Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

The Mediator multiprotein complex (‘Mediator’) is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways.

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinasedependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals.KEYWORDS Mediator complex; CDK8; MED23; MED15; EGFR; Notch P RECISE regulation of transcription is required to execute developmental programs such as proliferation and cell fate determination. The Mediator complex ("Mediator") is a conserved eukaryotic transcriptional coregulator of RNA polymerase II (Pol II) transcription (Malik and Roeder 2010;Poss et al. 2013). Mediator consists of 30 subunits that assemble into four modules. "Core" Mediator consists of three of the four modules: the head and middle modules, which contact Pol II, and the tail module, which serves as a docking site for transcription factors. The fourth module, the dissociable cyclin dependent kinase 8 (CDK8) kinase module (CKM), interacts with transcription factors, core Mediator, chromatin, and the Pol II machinery to either repress or activate transcription (Malik and Roeder 2010;Nemet et al. 2014 transcription, tail and CKM subunits regulate specific transcriptional programs in animal development or physiology (Malik and Roeder 2010;Nemet et al. 2014). The CKM consists of enzymatic subunits CDK8 and cyclin C, and structural subunits MED12 and MED13 that tether the CKM to core Mediator (Tsai et al. 2013). C...

Zinc is essential for cellular functions as it is a catalytic and structural component of many proteins. In contrast, cadmium is not required in biological systems and is toxic. Zinc and cadmium levels are closely monitored and regulated as their excess causes cell stress. To maintain homeostasis, organisms induce metal detoxification gene programs through stress responsive transcriptional regulatory complexes. In Caenorhabditis elegans, the MDT-15 subunit of the evolutionarily conserved Mediator transcriptional coregulator is required to induce genes upon exposure to excess zinc and cadmium. However, the regulatory partners of MDT-15 in this response, its role in cellular and physiological stress adaptation, and the putative role for mammalian MED15 in the metal stress responses remain unknown. Here, we show that MDT-15 interacts physically and functionally with the Nuclear Hormone Receptor HIZR-1 to promote molecular, cellular, and organismal adaptation to cadmium and excess zinc. Using gain- and loss-of-function mutants and qRT-PCR and reporter analysis, we find that mdt-15 and hizr-1 cooperate to induce zinc and cadmium responsive genes. Moreover, the two proteins interact physically in yeast-two-hybrid assays and this interaction is enhanced by the addition of zinc or cadmium, the former a known ligand of HIZR-1. Functionally, mdt-15 and hizr-1 mutants show defective storage of excess zinc in the gut and are hypersensitive to zinc-induced reductions in egg-laying. Furthermore, mdt-15 but not hizr-1 mutants are hypersensitive to cadmium-induced reductions in egg-laying, suggesting potential divergence of regulatory pathways. Lastly, mammalian MDT-15 orthologs bind genomic regulatory regions of metallothionein and zinc transporter genes in a cadmium and zinc-stimulated fashion, and human MED15 is required to induce a metallothionein gene in lung adenocarcinoma cells exposed to cadmium. Collectively, our data show that mdt-15 and hizr-1 cooperate to regulate cadmium detoxification and zinc storage and that this mechanism is at least partially conserved in mammals.

26Zinc is essential for cellular functions as it is a catalytic and structural component of many 27 proteins. In contrast, cadmium is not required in biological systems and is toxic. Zinc and cadmium 28 levels are closely monitored and regulated as their excess causes cell stress. To maintain homeostasis, 29 organisms induce metal detoxification gene programs through stress responsive transcriptional 30 regulatory complexes. In Caenorhabditis elegans, the MDT-15 subunit of the evolutionarily conserved 31Mediator transcriptional coregulator is required to induce genes upon exposure to excess zinc and 32 cadmium. However, the regulatory partners of MDT-15 in this response, its role in cellular and 33 physiological stress adaptation, and the putative role mammalian for MED15 in the metal stress 34 responses remain unknown. Here, we show that MDT-15 interacts physically and functionally with the 35Nuclear Hormone Receptor HIZR-1 to promote molecular, cellular, and organismal adaptation to 36 excess metals. Using gain-and loss-of-function mutants and qPCR and reporter analysis, we find that 37 mdt-15 and hizr-1 cooperate to induce zinc and cadmium responsive genes. Moreover, the two proteins 38 interact physically in yeast-two-hybrid assays and this interaction is enhanced by the addition of zinc or 39 cadmium, the former a known ligand of HIZR-1. Functionally, mdt-15 and hizr-1 mutants show 40 defective storage of excess zinc in the gut, and at the organismal level, mdt-15 mutants are 41 hypersensitive to zinc-and cadmium-induced reductions in egg-laying. Lastly, mammalian MDT-15 42 orthologs bind genomic regulatory regions of metallothionein and zinc transporter genes in a metal-43 stimulated fashion, and human MED15 is required to induce a metallothionein gene in lung 44 adenocarcinoma cells exposed to cadmium. Collectively, our data show that mdt-15 and hizr-1 45 cooperate to regulate metal detoxification and zinc storage and that this mechanism appears to be at 46 least partially conserved in mammals. 48In their habitats, biological organisms encounter many metals, including essential 49 micronutrients such as iron, zinc, copper, and manganese, and toxic metals such as cadmium, mercury, 50 lead, and arsenic. Zinc is an essential trace element that plays a crucial role in numerous cellular and 51 physiological processes (1). It has a structural role in metabolic enzymes, growth factors, and 52 transcriptional regulators such as zinc finger proteins, and is also an enzymatic cofactor and a signaling 53 molecule (2,3). Accordingly, zinc is necessary for the function of approximately 10% of proteins in the 54 human proteome and approximately 8% of proteins in the nematode worm Caenorhabditis elegans (4). 55

Receptors expressed on the growth cone of outgrowing axons detect cues required for proper navigation. The pathway choices available to an axon are in part defined by the set of guidance receptors present on the growth cone. Regulated expression of receptors and genes controlling the localization and activity of receptors ensures that axons respond only to guidance cues relevant for reaching their targets. In genetic screens for axon guidance mutants, we isolated an allele of let-19/mdt-13, a component of the Mediator, a large ~30 subunit protein complex essential for gene transcription by RNA polymerase II. LET-19/MDT-13 is part of the CDK8 module of the Mediator. By testing other Mediator components, we found that all subunits of the CDK8 module as well as some other Mediator components are required for specific axon navigation decisions in a subset of neurons. Expression profiling demonstrated that let-19/mdt-13 regulates the expression of a large number of genes in interneurons. A mutation in the sax-3 gene, encoding a receptor for the repulsive guidance cue SLT-1, suppresses the commissure navigation defects found in cdk-8 mutants. This suggests that the CDK8 module specifically represses the SAX-3/ROBO pathway to ensure proper commissure navigation.

Despite many advances, the molecular links between energy metabolism and longevity are not well understood. Here, we have used the nematode model Caenorhabditis elegans to study the role of the yet-uncharacterized gene R148.3 in fat accumulation and lifespan. In wild-type worms, a R148.3p::GFP reporter showed enhanced expression throughout life in the pharynx, in neurons, and in muscles. Functionally, a protein fusing a predicted 22 amino acid N-terminal signal sequence (SS) of R148.3 to mCherry displayed robust accumulation in coelomyocytes, indicating that R148.3 is a secreted protein. Systematic depletion of R148.3 by RNA interference (RNAi) at L1 but not at young-adult stage enhanced triglyceride accumulation, which was associated with increased food uptake and lower expression of genes involved in lipid oxidation. However, RNAi of R148.3 at both L1 and young-adult stages robustly diminished mean and maximal lifespan of wild-type worms, and also abolished the long-lived phenotypes of eat-2 and daf-2/InsR mutants. Based on these data, we propose that R148.3 is an SS that modulates fat mass and longevity in an independent manner.

The ZFY protein is a member of one of the most interesting classes of polydactyl zinc finger proteins. It has a domain of 13 tandem zinc fingers that is organized with an internal repeat of odd-even finger pairs. It has been proposed that each finger pair interacts with six base pairs within a turn of the double helix, the downstream linker crossing the minor groove to place the next finger pair on the following turn of the DNA. Yet putative binding sites for the full-length ZFY protein appear to consist of a six-base AGGCCY consensus sequence that is present in one or two copies. In this study the equilibrium binding of two ZFY-derived zinc finger peptides to 4R DNA with tandem copies of the consensus sequence was investigated. The ZFY5 peptide contains fingers 5-13, including four odd-even finger pairs, and the ZFY11 peptide contains fingers 11-13 and has one odd-even finger pair. Both peptides bound to 4R DNA with equal affinities, forming a bimolecular complex that is mediated by the downstream AGGCCY motif. The additional odd-even finger pairs in ZFY5 made no measurable difference in the mechanism of DNA binding compared to ZFY11. The effects on the DNA-protein interaction of mutations in the 4R DNA and in the key alpha-helical residues of fingers 11-13 indicate that the binding of ZFY to DNA is mediated by the interaction of the GGCC core base pairs with fingers 12 and 13. These results demonstrate that the even-odd repeats in the ZFY zinc finger domain do not make significant contributions to DNA binding.