endocr Relat Cancer 25:R385-R404
Pooley, John R; Flynn, Ben P; Grøntved, Lars et al. (2017) Genome-Wide Identification of Basic Helix-Loop-Helix and NF-1 Motifs Underlying GR Binding Sites in Male Rat Hippocampus. Endocrinology 158:1486-1501
Goldstein, Ido; Paakinaho, Ville; Baek, Songjoon et al. (2017) Synergistic gene expression during the acute phase response is characterized by transcription factor assisted loading. Nat Commun 8:1849
Paakinaho, Ville; Presman, Diego M; Ball, David A et al. (2017) Single-molecule analysis of steroid receptor and cofactor action in living cells. Nat Commun 8:15896
Goldstein, Ido; Hager, Gordon L (2017) Dynamic enhancer function in the chromatin context. Wiley Interdiscip Rev Syst Biol Med :
Sawyer, Iain A; Hager, Gordon L; Dundr, Miroslav (2017) Specific genomic cues regulate Cajal body assembly. RNA Biol 14:791-803
Siersbæk, Majken; Varticovski, Lyuba; Yang, Shutong et al. (2017) High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss. Sci Rep 7:40220
Baek, Songjoon; Goldstein, Ido; Hager, Gordon L (2017) Bivariate Genomic Footprinting Detects Changes in Transcription Factor Activity. Cell Rep 19:1710-1722

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