Genomic instability is a hallmark of cancer. Inducing severe DNA damage and augmenting apoptosis to destroy cancer cells is thus a major chemotherapy approach, specifically for Triple Negative Breast Cancer cell (TNBC cell). We have uncovered a previously unrecognized role for a specific long non-coding RNA 152 (lncRNA152) induced by DNA damage reagent and function in protect genome stability, significantly overexpressed in TNBC cell. Our studies provide preliminary data that lncRNA152 overexpressed in TNBC cell and could potentially serve as a TNBC diagnostic biomarker. Also, lncRNA152-depleted cells are hypersensitive to the DNA damage inducer induced by the topoisomerase II inhibitor doxorubicin. lncRNA152 expression levels increase in response to DNA damage and are significantly higher in TNBC.

We conclude that lncRNA152 could serve as a TNBC diagnostic biomarker and protects TNBC cells from DNA damage, perceptively providing a powerful approach to improve chemotherapy response.

This dissertation discusses the 4D chromosomal dynamics relative to subnuclear organelles and transcription hubs during transcription activation, in an estrogen-dependent system. The phenomenon of transcriptional bursting has been observed across numerous model organisms, where gene transcription does not occur continuously over time but rather in periods of high levels followed by periods of inactivity. I aim to explore such phenomenon in our estrogen inducible model and attempt to decipher the spatial and temporal dynamics associated with transcription burst by using RNA and DNA live cell imaging techniques. In the first chapter, I introduce the concept of burst transcription and relevant literature associated with bursting and transcription regulation. In the second chapter, I have included a review I co-authored on enhancers and its biological and biophysical properties that impact transcription regulation. Chapter 3 is where I present the core of my graduate research work involving live cell visualization techniques. Lastly, Chapter 4 is another paper I assisted on, where we examined how acute E2 enhancer activation results in eRNA-mediated phase-separated condensates that allow for cooperative activation of the most robust ERα enhancers.

Discovery of transferable distal genetic elements capable of activating gene transcription was a milestone in the study of transcriptional control. These genetic elements were termed “enhancers” because of their ability to enhance proximal coding gene transcription. Since then, it was believed that the activation potential of enhancers is entirely intrinsic. However, most prior studies of enhancer elements have removed them from their native genomic context, transplanting them onto plasmids or artificial chromosomes, thereby removing any potential effects of other elements in the genome. We sought to ask the question of whether the strongest E2 regulated enhancers on human chromosome 21 might interact, even when located on opposite ends of the chromosome q arm. These interactions play a functional role in allowing all interacting enhancers to have synergistically high levels of robustness. We have found that a cohort of the most highly active “first tier” ERα bound enhancers on chromosome 21, distributed across a linear distance of over 30 mega-bases, exhibit induced proximity when treated with the ligand agonist estradiol-17β (E2). Using CRISPR-Cas9 technology to delete some of these enhancers, we found, these enhancers seem to confer additional robustness onto other interacting enhancers. We believe that this occurs due to the physical proximity between “first tier” enhancers gained in response to E2, and that these interactions are dependent upon eRNA and a host of protein factors. These individual first tier enhancers in effect form a previously unappreciated “spatially distributed super-enhancer network”. These interaction events reflect physical constraints placed upon chromosome 21 imposed by a very large heterochromatic “B” compartment, which seems to predominantly localize to the surface of the nucleolus arising from the ribosomal DNA repeat rich p-arm of the acrocentric chromosome 21. The euchromatin “A” compartments on either side of the “B” compartment are thus allowed to interact dynamically. These sorts of dynamic interactions between regions of the chromosome constitute, in part, the overall structure of the chromosome. Therefore, the robustness of individual enhancers in a chromosome cannot be disentangled from the contributions of all interacting enhancers and the subnuclear structures in which these interactions can occur.

This dissertation, by Yiren Hu, discusses how enhancer activity is regulated to drive rapid, coordinated transcriptional response to signals like estrogen and inflammatory stimuli. Enhancer elements play a central role in instructing precise spatiotemporal gene expression and are capable of activating target gene transcription from a long distance. How do enhancers achieve these functions? And how are enhancers regulated in response to multiple signals at the same time? To answer these questions, we took a high-throughput approach with the power of next-generation sequencing technologies to study the genome wide transcription landscape changes of human breast cancer cells as well as mouse immune cells in response to stimuli like estradiol and TNFα. We tackled enhancer regulatory mechanisms from three aspects and found that: 1) chromatin structure regulators Condensin Complex I and II play essential roles in activating Estrogen Receptor α-bound enhancers by balancing transcription co-activators versus co-repressors homeostasis (see Chapter 1) 2) histone demethylase JMJD6 activates Estrogen Receptor α-bound enhancers and controls coding gene Pol II pausing release by modulating mediator complex protein MED12 chromatin loading (see Chapter 2) 3) Estrogen Receptor α and histone demethylase KDM2B form a two-layer restriction mechanism to repress NFκB enhancers and corresponding pro-inflammatory genes (see Chapter 3 and 4).

This dissertation, by Thomas Barton Suter, discusses enhancer function driving cellular senescence, DNA damage repair, differentiation, and nuclear organization. Enhancers are a major regulatory feature of epigenomic regulation, and play a diverse array of functions. In the first chapter, I discuss the background of cellular senescence and the epigenomic modifications that are known to play a role in this process, including marks associated with enhancer function. In the second chapter, I provide primary evidence of the key role enhancers play in driving the senescence phenotype. In the third chapter, I discuss the role that DNA damage and repair plays in nuclear receptor activity at enhancers. In the fourth chapter, I discuss the mechanisms by which regions in embryonic stem cells can be marked for later activation in future cell fates. In the fifth chapter, I present a study showing the role enhancers can play in nucleating nuclear organization.

The transcription machinery requires spatiotemporal coordination of its regulatory elements for precise gene expression. It is believed that the dynamics in the nuclear architecture like enhancer:promoter looping interaction is essential for the regulation of the transcription machinery. However, the detail of the change in 4D nuclear architecture during transcription is poorly understood. Here we seek to visualize a pair of enhancer and its associated target gene based on a real time, live cell imaging that uses optimized CRISPR-Cas9 and MS2/PP7 system. Our prior work on measuring TFF1 transcription bursting in MCF7 cells confirmed the efficiency and sensitivity of our imaging techniques. In this study, we aim to visualize c-MYC enhancer(c-MYCe) and c-MYC gene in live HeLa cells based on our preliminary data proving that c-MYC enhancer is a functional enhancer. We plan to measure the relative timing of signal, the duration of activity, and the distribution of intensity over the course of activity. The spatial kinetics between c-MYCe and c-MYC over time will be measured, and their motion relative to transcriptional activity (on/off signal) will be examined. Collectively, we hope to advance the understanding of the precise 4D dynamics of the events underlying enhancer-mediated transcription regulation.

This dissertation, by Soohwan Oh, discusses the general mechanisms how enhancers bring target genes activation in various stimuli. Enhancers are cis-regulatory DNA sequences that can increase the transcription of its cognate target genes. Fine-tuned coordination between enhancers and promoters is a fundamental tenant of gene transcriptional control. While enhancer-promoter (E-P) looping is widely observed, the precise mechanism allowing enhancer-promoter inter-regulation via the loop remains unknown. Here we report two interesting findings of 1) enhancer activation and 2) enhancer-promoter specificity. Critical chromatin structure regulators Condensin Complex I and II exhibit an unexpected, dramatic estrogen-induced recruitment to estrogen receptor a (ER-a)-bound eRNA transcribing active enhancers in interphase breast cancer cells and play essential roles in modulating estrogen-regulated enhancer activation and coding gene transcriptional program. (see Chapter 1). In addition, we found genetic deletion or mutation of gene promoters often, surprisingly, results in a more active state of their cognate enhancers. Intriguingly, the super-active enhancers switch their promoter target, causing other genes in the same contact domain to be activated, a target switching process that we referred to as “enhancer release and retargeting” (ERR). Integrative analyses of cancer mutations, the GTEx database and disease risk loci elucidated that ERR events represent a remarkably common, if overlooked, mechanism underlying disease-associated gene activation. (see Chapter 2)

FOXA1 is a DNA-binding protein that can regulate the estrogen transcriptional program. Although recent work has elucidated specific activities of the FOXA1 C-terminal domain, there is a dearth of information on the contribution of the N-terminal domain to FOXA1 function. By coupling FOXA1 shRNA knockdown with the expression of transgenic FOXA1 mutants, we show that the N-terminal domain is important for FOXA1’s ability to assist ER-α in activating estrogen-dependent transcription at promoters and enhancers. FOXA1 binding-site mutants showed that FOXA1-binding was critical for the recruitment of the transcription factor AP2-γ. The binding of AP2-γ was also affected by deletion of the FOXA1 N-terminal intrinsically-disordered domain. Knockdown and rescue experiments also showed that the FOXA1 N-terminus regulated H3K27 acetylation at estrogen-activated enhancers prior to ER-α recruitment without affecting the recruitment of FOXA1 or ER-α. Subsequently, proximity ligation mass spectrometry experiments suggested that FOXA1 was not only associated with a particular suite transcription factors, but also the Nuclear Remodeling and Deacetylase complex (NuRD). Estrogen-regulated enhancer FOXA1 binding-site deletion mutants were also shown to have decreased recruitment of the NuRD components CHD4 and HDAC2 compared to wild-type cells. Taken together, this data suggests that FOXA1 can regulate estrogen-dependent transcription through its N-terminus by recruiting AP2-γ and the NuRD complex.

Nuclear receptor co-repressors NCoR and SMRT are transcriptional regulatory proteins that interact with different transcription factors and epigenetic modifiers. Here, we study how NCoR/SMRT co-repressor complex regulates neuronal gene expression. In the lab, conditional knock-out mice have been developed to delete the NCoR and SMRT genes specifically in excitatory neurons of the postnatal forebrain. The conditional deletion of these genes is necessary because the traditional null mice die during embryonic development. Preliminary behavioral data show that the genetic deletion of both genes leads to stress-related behaviors. Hence, we hypothesize that NCoR/SMRT complex regulates the genomic action of glucocorticoid receptor (GR), a nuclear hormone receptor that functions as a cellular sensor for stress and it is known to interact with the NCoR/SMRT complex. To identify GR target genes regulated by NCoR/SMRT complex in the brain, we used NCoR/SMRT double knock-out mice and a transcriptomic approach known as Global Run-On sequencing. Subsequent bioinformatics analysis of sequencing data showed that the expression of hundreds of genes is altered upon genetic deletion of NCoR and SMRT. Moreover, biological processes linked to glucocorticoid signaling, such as immune response and circadian rhythm, were found to be dysregulated in the absence of NCoR and SMRT. These results provide evidence that NCoR/SMRT complex mediates the transcriptional regulation of key molecular pathways linked to the function of glucocorticoids in the brain.