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 solitary long terminal repeats (LTRs) of ERV-9 endogenous retrovirus contain the U3, R, and U5 regions but no internal viral genes. They are middle repetitive DNAs present at 2,000 to 4,000 copies in primate genomes. Sequence analyses of the 5 boundary area of the erythroid ␤-globin locus control region (␤-LCR) and the intron of the embryonic axin gene show that a solitary ERV-9 LTR has been stably integrated in the respective loci for at least 15 million years in the higher primates from orangutan to human. The solitary long terminal repeats (LTRs) of human endogenous retroviruses comprise approximately 5% of the human genome and belong to the category of middle repetitive DNAs characterized as retrotransposons (14,19,24,35). These solitary LTRs contain the U3 enhancer and promoter region, the transcribed R region whose 5Ј end marks the initiation site of retroviral RNA synthesis, and the U5 region (27) but no internal gag, pol, and env genes. During primate evolution, the LTRs were apparently self-replicated and inserted into various host chromosomal sites. The functional roles in the host genomes of these transposed, repetitive DNAs are not clear. The LTR retrotransposons have been suggested to be selfish DNAs that do not serve relevant host functions (8). However, recent findings indicate that the solo LTRs can provide enhancers and promoters for cis-linked genes and regulate host gene transcription (9,10,22,25,26,29).The human genome contains approximately 50 copies of the ERV-9 endogenous retrovirus and an additional 3,000 to 4,000 copies of solitary ERV-9 LTRs (15,17,19,35,36). Compared with the LTRs of other families of endogenous retroviruses, the ERV-9 LTRs exhibit an unusual sequence feature: the U3 regions contain from 5 to 17 tandem repeats of 37 to 41 bases (17, 18) with recurrent GATA (23), CCAAT (28), and CCACC (21) motifs potentially capable of binding to cognate transcription factors expressed in embryonic and hematopoietic cells.This suggests that the ERV-9 enhancer and promoter could be active in those cells.To gain further insight into the stability and functional significance of the ERV-9 LTRs, here we have mapped the erythroid ␤-globin and embryonic axin gene loci in primates by using human primers in PCR. We found a solitary ERV-9 LTR that is conserved in identical locations in the 5Ј boundary area of the ␤-globin gene locus and in the axin gene in the higher primates orangutan, gorilla, chimpanzee, and human, whose ancestors diverged over an evolutionary period of 15 million years (12). In the lower primates gibbon and monkey, whose ancestors diverged from the human ancestor 18 and 25 million years ago, respectively (12), the globin and axin LTRs are absent in the respective gene loci. However, other ERV-9 LTRs are detectable in the monkey genome. These results indicate that copies of the ERV-9 LTRs present in the lower primates were inserted into the globin and axin gene loci in the common ancestor of the higher primates 15 to 18 millions years ago and have remained stably integrate...

Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) compose >40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions is not known. Here we show that an LTR retrotransposon of ERV-9 human endogenous retrovirus located 40-70 kb upstream of the human fetal γ-and adult β-globin genes serves a long-range, host function. The ERV-9 LTR contains multiple CCAAT and GATA motifs and competitively recruits a high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. The LTR complex transcribes intergenic RNAs unidirectionally through the intervening DNA to loop with and modulate transcription factor occupancies at the far downstream globin promoters, thereby modulating globin gene switching by a competitive mechanism.

In the human ε−globin gene locus, the HS2 enhancer in the Locus Control Region regulates transcription of the embryonic ε-globin gene located over 10 kb away. The mechanism of long-range HS2 enhancer function was not fully established. Here we show that the HS2 enhancer complex containing the enhancer DNA together with RNA polymerase II (pol II) and TBP tracks along the intervening DNA, synthesizing short, polyadenylated, intergenic RNAs to ultimately loop with the ε-globin promoter. Guided by this facilitated tracking and transcription mechanism, the HS2 enhancer delivers pol II and TBP to the cis-linked globin promoter to activate mRNA synthesis from the target gene. An insulator inserted in the intervening DNA between the enhancer and the promoter traps the enhancer DNA and the associated pol II and TBP at the insulator site, blocking mid-stream the facilitated tracking and transcription mechanism of the enhancer complex, thereby blocking long-range enhancer function.

The HS2 enhancer in the ␤-globin locus control region regulates transcription of the globin genes 10 -50 kb away. How the HS2 enhancer acts over this distance is not clearly understood. Earlier studies show that in erythroid cells the HS2 enhancer initiates synthesis of intergenic RNAs from sites within and downstream of the enhancer, and the enhancer-initiated RNAs are transcribed through the intervening DNA into the cis-linked promoter and gene. To investigate the functional significance of the enhancer-initiated transcription, here we inserted the lac operator sequence in the intervening DNA between the HS2 enhancer and the ⑀-globin promoter in reporter plasmids and integrated the plasmids into erythroid K562 cells expressing the lac repressor protein. We found that the interposed lac operator/repressor complex blocked the elongation of enhancerinitiated transcription through the intervening DNA and drastically reduced HS2 enhancer function as measured by the level of mRNA synthesized from the ⑀-globin promoter. The results indicate that the tracking and transcription mechanism of the HS2 enhancer-assembled transcriptional machinery from the enhancer through the intervening DNA into the cis-linked promoter can mediate enhancer-promoter interaction over a long distance. The locus control region (LCR)1 of the human ␤-globin gene domain, defined by four erythroid specific DNase I hypersensitive sites (HS1, -2, -3, and -4) and a ubiquitous HS5 site (1-3), regulates transcription of the far downstream embryonic ⑀-globin, fetal G␥-globin and A␥-globin, and the adult ␦-globin and ␤-globin genes during erythroid cell differentiation. It is not fully understood whether the LCR acts over the long distance by a looping mechanism in which the LCR complex (the LCR DNA and its associated transcription factors) loops over the intervening DNA to directly interact with the globin promoters or by a tracking mechanism in which the LCR complex or its protein components track along the intervening DNA to reach and activate the downstream promoters (4 -7).In the LCR, the HS2 site located 11 and 55 kb 5Ј, respectively, of the ⑀-and ␤-globin genes possesses strong enhancer activity (8) and regulates transcription of the ␤-like globin genes over a long distance (9, 10). In the endogenous genome of erythroid cells and in recombinant constructs transfected into erythroid cells, the HS2 enhancer recruits erythroid and general transcription factors (11-13) in the assembly of a pol II transcription complex that synthesizes intergenic RNAs from sites within the enhancer in the direction of the downstream gene (14 -17). In plasmids integrated into erythroid cells, this genetropic HS2 enhancer transcription, like HS2 enhancer function, is detected regardless of the orientation, position, and distance of the enhancer relative to the cis-linked promoter and gene (15). Moreover, splitting the HS2 enhancer at the enhancer core, thereby preventing assembly of the intact enhancer complex, diminishes both enhancer transcription at the enhancer site...

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Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms, mouse and human cell infection models and human biopsy samples were employed. We show that the ‘keystone’ pathogen Porphyromonas gingivalis, disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + Tregs through an anti-apoptotic pathway. This pathway, involving pAKT1, pFOXO1, FOXP3, IDO1 and BIM, is activated in humans with CP and in mice orally infected with Mfa1 expressing P. gingivalis strains. Mechanistically, activation of this pathway, demonstrating FOXP3 as a direct FOXO1-target gene, was demonstrated by ChIP-assay in human CP gingiva. Expression of oncogenic but not tumor suppressor markers is consistent with tumor cell proliferation demonstrated in OSCC-P. gingivalis cocultures. Importantly, FimA + P. gingivalis strain MFI invades OSCCs, inducing inflammatory/angiogenic/oncogenic proteins stimulating OSCCs proliferation through CXCR4. Inhibition of CXCR4 abolished Pg-MFI-induced OSCCs proliferation and reduced expression of oncogenic proteins SDF-1/CXCR4, plus pAKT1-pFOXO1. Conclusively, P. gingivalis, through Mfa1 and FimA fimbriae, promotes immunosuppression and oncogenic cell proliferation, respectively, through a two-hit receptor-ligand process involving DC-SIGN+hi/CXCR4+hi, activating a pAKT+hipFOXO1+hiBIM−lowFOXP3+hi and IDO+hi- driven pathway, likely to impact the prognosis of oral cancers in patients with periodontitis.

The solitary LTRs of ERV-9 human endogenous retrovirus are middle repetitive DNAs associated with 3,000 -4,000 human gene loci including the ␤-globin gene locus where the ERV-9 LTR is juxtaposed to the locus control region (␤-LCR) far upstream of the globin genes. The ERV-9 LTRs are conserved during primate evolution, but their function in the primate genomes is unknown. Here, we show that in transgenic zebrafish harboring the ␤-globin ERV-9 LTR coupled to the GFP gene, the LTR enhancer was active and initiated synthesis of GFP mRNA in oocytes but not in spermatozoa, and GFP expression in the embryos was maternally inherited. The LTR enhancer was active also in stem͞ progenitor cell regions of adult tissues of transgenic zebrafish. In human tissues, ERV-9 LTR enhancer was active also in oocytes and stem͞progenitor cells but not in spermatozoa and a number of differentiated, adult somatic cells. Transcriptional analyses of the human ␤-globin gene locus showed that the ␤-globin ERV-9 LTR enhancer initiated RNA synthesis from the LTR in the direction of the downstream ␤ locus control region and globin genes in ovary and erythroid progenitor cells. The findings suggest that, during oogenesis, ERV-9 LTR enhancers in the human genome could activate the cis-linked gene loci to synthesize maternal mRNAs required for early embryogenesis. Alternatively, the ERV-9 LTR enhancers, in initiating RNA syntheses into the downstream genomic DNAs, could transcriptionally potentiate and preset chromatin structure of the cis-linked gene loci in oocytes and adult stem͞progenitor cells.