In this paper, we combine genetic, proteomic and genomic approaches to determine how a cGMP dependent protein kinase, EGL-4, modulates gene expression. Activated EGL-4 preferentially associates with a conserved SAEG-1/SAEG-2 histone deacetylase complex, which in turn represses a novel gene that regulates egg-laying rate (). Our results demonstrate that in additional to cytoplasmic roles in Drosophila
and mammals 
, cGMP dependent kinase can act in the nucleus to elicit long-term transcriptional changes that affect multiple physiological processes such as foraging behavior, egg laying rate and body length in C. elegans
Genomic effects of activated nuclear EGL-4 are mediated by the SAEG-1/SAEG-2/HDA-2 complex.
We propose that EGL-4 plays a central role in linking sensory input regarding environmental conditions to long-term physiological responses by altering gene expression of intracellular and extracellular signaling molecules. Although nuclear function of EGL-4 and mammalian PKG had been suggested in the past, in part through the demonstration of EGL-4/PKG nuclear translocation 
, the effectors of nuclear EGL-4/PKG activity in vivo
had been ill-defined. Given the ubiquitous expression of EGL-4, SAEG-1 and SAEG-2, and the requirement of SAEG-1 and SAEG-2 by EGL-4 in multiple physiological processes, our results support a model in which the SAEG-1/SAEG-2 complex mediates EGL-4 activity in the nucleus in diverse tissues. Nevertheless, we noted that loss of saeg-1
function was not equivalent to a complete loss of egl-4
function (). This suggests that EGL-4 activity can be mediated by alternative nuclear or cytoplasmic pathways in parallel of SAEG-1 and SAEG-2 (). For example, EGL-4 has been reported to modulate gene expression of two chemosensory receptors, str-1
, through inhibition of the histone deacetylase HDA-4 
. It is plausible that EGL-4 engages different histone deacetylase complexes in a context dependent manner.
We took a non-biased genomic approach to identify EGL-4 activity responsive genes. Based on our model, we reasoned that such genes should be repressed upon EGL-4 activation in a SAEG-1/SAEG-2 complex dependent manner. In addition to Y45F10C.2, which regulates egg laying rate, we also identified a group of genes that are involved in lipid metabolism, lipid and sugar transport, and extracellular signaling (; YAH, LCH and HYM, unpublished data). Our results are consistent with the observation that alteration in PKG activity in Drosophila
also led to changes in lipid and carbohydrate storage and metabolism 
How does EGL-4 regulate egg-laying has been an open question since the original isolation of egl-4
mutants almost 30 years ago 
. Although the neuronal circuit that controls egg-laying muscles is well-defined 
, additional mechanisms have been proposed to modulate egg-laying rate in response to environmental stimuli. EGL-4 may be involved in the latter since pharmacological studies failed to assign a role for EGL-4 in the neuronal circuit that controls the egg-laying muscles or at the muscles themselves 
. Here, we provide experimental evidence that EGL-4 controls egg-laying, at least in part, by modulating the expression of Y45F10C.2, a putative secreted protein from the uterine epithelium. The DUF1505 family, to which Y45F10C.2 belongs, appears to have undergone expansion through gene duplication in C. elegans
. However, Y45F10C.2 and its duplicate, C08F11.12 are the only family members whose expression is regulated by EGL-4 activity (NXU and HYM, unpublished data). The molecular target of Y45F10C.2 is unknown. It is plausible that Y45F10C.2 can bind to novel cell surface receptors that inhibit egg laying or modulate the function of serotonin, acetylcholine or neuropeptide receptors at the neuromuscular circuit for egg laying.
Our results support a surprising in vivo
role for the mammalian orthologs of SAEG-1 and SAEG-2. In cell culture systems, TRERF1 has been reported to activate CYP11A1, a gene required for steroidogenesis 
. TRERF1 also interacts with Dnttip1 and together, they have been implicated in V(D)J recombination by antagonizing the terminal deoxynucleotidyltransferase (TdT) 
. Another SAEG-1 ortholog, ZNF541, has been implicated in chromatin remodeling during spermatogenesis in mice 
. While it is hard to reconcile such diverse functions of TRERF1, ZNF541 and Dnttip1, our results implicate that together with HDAC1 or HDAC2, TRERF1 and Dnttip1 may constitute a PKG effector complex in tissues where they are co-expressed, such as the olfactory bulb in mice (Allen Brain Atlas). Notably, EGL-4 is known to be required for chemotaxis and olfactory adaptation in sensory neurons in C. elegans 
. It is plausible that PKG may modulate gene expression in the olfactory bulb via the TRERF1/Dnttip1/HDAC complex upon olfactory stimulation.
While the intracellular cGMP level is tightly regulated by the opposing action of guanylyl cyclase (GC) and phosphodiesterase (PDE) physiologically 
, NO-releasing organic nitrates and PDE inhibitors have been developed to increase cGMP level pharmacologically 
. This is because the NO-cGMP-PKG pathway is critical for relaxation of smooth muscles and activation of this pathway provides effective therapy for erectile dysfunction, pulmonary hypertension and potentially other diseases associated with abnormal smooth muscle tone. Despite the wide spread use of nitrates and PDE inhibitors, little is known about the effects of sustained elevation of cGMP level and presumably prolonged activation of PKG. Our results suggest that the potential genomic effects of PKG activation should be considered when administering nitrates and PDE inhibitors. Furthermore, the conserved SAEG-1/SAEG-2 histone deacetylase complex represents new molecular targets for the development of chemicals that specifically target cytoplasmic versus nuclear PKG activity.
In summary, our results provide a molecular framework on how nuclear EGL-4/PKG activity triggers wide-spread physiological responses. In C. elegans, we expect to identify additional EGL-4 responsive genes that regulate foraging behavior and body length. It is plausible that the SAEG-1/SAEG-2 histone deacetylase complex may engage tissue specific transcription factors, which may be identified through isolation of tissue specific genetic suppressors of the egl-4(gf) mutant. Given the deep conservation of EGL-4, SAEG-1 and SAEG-2, our model should be readily applicable in mammals and expand the mode of action of cGMP signaling and its downstream kinase PKG.