The temporal dynamics of lncRNA Firre-mediated epigenetic and transcriptional regulation.

Numerous studies have now demonstrated that lncRNAs can influence gene expression programs leading to cell and organismal phenotypes. Typically, lncRNA perturbations and concomitant changes in gene expression are measured on the timescale of many hours to days. Thus, we currently lack a temporally grounded understanding of the primary, secondary, and tertiary relationships of lncRNA-mediated transcriptional and epigenetic regulation-a prerequisite to elucidating lncRNA mechanisms. To begin to address when and where a lncRNA regulates gene expression, we genetically engineer cell lines to temporally induce the lncRNA Firre. Using this approach, we are able to monitor lncRNA transcriptional regulatory events from 15 min to four days. We observe that upon induction, Firre RNA regulates epigenetic and transcriptional states in trans within 30 min. These early regulatory events result in much larger transcriptional changes after 12 h, well before current studies monitor lncRNA regulation. Moreover, Firre-mediated gene expression changes are epigenetically remembered for days. Overall, this study suggests that lncRNAs can rapidly regulate gene expression by establishing persistent epigenetic and transcriptional states.

DNMT1 inhibition by pUG-fold quadruplex RNA.

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

Evolutionary divergence of Firre localization and expression.

Long noncoding RNAs (lncRNAs) are rapidly evolving and thus typically poorly conserved in their sequences. How these sequence differences affect the characteristics and potential functions of lncRNAs with shared synteny remains unclear. Here we show that the syntenically conserved lncRNA Firre displays distinct expression and localization patterns in human and mouse. Single molecule RNA FISH reveals that in a range of cell lines, mouse Firre (mFirre) is predominantly nuclear, while human FIRRE (hFIRRE) is distributed between the cytoplasm and nucleus. This localization pattern is maintained in human/mouse hybrid cells expressing both human and mouse Firre, implying that the localization of the lncRNA is species autonomous. We find that the majority of hFIRRE transcripts in the cytoplasm are comprised of isoforms that are enriched in RRD repeats. We furthermore determine that in various tissues, mFirre is more highly expressed than its human counterpart. Our data illustrate that the rapid evolution of syntenic lncRNAs can lead to variations in lncRNA localization and abundance, which in turn may result in disparate lncRNA functions even in closely related species.

Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

Myosin 7b is a regulatory long noncoding RNA (lncMYH7b) in the human heart.

Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, ß-myosin heavy chain (ß-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike ß-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of ß-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate ß-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition.

Revealing Dynamic Protein Acetylation across Subcellular Compartments.

Protein acetylation is a widespread post-translational modification implicated in many cellular processes. Recent advances in mass spectrometry have enabled the cataloging of thousands of sites throughout the cell; however, identifying regulatory acetylation marks have proven to be a daunting task. Knowledge of the kinetics and stoichiometry of site-specific acetylation is an important factor to uncover function. Here, an improved method of quantifying acetylation stoichiometry was developed and validated, providing a detailed landscape of dynamic acetylation stoichiometry within cellular compartments. The dynamic nature of site-specific acetylation in response to serum stimulation was revealed. In two distinct human cell lines, growth factor stimulation led to site-specific, temporal acetylation changes, revealing diverse kinetic profiles that clustered into several groups. Overlap of dynamic acetylation sites among two different human cell lines suggested similar regulatory control points across major cellular pathways that include splicing, translation, and protein homeostasis. Rapid increases in acetylation on protein translational machinery suggest a positive regulatory role under progrowth conditions. Finally, higher median stoichiometry was observed in cellular compartments where active acetyltransferases are well described. Data sets can be accessed through ProteomExchange via the MassIVE repository (ProteomExchange: PXD014453; MassIVE: MSV000084029).

Mechanisms and Dynamics of Protein Acetylation in Mitochondria.

Reversible protein acetylation is a major regulatory mechanism for controlling protein function. Through genetic manipulations, dietary perturbations, and new proteomic technologies, the diverse functions of protein acetylation are coming into focus. Protein acetylation in mitochondria has taken center stage, revealing that 63% of mitochondrially localized proteins contain lysine acetylation sites. We summarize the field and discuss salient topics that cover spurious versus targeted acetylation, the role of SIRT3 deacetylation, nonenzymatic acetylation, and molecular models for regulatory acetylations that display high and low stoichiometry.

SIRT3 mediates multi-tissue coupling for metabolic fuel switching.

SIRT3 is a member of the Sirtuin family of NAD(+)-dependent deacylases and plays a critical role in metabolic regulation. Organism-wide SIRT3 loss manifests in metabolic alterations; however, the coordinating role of SIRT3 among metabolically distinct tissues is unknown. Using multi-tissue quantitative proteomics comparing fasted wild-type mice to mice lacking SIRT3, innovative bioinformatic analysis, and biochemical validation, we provide a comprehensive view of mitochondrial acetylation and SIRT3 function. We find SIRT3 regulates the acetyl-proteome in core mitochondrial processes common to brain, heart, kidney, liver, and skeletal muscle, but differentially regulates metabolic pathways in fuel-producing and fuel-utilizing tissues. We propose an additional maintenance function for SIRT3 in liver and kidney where SIRT3 expression is elevated to reduce the acetate load on mitochondrial proteins. We provide evidence that SIRT3 impacts ketone body utilization in the brain and reveal a pivotal role for SIRT3 in the coordination between tissues required for metabolic homeostasis.

Site-specific reactivity of nonenzymatic lysine acetylation.

Protein acetylation of lysine ε-amino groups is abundant in cells, particularly within mitochondria. The contribution of enzyme-catalyzed and nonenzymatic acetylation in mitochondria remains unresolved. Here, we utilize a newly developed approach to measure site-specific, nonenzymatic acetylation rates for 90 sites in eight native purified proteins. Lysine reactivity (as second-order rate constants) with acetyl-phosphate and acetyl-CoA ranged over 3 orders of magnitude, and higher chemical reactivity tracked with likelihood of dynamic modification in vivo, providing evidence that enzyme-catalyzed acylation might not be necessary to explain the prevalence of acetylation in mitochondria. Structural analysis revealed that many highly reactive sites exist within clusters of basic residues, whereas lysines that show low reactivity are engaged in strong attractive electrostatic interactions with acidic residues. Lysine clusters are predicted to be high-affinity substrates of mitochondrial deacetylase SIRT3 both in vitro and in vivo. Our analysis describing rate determination of lysine acetylation is directly applicable to investigate targeted and proteome-wide acetylation, whether or not the reaction is enzyme catalyzed.

Stoichiometry of site-specific lysine acetylation in an entire proteome.

Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.