Metabolic Regulation of Gene Expression through Differential Histone Acylation: The Regulation and Function of Histone Crotonylation

Histone lysine acetylation (Kac) plays a critical role in gene regulation by affecting the accessibility of the DNA wrapped around histones and by recruiting effector complexes. Three major classes of proteins are associated with Kac, namely “writers,” enzymes that covalently modify specific lysine residues, “readers,” protein domains that specifically bind modified residues, and “erasers,” enzymes that catalyze the removal of the modification. While histone acetylation is well characterized within this paradigm, little is known about the regulation and function of an expanding list of histone lysine acylations. Lysine propionylation, butyrylation, and crotonylation were all discovered by proteomics-based approaches as I started in the Allis Lab. With reports suggesting that lysine crotonylation (Kcr) was the most functionally distinct from Kac I embarked to purify and identify the writers, readers, and erasers and thereby characterize the regulation and function of histone Kcr. To identify writers of Kcr I purified a histone crotonyltransferase (HCT) activity from nuclear extract by fractionation, which resulted in the purification of p300, a well-studied transcriptional co-activator and histone acetyltransferase (HAT). Together with colleagues in the Roeder lab, we established that p300’s HCT activity directly stimulates transcription to a greater degree than p300’s HAT activity. This work is discussed in Chapter 2. I developed several genetic and chemical approaches to manipulate the cellular concentrations of acetyl-CoA and crotonyl-CoA and established that acyl-CoA metabolism determines the state of differential histone acylation (Kac versus Kcr) thereby coupling the metabolic state to gene regulation. This work is discussed in Chapter 3. With these methods, I next studied the impact of Kcr in the macrophage inflammatory response, a classic model of signal-dependent gene activation. Through bioinformatics analysis of RNA-seq and ChIP-seq of macrophages in various conditions, I established that increased histone Kcr leads to enhanced expression of p300-regulated genes. This work is discussed in Chapter 4. These data suggested that there was a reader for Kcr with positive regulatory activity. In collaboration with colleagues in the Li lab, we identified the YEATS domain as a novel Kcr reader. Through a series of structural, biophysical, bioinformatic, and genetic studies we showed that AF9 and the YEATS-Kcr interaction is responsible for the enhanced expression of increased Kcr. This work is discussed in Chapter 5. As discussed in Chapter 6, I have also identified and characterized several decrotonylase (eraser) activities. The regulation of histone crotonylation or the functional consequence of a histone being acetylated versus crotonylated (differential acylation) has remained unclear since the discovery of the modification was reported. In my thesis work, I have demonstrated that the differential acylation state of histones is an integration of environmental and metabolic information, which serves a functional role in the regulation of gene expression

SnapShot: Histone Modifications

Histone proteins are decorated by a variety of protein posttranslational modifications called histone marks that modulate chromatin structure and function, contributing to the cellular gene expression program. This SnapShot summarizes the reported human, mouse, and rat histone marks, including recently identified lysine acylation marks

Coactivator condensation at super-enhancers links phase separation and gene control

Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.National Institutes of Health (U.S.) (Grant GM123511)National Institutes of Health (U.S.) (Grant P01-CA042063)National Science Foundation (U.S.) (Grant PHY-1743900)National Cancer Institute (U.S.) (Grant P30-CA14051

Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference

Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-β, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator β-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity

Coactivator condensation at super-enhancers links phase separation and gene control

Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes

Fibronectin Matrix Assembly Suppresses Dispersal of Glioblastoma Cells

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12–15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal

Biomolecular Condensates in the Nucleus

© 2020 Elsevier Ltd Nuclear processes such as DNA replication, transcription, and RNA processing each depend on the concerted action of many different protein and RNA molecules. How biomolecules with shared functions find their way to specific locations has been assumed to occur largely by diffusion-mediated collisions. Recent studies have shown that many nuclear processes occur within condensates that compartmentalize and concentrate the protein and RNA molecules required for each process, typically at specific genomic loci. These condensates have common features and emergent properties that provide the cell with regulatory capabilities beyond canonical molecular regulatory mechanisms. We describe here the shared features of nuclear condensates, the components that produce locus-specific condensates, elements of specificity, and the emerging understanding of mechanisms regulating these compartments