We have mapped the distribution of the major and minor DNase I-hypersensitive sites in the human "fi-like-globin" gene domain. The minor DNase I-hypersensitive sites map close to the 5' end of each of the 13-like-globin genes. Their presence is specifically associated with the transcription of the immediate downstream fi-like-globin genes.The major DNase I-hypersensitive sites map in what appear to be the 5' and 3' boundary areas of the human fi-like-globin gene domain, a region estimated to span at least 90 kilobases of DNA. These major sites are present in various erythroid cells, which express predominantly either the embryonic, the fetal, or the adult (3-like-globin genes, and seem to be involved in derming the active j8-like-globin gene domain in cells of erythroid lineage. The four major DNase I-hypersensitive sites in the 5' boundary area, when correlated with sequencing data, are shown to be located in DNA regions containing enhancer core-like sequences and alternating purine and pyrimidine bases.The human "/3-like-globin" genes (hemoglobin P-chain gene cluster) encode, respectively, one embryonic (e), two fetal (G y and Ay), and two adult (8 and /3) globin chains. These genes have been shown to reside within "50 kilobases (kb) of chromosomal DNA in the transcriptional order 5' e-y.GAy. EXPERIMENTAL PROCEDURES Cells were grown as described (7). Human bone marrow cells were collected from cancer patients with normal marrow who were to undergo chemotherapy and bone marrow reinfusion. Isolated by dextran column chromatography, -25% of the nucleated cells were erythroid.DNase I-digestion, gel electrophoresis, RNA isolation, blotting, and hybridization were carried out as described (7). RESULTS Globin Gene Transcription in K562,HEL, Adult Human Marrow, and HL60 Cells. Nuclear and cytoplasmic RNAs were isolated from cells, and individual globin gene transcription was detected by "dot-blot" hybridization with e-,

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.

We have identified an erythroid-specific enhancer element far upstream of the human "l,-like globin" genes, at 10.2-11.0 kilobases 5' ofthe embryonic e-globin gene, and thus at 53-54 kilobases 5' of the adult .8-globin gene. It is capable of enhancing the expression of a cis-linked test gene by up to 300-fold. This enhancer element is apparently developmental-stage-independent, as it is functional at the embryonic and the adult developmental stages in erythroid cells that are expressing the respective f-like globin genes. The enhancer and globin promoter sequences work in synergy and are capable of conferring on 'a cis-linked gene the high transcriptional efficiency (enhancer function), erythroid specificity (enhancer and promoter functions), and developmental-stage specificity (promoter Using DNase I-hypersensitivity mapping to identify the cis control elements situated both near and far from the human "p-like globin" gene cluster (5' e Gy-Ay-p 3'), we have found minor DNase I-hypersensitive sites located in the promoter regions near actively transcribed globin genes and major DNase I-hypersensitive sites located very far upstream and downstream ofthe ,-like globin gene domain (6, 7). The 5' and 3' major hypersensitive sites appear to be erythroid specific and developmentally stable; i.e., they remain hypersensitive throughout various stages of erythroid cell development. The more proximal minor hypersensitive sites near the 5' end of globin genes appear only at specific developmental stages. The above characteristics of the major and minor sites have led us (7), as well as others (8), to postulate that transcriptional activation of the human P-like globin genes may consist of at least two separate but synergistic activation steps mediated by distant cis regulatory sequences (major sites) and sequences much closer to the globin genes (minor sites).The functional characteristics of the far upstream regulatory sequences (i.e., tissue specificity and stimulation ofgene expression over long distances) are the properties of transcriptional enhancer sequences (9-11). We report here that a 0.8-kilobase (kb) DNA fragment directly underlying one of the far upstream major DNase I-hypersensitive sites, at -10.2 to -11.0 kb 5' of the embryonic e-globin gene and therefore between -53 and -54 kb 5' of the adult p-globin gene, possesses enhancer activity. When coupled to either the viral simian virus 40 (SV40) promoter or to the e-globin promoter, it stimulates the expression of a linked chloramphenicol acetyltransferase (CAT) gene (12) by up to 300-fold. Coupled to the globin promoter, this enhancer sequence shows strict erythroid specificity. Furthermore, the enhancer element appears to be functional in erythroid cells at embryonic and adult developmental stages, a finding consistent with the developmental-stage stability of its DNase I hypersensitivity and its proposed crucial role in globin gene activation throughout erythroid development. MATERIALS AND METHODSConstruction of the Recombinant CAT Plasmids. CAT pl...