The ubiquitin fold modifier 1 (Ufm1) is the most recently discovered ubiquitin-like modifier whose conjugation (ufmylation) system is conserved in multicellular organisms. Ufm1 is known to covalently attach with cellular protein(s) via a specific E1-activating enzyme (Uba5) and an E2-conjugating enzyme (Ufc1), but its E3-ligating enzyme(s) as well as the target protein(s) remain unknown. Herein, we report both a novel E3 ligase for Ufm1, designated Ufl1, and an Ufm1-specific substrate ligated by Ufl1, C20orf116. Ufm1 was covalently conjugated with C20orf116. Although Ufl1 has no obvious sequence homology to any other known E3s for ubiquitin and ubiquitin-like modifiers, the C20orf116⅐Ufm1 formation was greatly accelerated by Ufl1. The C20orf116⅐Ufm1 conjugate was cleaved by Ufm1-specific proteases, implying the reversibility of ufmylation. The conjugation was abundant in the liver and lungs of Ufm1-transgenic mice, fractionated into membrane fraction, and impaired in Uba5 knock-out cells. Intriguingly, immunological analysis revealed localizations of Ufl1 and C20orf116 mainly to the endoplasmic reticulum. Our results provide novel insights into the Ufm1 system involved in cellular regulation of multicellular organisms.

Biological roles for UFM1, a ubiquitin-like protein, are largely unknown, and therefore we screened for targets of ufmylation. Here we show that ufmylation of the nuclear receptor coactivator ASC1 is a key step for ERα transactivation in response to 17β-estradiol (E2). In the absence of E2, the UFM1-specific protease UfSP2 was bound to ASC1, which maintains ASC1 in a nonufmylated state. In the presence of E2, ERα bound ASC1 and displaced UfSP2, leading to ASC1 ufmylation. Polyufmylation of ASC1 enhanced association of p300, SRC1, and ASC1 at promoters of ERα target genes. ASC1 overexpression or UfSP2 knockdown promoted ERα-mediated tumor formation in vivo, which could be abrogated by treatment with the anti-breast cancer drug tamoxifen. In contrast, expression of ufmylation-deficient ASC1 mutant or knockdown of the UFM1-activating E1 enzyme UBA5 prevented tumor growth. These findings establish a role for ASC1 ufmylation in breast cancer development by promoting ERα transactivation.

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.

Heterodimers of BMAL1 and CLOCK drive rhythmic expression of clock-controlled genes, thereby generating circadian physiology and behavior. Posttranslational modifications of BMAL1 play a key role in modulating the transcriptional activity of the CLOCK/BMAL1 complex during the circadian cycle. Recently, we demonstrated that circadian activation of the heterodimeric transcription factor is accompanied by ubiquitindependent proteolysis of BMAL1. Here we show that modification by SUMO localizes BMAL1 exclusively to the promyelocytic leukemia nuclear body (NB) and simultaneously promotes its transactivation and ubiquitindependent degradation. Under physiological conditions, BMAL1 was predominantly conjugated to poly-SUMO2/3 rather than SUMO1, and the level of these conjugates underwent rhythmic variation, peaking at times of maximum E-box-mediated circadian transcription. Interestingly, mutation of the sumoylation site (Lys 259 ) of BMAL1 markedly inhibited both its ubiquitination and its proteasome-mediated proteolysis, and these effects were reversed by covalent attachment of SUMO3 to the C terminus of the mutant BMAL1. Consistent with this, SUSP1, a SUMO protease highly specific for SUMO2/3, abolished ubiquitination, as well as sumoylation of BMAL1, while the ubiquitin protease UBP41 blocked BMAL1 ubiquitination but induced accumulation of polysumoylated BMAL1 and its localization to the NB. Furthermore, inhibition of proteasome with MG132 elicited robust nuclear accumulation of SUMO2/3-and ubiquitin-modified BMAL1 that was restricted to the transcriptionally active stage of the circadian cycle. These results indicate that dual modification of BMAL1 by SUMO2/3 and ubiquitin is essential for circadian activation and degradation of the CLOCK/BMAL1 complex.Circadian rhythms are fundamental mechanisms that optimize the physiology and behavior of most organisms. Extensive genetic and molecular studies have shown that these biological rhythms are driven by molecular clockwork composed of autoregulatory transcription-translation feedback loops involving several clock gene products (27,36). In mammals, the heterodimeric transcription factor CLOCK/ BMAL1 activates transcription of its own negative regulators, including cryptochromes (CRYs) and periods (PERs), and the products of the newly synthesized transcripts form complexes that block their own transcription by binding to the heterodimeric transcription activator. Of the negative regulators, the CRYs play a central role in this feedback inhibition by interacting directly with the C terminus of BMAL1 (15,26,29).There is, however, some evidence that this canonical feedback mechanism is insufficient to account for the robust oscillation of the circadian clock. During the circadian cycle, the mRNA profiles of the key negative regulators, the CRYs, exhibit weak oscillations compared to those of other clock genes controlled by the CLOCK/BMAL1 complex (14,31,35). Moreover, the abundance of BMAL1 and CLOCK proteins in the nucleus reaches a minimum at the time that expression of...

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