Ageing populations pose one of the main public health crises of our time. Reprogramming gene expression by altering the activities of sequence-specific transcription factors (TFs) can ameliorate deleterious effects of age. Here we explore how a circuit of TFs coordinates pro-longevity transcriptional outcomes, which reveals a multi-tissue and multi-species role for an entire protein family: the E-twenty-six (ETS) TFs. In Drosophila , reduced insulin/IGF signalling (IIS) extends lifespan by coordinating activation of Aop , an ETS transcriptional repressor, and Foxo , a Forkhead transcriptional activator. Aop and Foxo bind the same genomic loci, and we show that, individually, they effect similar transcriptional programmes in vivo . In combination, Aop can both moderate or synergise with Foxo , dependent on promoter context. Moreover, Foxo and Aop oppose the gene-regulatory activity of Pnt , an ETS transcriptional activator. Directly knocking down Pnt recapitulates aspects of the Aop / Foxo transcriptional programme and is sufficient to extend lifespan. The lifespan-limiting role of Pnt appears to be balanced by a requirement for metabolic regulation in young flies, in which the Aop-Pnt-Foxo circuit determines expression of metabolic genes, and Pnt regulates lipolysis and responses to nutrient stress. Molecular functions are often conserved amongst ETS TFs, prompting us to examine whether other Drosophila ETS-coding genes may also affect ageing. We show that five out of eight Drosophila ETS TFs play a role in fly ageing, acting from a range of organs and cells including the intestine, adipose and neurons. We expand the repertoire of lifespan-limiting ETS TFs in C . elegans , confirming their conserved function in ageing and revealing that the roles of ETS TFs in physiology and lifespan are conserved throughout the family, both within and between species.

13 14 Increasing average population age, and the accompanying burden of ill health, is one of the public health 15 crises of our time. Understanding the basic biology of the ageing process may help ameliorate the 16 pathologies that characterise old age. Ageing can be modulated, often through changes in gene expression 17 where regulation of transcription plays a pivotal role. Activities of Forkhead transcription factors (TFs) are 18 known to extend lifespan, but detailed knowledge of the broader transcriptional networks that promote 19 longevity is lacking. This study focuses on the E twenty-six (ETS) family of TFs. This family of TFs is large, 20 conserved across metazoa, and known to play roles in development and cancer, but the role of its 21 members in ageing has not been studied extensively. In Drosophila, an ETS transcriptional repressor, Aop, 22 and an ETS transcriptional activator, Pnt, are known to genetically interact with Foxo and activating Aop is 23 sufficient to extend lifespan. Here, it is shown that Aop and Foxo effect a related gene-expression 24 programme. Additionally, Aop can modulate Foxo's transcriptional output to moderate or synergise with 25Foxo activity depending on promoter context, both in vitro and in vivo. In vivo genome-wide mRNA 26 expression analysis in response to Aop, Pnt or Foxo indicated, and further experiments confirmed, that 27 combinatorial activities of the three TFs dictate metabolic status, and that direct reduction of Pnt activity is 28 sufficient to promote longevity. The role of ETS factors in longevity was not limited to Pnt and Aop. 29 Knockdown of Ets21c or Eip74EF in distinct cell types also extended lifespan, revealing that lifespan is 30 limited by transcription from the ETS binding site in multiple cellular contexts. Reducing the activity of the 31 C. elegans ETS TF Lin-1 also extended lifespan, a finding that corroborates established evidence of roles 32 of this TF family in ageing. Altogether, these results reveal the ETS family of TFs as pervasive and 33 evolutionarily conserved brokers of longevity. 34 35 36 Ageing is characterised by a steady systematic decline in biological function and increased 37 likelihood of disease. Understanding the basic biology of ageing therefore promises to help alleviate the 38 personal and societal burdens resulting from the increasing proportion of older people in our populations. 39The pursuit of this goal has revealed that ageing is plastic, and healthy lifespan can be extended by 40 manipulating specific genes, including those encoding components of nutrient signalling pathways 1 . Such 41 interventions often act through changes in gene expression, with transcriptional regulation playing a critical 42 role 2-5 . Sequence-specific transcription factors (TFs) are the primary coordinators of transcriptional 43 programmes 6 . Hence, understanding their function in adult animals will provide insight into how gene 44 expression can be altered to optimise physiology towards promoting lasting health into late life. 4...