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.

The histone deacetylase inhibitors (HDACIs) butyrate and trichostatin A activate ␥-globin expression via a p38 mitogenactivating protein kinase (MAPK)-dependent mechanism. We hypothesized that downstream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNAprotein interactions occurred in the CRE in the G ␥-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced G ␥-promoter IntroductionThe growth factor erythropoietin (Epo) exerts its effects on commitment, proliferation, and differentiation of erythroid progenitors and globin chain synthesis through Janus kinase 2/Stat5 signaling and crosstalk with mitogen-activated protein kinase (MAPK) pathways. 1-3 p38 MAPK signaling is required for Epo mRNA stability and hemoglobin synthesis. 4,5 The reversible inhibition of p38 MAPK using SB203580 blocked Epo-dependent accumulation of mouse globin chains, 6 and studies in p38␣ Ϫ/Ϫ knockout mice showed a failure of definitive ␤ maj -globin gene expression. These studies confirm an Epo-p38 MAPK-dependent mechanism for hemoglobin synthesis. 7 The HDACI sodium butyrate (NaB) induces differentiation in erythroleukemia cells via Stat5 8,9 and p38 MAPK signaling. 10,11 Butyrate is a clinically useful fetal hemoglobin (HbF) inducer which has been used to treat individuals with sickle cell disease 12 and thalassemia 13 ; however, the molecular mechanism for NaB-mediated HbF induction is poorly understood. Recent data from Weinberg et al 14 showed that HbF induction by arginine butyrate is due in part to posttranslational mechanisms and increased ␥-globin mRNAtranslation.Several HDACIs, including trichostatin A (TSA), 10, and scriptaid,15,16 induce ␥-globin expression via p38 MAPK signaling. These studies suggest that different pharmacologic agents converge on the p38 MAPK pathway to activate ␥-globin expression. Four major MAPK pathways have been characterized: ERK1/2, ERK5/BMK1, cJun amino-terminal signal kinases (JNK), and p38. [17][18][19][20] Studies using erythroid progenitors, 21,22 knockout mice, 7 and K562 stable lines 10 suggest that p38␣ is the primary mediator of globin gene regulation.The downstream effector molecules of p38 MAPK signaling include MAPK-activated protein kinases 1 and 2, 23,24 PRAK, 25 ATF-1-4, CREB1, CREB2, and CREM. 26,27 Commonly, p38 phosphorylates ATF-2 and CREB to augment gene transcription. We recently demonstrated a p38 MAPK-dependent mechanism for NaB and TSA-induced ␥-globin expression. 10 Mechanistically, both agents bind a central zinc atom in HDAC to produce hyperacetylation of histone H3 (H3) and H4 28,29 to activate...

Current chemotherapeutic and butyrate therapeutics that induce fetal hemoglobin expression generally also suppress erythropoiesis, limiting the production of cells containing fetal hemoglobin (F cells). Recently, selected short-chain fatty acid derivatives (SCFADs) were identified that induce endogenous ␥-globin expression in K562 cells and human burst-forming units-erythroid and that increase proliferation of human erythroid progenitors and a multilineage interleukin-3-dependent hematopoietic cell line. In this report, ␥-globin inducibility by these SCFADs was further demonstrated in mice transgenic for the locus control region and the entire ␤-globin gene locus in a yeast artificial chromosome and in 2 globin promoter-reporter assays. Conditioned media experiments strongly suggest that their proliferative activity is a direct effect of the test compounds. Investigation of potential mechanisms of action of these SCFADs demonstrates that these compounds induce prolonged expression of the growth-promoting genes c-myb and c-myc. Both butyrate and specific growth-stimulatory SCFADs induced prolonged signal transducer and activator of transcription (STAT)-5 phosphorylation and activation, and c-cis expression, persisting for more than 120 minutes, whereas with IL-3 alone phosphorylation disappeared within minutes. In contrast to butyrate treatment, the growth-stimulating SCFADs did not result in bulk histone H4 hyperacetylation or induction of p21 Waf/Cip , which mediates the suppression of cellular growth by butyrate. These findings suggest that the absence of bulk histone hyperacetylation and p21 induction, but prolonged induction of cis, myb, myc, and IntroductionButyrate analogs have been of interest as potential therapeutics for the ␤-globin disorders following the demonstration that butyrate could transcriptionally activate developmentally silenced fetal and embryonic globin genes in many experimental conditions. [1][2][3][4][5][6][7][8][9][10][11] In clinical trials, arginine butyrate stimulated hemoglobin F (HbF) synthesis to levels above 20% and induced a nearly 2-fold increase in the amount of HbF/cell and in proportions of red blood cells expressing HbF (F reticulocytes) in patients with sickle cell anemia. [10][11][12] With constant use, however, the activity of the drug in maintaining substantial increases in HbF-containing erythrocytes gradually decreased. 13 We hypothesized that this tachyphylaxis was due to the coincident cellular growth-inhibitory properties of butyrate and developed intermittent or "pulse" regimens (4 days of use per month) to surmount this problem. 12 Although this regimen proved effective, it also imposed severe limitations on the amount of drug that can be delivered to the patient. An orally bioavailable transcriptional inducer of ␥-globin that does not have cellular growth-inhibitory properties and could be administered more frequently to stimulate a further increase in the proportion of HbF-containing cells (F cells) would be therapeutically advantageous for ␤-globin disord...

Orally bioactive compounds that induce ␥ globin gene expression at tolerable doses are needed for optimal treatment of the ␤-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce ␥ globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce ␥ globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which ␥ globin can be induced. Selected shortchain fatty acid derivatives (SCFADs) were recently found to induce ␥ globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human ␤ globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, ␥ globin mRNA increased 2-fold, reticulocytes increased 3-to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2-to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate ␥ globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the ␤ hemoglobinopathies and useful for the oral treatment of other anemias. (Blood. 2002;100:4640-4648)

Moreover, digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus, and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9, two critical components for Tat transactivation function on HIV-1 long terminal repeat promoter. Taken together, these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction, and suggest that Tip110 is an important cellular factor for HIV-1 gene expression and viral replication.

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