The samples were ground to powder in liquid nitrogen, and the chromatin complexes were isolated and sonicated and then incubated with polyclonal HY5 antibodies (Osterlund et al., 2000). in promoting photomorphogenesis, directly binds ACGT-containing elements a few base pairs away from the FHY3/FAR1 binding sites in theFHY1/FHLpromoters. We demonstrate that HY5 physically interacts with FHY3/FAR1 through their respective DNA binding domains and negatively regulates FHY3/FAR1-activatedFHY1/FHLexpression LOR-253 under far-red light. Together, our data show that HY5 plays a role in negative feedback regulation of phyA signaling by attenuating FHY3/FAR1-activatedFHY1/FHLexpression, providing a mechanism LOR-253 for fine-tuning phyA signaling homeostasis. == INTRODUCTION == Phytochromes are red (R)/far-red Tm6sf1 (FR) light photoreceptors that play fundamental roles in photoperception of the light environment and the subsequent adaptation of plant growth and development (Quail et al., 1995;Whitelam and Devlin, 1997;Wang and Deng, 2003;Bae and Choi, 2008). There are five distinct phytochromes inArabidopsis thaliana, designated phytochrome A (phyA) to phyE. PhyA is light-labile and is the primary photoreceptor responsible for perceiving and mediating various responses to FR light, whereas phyB-phyE are light-stable, and phyB is the predominant phytochrome regulating responses to R light (Sharrock and Quail, 1989;Somers et al., 1991;Nagatani et al., 1993;Parks and Quail, 1993;Reed et al., 1993;Whitelam et al., 1993). Phytochromes are synthesized in the cytosol in their LOR-253 inactive Pr form. Upon light irradiation, phytochromes are converted from the R lightabsorbing Pr forms to the FR lightabsorbing Pfr forms, and the Pfr forms of phytochromes (which are generally considered to be the biologically active forms) are translocated from the cytosol into the nucleus, triggering a signaling cascade that alters the expression of target genes and ultimately leads to the modulation of the biological responses (Sakamoto and Nagatani, 1996;Kircher et al., 2002;Quail, 2002;Jiao et al., 2007). Thus, light-regulated translocation of the photoreceptors from the cytosol into the nucleus is a key event in the phytochrome signaling cascade. PhyA nuclear import is rapid and can be induced by either FR or R light; however, phyB nuclear import is relatively slow, only occurs in R light, and can be reversed by FR light (Kircher et al., 1999;Nagatani, 2004;Kevei et al., 2007;Fankhauser and Chen, 2008). The C-terminal half of phyB contains a putative nuclear localization signal (NLS), which is masked by the N-terminal half in darkness. Light triggers a conformational change in phyB, potentially unmasking the NLS and thus allowing its nuclear import (Chen et al., 2005;Fankhauser and Chen, 2008). By contrast, phyA does not contain any known NLS. Therefore, phyA translocation appears to depend on other components. Recently, it has been shown that two small plant-specific proteins, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL), are essential for nuclear accumulation of light-activated phyA and subsequent light responses (Hiltbrunner et al., 2005,2006;Rsler et al., 2007). A database search for FHY1/FHL homologs identified FHY1-like proteins in numerous plant species, and the only motifs conserved among these LOR-253 FHY1-like proteins are the NLS in their N-terminal region and the phyA binding domain at the C terminus, suggesting that the phyA nuclear import mechanism discovered inArabidopsismight LOR-253 be conserved in higher plants (Genoud et al., 2008). Two transposase-derived transcription factors, FHY3 and FAR-RED IMPAIRED RESPONSE1 (FAR1), act together to activate directly the transcription ofFHY1andFHL, thus indirectly regulating phyA nuclear accumulation and phyA responses (Lin et al., 2007,2008). Genetic and molecular studies have led to the identification of numerous signaling intermediates that are either specific for individual photoreceptors or shared by multiple types of photoreceptors. Many of these intermediates are transcription factors or transcriptional regulators (for reviews, seeQuail, 2002;Wang and Deng, 2003;Jiao et al., 2007). In addition to FHY3 and FAR1, a group of basic helix-loop-helix class transcription factors, also known as PHYTOCHROME INTERACTING FACTORS (PIFs), including PIF1, PIF3, PIF4, and PIF5, have been shown to bind photoactivated phytochromes directly and play central roles in phytochrome signaling networks (Ni et al., 1998;Huq and Quail, 2002;Kim et al., 2003;Huq et al., 2004;Khanna et al.,.