The Ufm1 conjugation system is an ubiquitin-like modification system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. The biological importance of this system is highlighted by its essential role in embryogenesis and erythroid development, but the underlying mechanism is poorly understood. UFBP1 (Ufm1 binding protein 1, also known as DDRGK1, Dashurin and C20orf116) is a putative Ufm1 target, yet its exact physiological function and impact of its ufmylation remain largely undefined. In this study, we report that UFBP1 is indispensable for embryonic development and hematopoiesis. While germ-line deletion of UFBP1 caused defective erythroid development and embryonic lethality, somatic ablation of UFBP1 impaired adult hematopoiesis, resulting in pancytopenia and animal death. At the cellular level, UFBP1 deficiency led to elevated ER (endoplasmic reticulum) stress and activation of unfolded protein response (UPR), and consequently cell death of hematopoietic stem/progenitor cells. In addition, loss of UFBP1 suppressed expression of erythroid transcription factors GATA-1 and KLF1 and blocked erythroid differentiation from CFU-Es (colony forming unit-erythroid) to proerythroblasts. Interestingly, depletion of Uba5, a Ufm1 E1 enzyme, also caused elevation of ER stress and under-expression of erythroid transcription factors in erythroleukemia K562 cells. By contrast, knockdown of ASC1, a newly identified Ufm1 target that functions as a transcriptional co-activator of hormone receptors, led to down-regulation of erythroid transcription factors, but did not elevate basal ER stress. Furthermore, we found that ASC1 was associated with the promoters of GATA-1 and Klf1 in a UFBP1-dependent manner. Taken together, our findings suggest that UFBP1, along with ASC1 and other ufmylation components, play pleiotropic roles in regulation of hematopoietic cell survival and differentiation via modulating ER homeostasis and erythroid lineage-specific gene expression. Modulating the activity of this novel ubiquitin-like system may represent a novel approach to treat blood-related diseases such as anemia.

The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis. Cell Death and Differentiation (2015) 22, 1922-1934 doi:10.1038/cdd.2015 .51; published online 8 May 2015The Ufm1 (Ubiquitin-fold modifier 1) conjugation system is a novel ubiquitin-like (Ubl) modification system that shares biochemical features with other Ubl systems.1 Ufm1 modifies its target proteins through a biochemical pathway catalyzed by specific E1 (Uba5), E2 (Ufc1) and E3 enzyme(s) even though the identities of E3 ligases remain mostly elusive. Genetic study from Uba5 knockout (KO) mice has shown that Uba5 is indispensable for embryonic erythropoiesis, highlighting the pivotal role of this novel Ubl system in animal development. 2Yet its role in adult erythropoiesis and other developmental processes is largely unexplored and the underlying molecular mechanism remains poorly understood.Regulator of C53 and DDRGK1 (also known as KIAA0776, Ufl1, NLBP and Maxer, referred to as RCAD hereafter) has recently been identified by independent studies as an important regulator of several signaling pathways, including protein ufmylation, NF-κB signaling and unfolded protein response (UPR).3-9 Endogenous RCAD forms a complex with two proteins: C53 (also known as LZAP and Cdk5rap3) 5,6,10 and DDRGK1 (also designated as C20orf116, Dashurin and UFBP1), 3,6,7,11 and regulates the stability of its binding partners. 5,6 Intriguingly, Tatsumi et al. 3 found that Ufl1 (same as RCAD) promoted ufmylation of DDRGK1, suggesting that RCAD may function as an E3 ligase for ufmylation of DDRGK1. In line with its role in ufmylation, knockdown of endogenous RCAD resulted in attenuated ufmylation of endogen...

Background:Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood.Methods:In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder.Results:We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively. We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice. The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7.Conclusions:Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice.