Joaquín M. Espinosa

The Histone Deacetylase Sirt6 Regulates Glucose Homeostasis via Hif1α

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes.

We investigated co-transcriptional recruitment of pre-mRNA processing factors to human genes. Capping factors associate with paused RNA polymerase II (pol II) at the 5′ ends of quiescent genes. They also track throughout actively transcribed genes and accumulate with paused polymerase in the 3′ flanking region. The 3′ processing factors cleavage stimulation factor and cleavage polyadenylation specificity factor are maximally recruited 0.5–1.5 kilobases downstream of poly(A) sites where they coincide with capping factors, Spt5, and Ser2-hyperphosphorylated, paused pol II. 3′ end processing factors also localize at transcription start sites, and this early recruitment is enhanced after polymerase arrest with the elongation factor DRB. These results suggest that promoters may help specify recruitment of 3′ end processing factors. We propose a dual-pausing model wherein elongation arrests near the transcription start site and in the 3′ flank to allow co-transcriptional processing by factors recruited to the pol II ternary complex.

CDK8 is a positive regulator of transcriptional elongation within the serum response network

The Mediator complex allows communication between transcription factors and RNA polymerase II (RNAPII). Cyclin-dependent kinase 8 (CDK8), the kinase found in some variants of Mediator, has been characterized mostly as a transcriptional repressor. Recently, CDK8 was demonstrated to be a potent oncoprotein. Here we show, using a human tumor cell line, that CDK8 is a positive regulator of genes within the serum response network, including several members of the activator protein 1 and early growth response family of oncogenic transcription factors. Mechanistic studies show that CDK8 is not required for RNAPII recruitment or promoter escape. Instead, CDK8 depletion leads to the appearance of slower elongation complexes carrying hypophosphorylated RNAPII. CDK8-Mediator regulates precise steps in the assembly of the RNAPII elongation complex, including the recruitment of positive transcription elongation factor b and BRD4. Furthermore, CDK8-Mediator specifically interacts with positive transcription elongation factor b. Thus, we have uncovered a role for CDK8 in transcriptional regulation that may contribute to its oncogenic effects.

The Human CDK8 Subcomplex Is a Histone Kinase That Requires Med12 for Activity and Can Function Independently of Mediator

ABSTRACT The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable “CDK8 subcomplex” in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12—but not Med13—to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex—unlike the recombinant submodule—is unable to phosphorylate the Pol II CTD.

HIF1A Employs CDK8-Mediator to Stimulate RNAPII Elongation in Response to Hypoxia

The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the super elongation complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.

CDK8: a positive regulator of transcription.

CDK8 belongs to a group of cyclin-dependent kinases involved in transcriptional regulation from yeast to mammals. CDK8 associates with the Mediator complex, but functions outside of Mediator are also likely. Historically, CDK8 has been described mostly as a transcriptional repressor, but a growing body of research provides unequivocal evidence for various roles of CDK8 in gene activation. Several transcriptional programs of biomedical importance employ CDK8 as a co-activator, including the p53 network, the Wnt/β-catenin pathway, the serum response network, and those governed by SMADs and the thyroid hormone receptor, thus highlighting the importance of further investigation into this enigmatic transcriptional regulator.CDK8 belongs to a group of cyclin-dependent kinases involved in transcriptional regulation from yeast to mammals. CDK8 associates with the Mediator complex, but functions outside of Mediator are also likely. Historically, CDK8 has been described mostly as a transcriptional repressor, but a growing body of research provides unequivocal evidence for various roles of CDK8 in gene activation. Several transcriptional programs of biomedical importance employ CDK8 as a co-activator, including the p53 network, the Wnt/β-catenin pathway, the serum response network, and those governed by SMADs and the thyroid hormone receptor, thus highlighting the importance of further investigation into this enigmatic transcriptional regulator.

Mechanisms of regulatory diversity within the p53 transcriptional network

p53 is arguably the most intensively studied protein to date, yet there is much that we ignore about its function as a transcription factor. The p53-dependent transcriptional program is remarkably flexible, as it varies with the nature of p53-activating stimuli, the cell type and the duration of the activation signal. This flexibility may allow cells to mount alternative responses to p53 activation, such as cell cycle arrest or apoptosis. Here, I organize the available data into two alternative models to explain how this regulatory diversity is achieved.

Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms

The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms. DOI: http://dx.doi.org/10.7554/eLife.02200.001

Gene-specific repression of the p53 target gene PUMA via intragenic CTCF–Cohesin binding

The p53 transcriptional program orchestrates alternative responses to stress, including cell cycle arrest and apoptosis, but the mechanism of cell fate choice upon p53 activation is not fully understood. Here we report that PUMA (p53 up-regulated modulator of apoptosis), a key mediator of p53-dependent cell death, is regulated by a noncanonical, gene-specific mechanism. Using chromatin immunoprecipitation assays, we found that the first half of the PUMA locus (approximately 6 kb) is constitutively occupied by RNA polymerase II and general transcription factors regardless of p53 activity. Using various RNA analyses, we found that this region is constitutively transcribed to generate a long unprocessed RNA with no known coding capacity. This permissive intragenic domain is constrained by sharp chromatin boundaries, as illustrated by histone marks of active transcription (histone H3 Lys9 trimethylation [H3K4me3] and H3K9 acetylation [H3K9Ac]) that precipitously transition into repressive marks (H3K9me3). Interestingly, the insulator protein CTCF (CCCTC-binding factor) and the Cohesin complex occupy these intragenic chromatin boundaries. CTCF knockdown leads to increased basal expression of PUMA concomitant with a reduction in chromatin boundary signatures. Importantly, derepression of PUMA upon CTCF depletion occurs without p53 activation or activation of other p53 target genes. Therefore, CTCF plays a pivotal role in dampening the p53 apoptotic response by acting as a gene-specific repressor.

Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3

The human Mediator complex is generally required for expression of protein‐coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative ‘T/G‐Mediator’ complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G‐Mediator, cdk8 phosphorylates serine‐10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co‐occupancy of T/G‐Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G‐Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8‐Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G‐Mediator directs early events—such as modification of chromatin templates—in transcriptional activation.