Histone oxidation as a new mechanism of metabolic control over gene expression.

The emergence of aerobic respiration created unprecedented bioenergetic advantages, while imposing the need to protect critical genetic information from reactive byproducts of oxidative metabolism (i.e., reactive oxygen species, ROS). The evolution of histone proteins fulfilled the need to shield DNA from these potentially damaging toxins, while providing the means to compact and structure massive eukaryotic genomes. To date, several metabolism-linked histone post-translational modifications (PTMs) have been shown to regulate chromatin structure and gene expression. However, whether and how PTMs enacted by metabolically produced ROS regulate adaptive chromatin remodeling remain relatively unexplored. Here, we review novel mechanistic insights into the interactions of ROS with histones and their consequences for the control of gene expression regulation, cellular plasticity, and behavior.

In Science Journals.

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Transcriptional elongation control of hypoxic response.

The release of paused RNA polymerase II (RNAPII) from promoter-proximal regions is tightly controlled to ensure proper regulation of gene expression. The elongation factor PTEF-b is known to release paused RNAPII via phosphorylation of the RNAPII C-terminal domain by its cyclin-dependent kinase component, CDK9. However, the signal and stress-specific roles of the various RNAPII-associated macromolecular complexes containing PTEF-b/CDK9 are not yet clear. Here, we identify and characterize the CDK9 complex required for transcriptional response to hypoxia. Contrary to previous reports, our data indicate that a CDK9 complex containing BRD4 but not AFF1/4 is essential for this hypoxic stress response. We demonstrate that BRD4 bromodomains (BET) are dispensable for the release of paused RNAPII at hypoxia-activated genes and that BET inhibition by JQ1 is insufficient to impair hypoxic gene response. Mechanistically, we demonstrate that the C-terminal region of BRD4 is required for Polymerase-Associated Factor-1 Complex (PAF1C) recruitment to establish an elongation-competent RNAPII complex at hypoxia-responsive genes. PAF1C disruption using a small-molecule inhibitor (iPAF1C) impairs hypoxia-induced, BRD4-mediated RNAPII release. Together, our results provide insight into potentially targetable mechanisms that control the hypoxia-responsive transcriptional elongation.

Therapeutic targeting of BET bromodomain and other epigenetic acetylrecognition domain-containing factors.

Development of cancer therapies targeting chromatin modifiers and transcriptional regulatory factors is rapidly expanding to include new targets and novel targeting strategies. At the same time, basic molecular research continues to refine our understanding of the epigenetic mechanisms regulating transcription, gene expression, and oncogenesis. This mini-review focuses on cancer therapies targeting the chromatin-associated factors that recognize histone lysine acetylation. Recently reported safety and efficacy are discussed for inhibitors targeting the bromodomains of bromodomain and extraterminal domain (BET) family proteins. In light of recent results indicating that the transcriptional regulator BRD4-PTEFb can function independently of BRD4's bromodomains, the clinical trial performance of these BET inhibitors is placed in a broader context of existing and potential strategies for targeting BRD4-PTEFb. Recently developed therapies targeting bromodomain-containing factors within the SWI/SNF (BAF) family of chromatin remodeling complexes are discussed, as is the potential for targeting the bromodomain-containing transcription factor TAF1 and the YEATS acetylrecognition domain-containing factor GAS41. Recent findings regarding the selectivity and combinatorial specificity of acetylrecognition are highlighted. In conclusion, the potential for further development is discussed with a focus on proximity-based therapies targeting this class of epigenetic factors.

Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis.

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

NELF coordinates Pol II transcription termination and DNA replication initiation.

Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD , a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict. HIGHLIGHTS: Expression of NELFCD transcript is up-regulated in colorectal tumors NELF-C protein is mandatory for the transition between G1-S phases during cell cycleNELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausingNELF-C loss leads Pol II to invade DNA replication initiation zones.

In Science Journals.

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Somatic mutations of MLL4/COMPASS induce cytoplasmic localization providing molecular insight into cancer prognosis and treatment.

Cancer genome sequencing consortiums have recently catalogued an abundance of somatic mutations, across a wide range of human cancers, in the chromatin-modifying enzymes that regulate gene expression. Defining the molecular mechanisms underlying the potentially oncogenic functions of these epigenetic mutations could serve as the basis for precision medicine approaches to cancer therapy. MLL4 encoded by the KMT2D gene highly mutated in a large number of human cancers, is a key histone lysine monomethyltransferase within the Complex of Proteins Associated with Set1 (COMPASS) family that regulates gene expression through enhancer function, potentially functioning as a tumor suppressor. We report that the KMT2D mutations which cause MLL4 protein truncation also alter MLL4's subcellular localization, resulting in loss-of-function in the nucleus and gain-of-function in the cytoplasm. We demonstrate that isogenic correction of KMT2D truncation mutation rescues the aberrant localization phenotype and restores multiple regulatory functions of MLL4, including COMPASS integrity/stabilization, histone H3K4 mono-methylation, enhancer activation, and therefore transcriptional regulation. Moreover, isogenic correction diminishes the sensitivity of KMT2D-mutated cancer cells to targeted metabolic inhibition. Using immunohistochemistry, we identified that cytoplasmic MLL4 is unique to the tissue of bladder cancer patients with KMT2D truncation mutations. Using a preclinical carcinogen model of bladder cancer in mouse, we demonstrate that truncated cytoplasmic MLL4 predicts response to targeted metabolic inhibition therapy for bladder cancer and could be developed as a biomarker for KMT2D-mutated cancers. We also highlight the broader potential for prognosis, patient stratification and treatment decision-making based on KMT2D mutation status in MLL4 truncation-relevant diseases, including human cancers and Kabuki Syndrome.

Transcriptional elongation control in developmental gene expression, aging, and disease.

The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.

ELOA3: A primate-specific RNA polymerase II elongation factor encoded by a tandem repeat gene cluster.

The biological role of the repetitive DNA sequences in the human genome remains an outstanding question. Recent long-read human genome assemblies have allowed us to identify a function for one of these repetitive regions. We have uncovered a tandem array of conserved primate-specific retrogenes encoding the protein Elongin A3 (ELOA3), a homolog of the RNA polymerase II (RNAPII) elongation factor Elongin A (ELOA). Our genomic analysis shows that the ELOA3 gene cluster is conserved among primates and the number of ELOA3 gene repeats is variable in the human population and across primate species. Moreover, the gene cluster has undergone concerted evolution and homogenization within primates. Our biochemical studies show that ELOA3 functions as a promoter-associated RNAPII pause-release elongation factor with distinct biochemical and functional features from its ancestral homolog, ELOA. We propose that the ELOA3 gene cluster has evolved to fulfil a transcriptional regulatory function unique to the primate lineage that can be targeted to regulate cellular hyperproliferation.

Iron drives anabolic metabolism through active histone demethylation and mTORC1.

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

In Science Journals.

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Distinct layers of BRD4-PTEFb reveal bromodomain-independent function in transcriptional regulation.

The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity.

Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis.

Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. We report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing, we identify specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determine that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we show that removal of Glucose transporter 1 and Lactate dehydrogenase A gene activity from developing retinal progenitors arrests eye morphogenesis. Surprisingly, we uncover that lactate-mediated upregulation of key eye-field transcription factors is controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase activity. Our results identify an unexpected bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.

Therapeutic targeting of metabolic vulnerabilities in cancers with MLL3/4-COMPASS epigenetic regulator mutations.

Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases, including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity toward increased purine synthesis in MLL3/4-KO mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA-Seq identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4-KO cells. Mechanistically, we demonstrated that compensation by MLL1/COMPASS was underlying these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vitro and in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.

Epigenetic moonlighting: Catalytic-independent functions of histone modifiers in regulating transcription.

The past three decades have yielded a wealth of information regarding the chromatin regulatory mechanisms that control transcription. The "histone code" hypothesis-which posits that distinct combinations of posttranslational histone modifications are "read" by downstream effector proteins to regulate gene expression-has guided chromatin research to uncover fundamental mechanisms relevant to many aspects of biology. However, recent molecular and genetic studies revealed that the function of many histone-modifying enzymes extends independently and beyond their catalytic activities. In this review, we highlight original and recent advances in the understanding of noncatalytic functions of histone modifiers. Many of the histone modifications deposited by these enzymes-previously considered to be required for transcriptional activation-have been demonstrated to be dispensable for gene expression in living organisms. This perspective aims to prompt further examination of these enigmatic chromatin modifications by inspiring studies to define the noncatalytic "epigenetic moonlighting" functions of chromatin-modifying enzymes.