In our current research, we are probing the molecular mechanisms responsible for orchestrating and integrating genome-wide transcriptional responses to diverse signaling pathways critical for developmental, physiological, and pathological regulation. We focus on previously unsuspected aspects of enhancer function, chromosomal structure, and subnuclear architectural interactions. These strategies, which underlie genome-wide transcriptional responses in the endocrine and central nervous systems and are critical for physiological and behavioral processes in all vertebrates, are orchestrated by the network of genomic enhancers, ~50 percent of which are cell-type specific. Despite their discovery more than 35 years ago, the fundamental principles by which enhancers are activated and regulate their coding gene transcriptional targets in metazoans have remained poorly understood. Our recent findings have substantially altered concepts regarding the roles of noncoding RNAs (ncRNAs), DNA repeats, mechanisms of enhancer activation and function, and nuclear architecture as critical aspects of regulated gene expression programs. This work has uncovered unexpected aspects of enhancer function, highlighting their functioning as regulated transcription units in dynamic alterations in nuclear architecture, by specific epigenomic strategies, linking transcriptional regulation to mechanisms underlying DNA damage/repair and inflammation, with therapeutic implications for many common diseases.
Enhancers and Mechanisms of "Epigenomic" Regulation
We found that 17b-estradiol (E2)-bound estrogen receptor a (ERa) on enhancers causes a global increase in enhancer RNA (eRNA) transcription on the ~1,000 enhancers adjacent to E2-upregulated coding genes. These induced eRNAs exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ERa binding. Cohesin, present on ERa-regulated enhancers even before ligand treatment, and only minimally responsive to ligand, is required for E2-induced gene activation by stabilizing E2/ERa/eRNA-induced enhancer-promoter looping and may serve as marks of protoregulatory enhancers.
A number of previously unsuspected complexes were revealed to be critical for enhancer activation events, including condensin I and condensin II, which exhibited dramatic E2-induced recruitment to ERα-bound active enhancers in interphase, regulating eRNA transcription and resulting in coding gene activation. A condensin-associated E3 ubiquitin ligase, HECTD1, recruited to active enhancers, was required for eRNA activation, revealing an enhancer-based role of interphase condensins in transcriptional regulation. Furthermore, functional estrogen (E2)-responsive enhancers were characterized by trans-recruitment/assembly by ERα at estrogen response element-containing enhancers of a large complex of diverse DNA-binding transcription factors, referred to as the MegaTrans complex. This complex proved to be a signature of the most potent functional enhancers, required for activation of eRNA transcription.
The MegaTrans complex appears to function, at least in part, by recruiting specific enzymatic machinery, exemplified by retinoic acid receptor (RAR)-dependent recruitment of DNA-dependent protein kinase, which is required for ligand-induced activation of enhancer transcription, raising the fundamental question of the relationship between regulated transcription and DNA damage/repair mechanisms. We have uncovered a molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis.
An additional aspect of enhancer function was revealed with evidence that JMJD6 and Brd4 regulate polymerase II (Pol II) promoter-proximal pausing in a large subset of genes based on their actions on distal enhancers, termed anti-pause enhancers (A-PEs). JMJD6 on A-PEs mediates both erasure of H4R3me2(s), a mark that is directly read by 7SK snRNA (small nuclear RNA), with decapping/demethylating of 7SK snRNA dismissing the 7SKsnRNA/HEXIM inhibitory complex, permitting activation of P-TEFb.
Enhancers and Subnuclear Architectural Structures
Investigation of a developmentally required POU-homeodomain transcription factor, Pit1/Pou1f1, has revealed that binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 and β-catenin is required for this tethering event. A naturally occurring, dominant-negative point mutation in human Pit1 (R271W), causing combined pituitary hormone deficiency (CPHD), results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the otherwise prerequisite association with β-catenin and Satb1. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of coactivators, exemplified by p300, causing both eRNA transcription and target gene activation. These studies have thus revealed a homeodomain transcription factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism required for enhancer-dependent activation of developmental gene transcriptional programs.
DNA Repeat Transcription as Integrators of Regulated Gene Response Programs
Although it is well established that liganded nuclear receptors regulate Pol II–dependent transcription units, their role in regulating DNA repeats has until recently remained largely unknown. We found that ~2–3 percent of the human DR2 Alu repeats, in proximity to activated Pol II transcription units, are bound and activated by RAR in human embryonic stem cells to generate Pol III–dependent RNAs. These transcripts are processed, initially in a Dicer-dependent fashion, into small RNAs (~28–65 nucleotides), referred to as riRNAs, that cause degradation of a subset of critical stem cell mRNAs, including Nanog, hence modulating exit from the proliferative stem cell state. This regulation requires Ago3-dependent accumulation of processed DR2 Alu transcripts and subsequent recruitment of Ago3-associated decapping complexes to the target mRNA. In this way, the RAR/Pol III–dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II–dependent neuronal transcriptional program. We also identified several long ncRNAs that impacted the androgen receptor (AR)-dependent transcriptional program in castration-resistant prostate cancer cell lines, licensing actions of AR at regulatory gene enhancers.
By establishing a cellular model that mimics authentic translocation events without proliferative selection, we uncovered mechanisms underlying nuclear receptor–dependent tumor translocations. This permitted new screening approaches to identify chemicals that prevent induced tumor translocations, leading to the discovery of a histone/protein demethylase inhibitor, SD70, synthesized by Sheng Ding's laboratory (Gladstone Institutes/University of California, San Francisco), as a potential therapeutic modality. In collaboration with the Ding lab, we devised a cell-based phenotypic screening strategy to explore potential interactions of drugs such as SD70 with the genome, based on synthesis of a derivatized form of SD70 that permits its application for a chromatin immunoprecipitation (ChIP)-sequencing-like approach, referred to as CHEM-seq. This revealed that SD70 largely colocalizes with AR on regulatory enhancers and inhibits the androgen-dependent AR program and prostate cancer cell growth, at least in part by functionally inhibiting the Jumonji domain–containing demethylase, KDM4C.
This work was supported in part by grants from the National Institutes of Health and the National Cancer Institute.
As of April 18, 2016