No abstract

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.

No abstract

BackgroundIn vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date.MethodsIndividual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software.ResultsTo determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24–48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with invasion potential. We also demonstrate, using lung cancers, that the zebrafish model can evaluate the metastatic ability of cancer cells isolated from primary tumors.ConclusionsThe zebrafish model described here offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells. Using this model it is possible to critically evaluate whether genetic manipulation of signaling pathways affects metastasis and whether primary tumors contain metastatic cells.