Hydroxyurea differentially modulates activator and repressors of γ-globin gene in erythroblasts of responsive and non-responsive patients with sickle cell disease in correlation with Index of Hydroxyurea Responsiveness.

Hydroxyurea (HU), the first of two drugs approved by the US Food and Drug Administration for treating patients with sickle cell disease (SCD), produces anti-sickling effect by re-activating fetal γ-globin gene to enhance production of fetal hemoglobin. However, approximately 30% of the patients do not respond to HU therapy. The molecular basis of non-responsiveness to HU is not clearly understood. To address this question, we examined HU-induced changes in the RNA and protein levels of transcription factors NF-Y, GATA-1, -2, BCL11A, TR4, MYB and NF-E4 that assemble the γ-globin promoter complex and regulate transcription of γ-globin gene. In erythroblasts cultured from peripheral blood CD34+ cells of patients with SCD, we found that HU-induced changes in the protein but not the RNA levels of activator GATA-2 and repressors GATA-1, BCL11A and TR4 correlated with HU-induced changes in fetal hemoglobin (HbF) levels in the peripheral blood of HU high and low responders. However, HU did not significantly induce changes in the protein or RNA levels of activators NF-Y and NF-E4. Based on HU-induced changes in the protein levels of GATA-2, -1 and BCL11A, we calculated an Index of Hydroxyurea Responsiveness (IndexHU-3). Compared to the HU-induced fold changes in the individual transcription factor protein levels, the numerical values of IndexHU-3 statistically correlated best with the HU-induced peripheral blood HbF levels of the patients. Thus, IndexHU-3 can serve as an appropriate indicator for inherent HU responsiveness of patients with SCD.

Hypermethylated LTR retrotransposon exhibits enhancer activity.

LTR retrotransposons are repetitive DNA elements comprising ∼10% of the human genome. They are silenced by hypermethylation of cytosines in CpG dinucleotides and are considered parasitic DNA serving no useful function for the host genome. However, hypermethylated LTRs contain enhancer and promoter sequences and can promote tissue-specific transcription of cis-linked genes. To resolve the apparent paradox of hypermethylated LTRs possessing transcriptional activities, we studied the ERV-9 LTR retrotransposon located at the 5' border of the transcriptionally active ß-globin gene locus in human erythroid progenitor and erythroleukemia K562 cells. We found that the ERV-9 LTR, containing 65 CpGs in 1.7 kb DNA, was hypermethylated (with > 90% methylated CpGs). Hypermethylated LTR possessed transcriptional enhancer activity, since in vivo deletion of the LTR by CRISPR-cas9 suppressed transcription of the globin genes by > 50%. ChIP-qPCR and ChIP-seq studies showed that the hypermethylated LTR enhancer spanning recurrent CCAATCG and GATA motifs associated respectively with key transcription factors (TFs) NF-Y and GATA-1 and -2 at reduced levels, compared with the unmethylated LTR in transfected LTR-reporter gene plasmids. Electrophoretic mobility shift assays with methylated LTR enhancer probe showed that the methylated probe bound both NF-Y and GATA-1 and -2 with lower affinities than the unmethylated enhancer probe. Thus, hypermethylation drastically reduced, but did not totally abolish, the binding affinities of the enhancer motifs to the key TFs to assemble the LTR-pol II transcription complex that activated transcription of cis-linked genes at reduced efficiency.

Long non-coding RNAs transcribed by ERV-9 LTR retrotransposon act in cis to modulate long-range LTR enhancer function.

LTR retrotransposons are repetitive DNA elements comprising ∼10% of the human genome. However, LTR sequences are disproportionately present in human long, non-coding RNAs (lncRNAs). Whether and how the LTR lncRNAs serve biological functions are largely unknown. Here we show that in primary human erythroblasts, lncRNAs transcribed from the LTR retrotransposons of ERV-9 human endogenous retrovirus activated transcription of key erythroid genes and modulated ex vivo erythropoiesis. To dissect the functional mechanism of ERV-9 lncRNAs, we performed genome-wide RNA and ChIRP analyses before and after global knockdown or locus-specific deletion of ERV-9 lncRNAs in human erythroblasts carrying ∼4000 copies of the ERV-9 LTRs and in transgenic mouse erythroblasts carrying a single copy of the primate-specific ERV-9 LTR in the 100 kb human ß-globin gene locus. We found that ERV-9 lncRNAs acted in cis to stabilize assembly of the ERV-9 LTR enhancer complex and facilitate long-range LTR enhancer function in activating transcription of downstream, cis-linked globin genes. Our findings suggested that LTR lncRNAs transcribed from many of the 4000 copies of ERV-9 LTR retrotransposons acted by a similar cis mechanism to modulate LTR enhancer function in activating transcription of downstream genes critical to cellular processes including erythropoiesis.

UFBP1, a Key Component of the Ufm1 Conjugation System, Is Essential for Ufmylation-Mediated Regulation of Erythroid Development.

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.

NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

The human embryonic, fetal and adult ß-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

Long-range function of an intergenic retrotransposon.

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 gamma- and adult beta-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.

A facilitated tracking and transcription mechanism of long-range enhancer function.

In the human epsilon-globin gene locus, the HS2 enhancer in the Locus Control Region regulates transcription of the embryonic epsilon-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 epsilon-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 long terminal repeat (LTR) of ERV-9 human endogenous retrovirus binds to NF-Y in the assembly of an active LTR enhancer complex NF-Y/MZF1/GATA-2.

The solitary ERV-9 long terminal repeat (LTR) located upstream of the HS5 site in the human beta-globin locus control region exhibits prominent enhancer activity in embryonic and erythroid cells. The LTR enhancer contains 14 tandemly repeated subunits with recurrent CCAAT, GTGGGGA, and GATA motifs. Here we showed that in erythroid K562 cells these DNA motifs bound the following three transcription factors: ubiquitous NF-Y and hematopoietic MZF1 and GATA-2. These factors and their target DNA motifs exhibited a hierarchy of DNA/protein and protein/protein binding affinities: NF-Y/CCAAT > NF-Y/GATA-2 > NF-Y/MZF1 > MZF1/GTGGGGA; GATA-2/GATA. Through protein/protein interactions, NF-Y bound at the CCAAT motif recruited MZF1 and GATA-2, but not Sp1 and GATA-1, and stabilized their binding to the neighboring GTGGGGA and GATA sites to assemble a novel LTR enhancer complex, NF-Y/MZF1/GATA-2. In the LTR-HS5-epsilonp-GFP plasmid integrated into K562 cells, mutation of the CCAAT motif in the LTR enhancer to abolish NF-Y binding inactivated the enhancer, closed down the chromatin structure of the epsilon-globin promoter, and silenced transcription of the green fluorescent protein gene. The results indicated that NF-Y bound at the CCAAT motifs assembled a robust LTR enhancer complex, which could act over the intervening DNA to remodel the chromatin structure and to stimulate the transcription of the downstream gene locus.

The HS2 enhancer of the beta-globin locus control region initiates synthesis of non-coding, polyadenylated RNAs independent of a cis-linked globin promoter.

The HS2 enhancer in the beta-globin locus control region (LCR) regulates transcription of the globin genes 10-50 kb away. Earlier studies show that a transcription mechanism initiated by the HS2 enhancer through the intervening DNA in the direction of the cis-linked promoter and gene mediates long-range enhancer function. Here, we further analyzed the enhancer-initiated RNAs and their mode of transcription from the HS2 enhancer in the endogenous genome of erythroid K562 cells, in plasmids integrated into K562 cells and in purified DNA used as template in in vitro transcription reactions. We found that the HS2 enhancer was able to initiate transcription autonomously in the absence of a cis-linked globin promoter. The enhancer-initiated, intergenic RNAs were different from the mRNA synthesized at the promoter in several aspects. The enhancer RNAs were synthesized not from a defined site but from multiple sites both within and as far as 1 kb downstream of the enhancer. The enhancer RNAs did not appear to contain a normal cap structure at the 5' ends. They were polyadenylated at multiple sites within 3 kb downstream of their initiation sites and were therefore shorter than 3 kb in lengths. The enhancer RNAs remained in discrete spots within the nucleus and were not processed into mRNA or translated into proteins. These particular features of enhancer-initiated transcription indicate that the transcriptional complex assembled by the enhancer was different from the basal transcription complex assembled at the promoter. The results suggest that in synthesizing non-coding, intergenic RNAs, the enhancer-assembled transcription complex could track through the intervening DNA to reach the basal promoter complex and activate efficient mRNA synthesis from the promoter.

HS2 enhancer function is blocked by a transcriptional terminator inserted between the enhancer and the promoter.

The HS2 enhancer in the beta-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 epsilon-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 enhancer-initiated transcription through the intervening DNA and drastically reduced HS2 enhancer function as measured by the level of mRNA synthesized from the epsilon-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 LTR enhancer of ERV-9 human endogenous retrovirus is active in oocytes and progenitor cells in transgenic zebrafish and humans.

The solitary LTRs of ERV-9 human endogenous retrovirus are middle repetitive DNAs associated with 3,000-4,000 human gene loci including the beta-globin gene locus where the ERV-9 LTR is juxtaposed to the locus control region (beta-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 beta-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 stem/progenitor cell regions of adult tissues of transgenic zebrafish. In human tissues, ERV-9 LTR enhancer was active also in oocytes and stem/progenitor cells but not in spermatozoa and a number of differentiated, adult somatic cells. Transcriptional analyses of the human beta-globin gene locus showed that the beta-globin ERV-9 LTR enhancer initiated RNA synthesis from the LTR in the direction of the downstream beta 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 stem/progenitor cells.

The ERV-9 LTR enhancer is not blocked by the HS5 insulator and synthesizes through the HS5 site non-coding, long RNAs that regulate LTR enhancer function.

A solitary long terminal repeat (LTR) of ERV-9 human endogenous retrovirus is located upstream of the HS5 site in the human beta-globin locus control region and possesses unique enhancer activity in erythroid K562 cells. In cells transfected with plasmid LTR-HS5-epsilonp-GFP, the LTR enhancer activates the GFP reporter gene and is not blocked by the interposed HS5 site, which has been reported to have insulator function. The LTR enhancer initiates synthesis of long RNAs from the LTR promoter through the intervening HS5 site into the epsilon-globin promoter and the GFP gene. Synthesis of the sense, long LTR RNAs is correlated with high level synthesis of GFP mRNA from the epsilon-globin promoter. Mutations of the LTR promoter and/or the epsilon-globin promoter show that (i) the LTR enhancer can autonomously initiate synthesis of LTR RNAs independent of the promoters and (ii) the LTR RNAs are not processed into GFP mRNA or translated into GFP. However, reversing the orientation of the LTR in plasmid (LTR)rev-HS5-epsilonp-GFP, thus reversing the direction of synthesis of LTR RNAs in the antisense direction away from the epsilon-globin promoter and GFP gene drastically reduces the level of GFP mRNA and thus LTR enhancer function. The results suggest that the LTR-assembled transcription machinery in synthesizing non-coding, LTR RNAs can reach the downstream epsilon-globin promoter to activate transcription of the GFP gene.

The solitary long terminal repeats of ERV-9 endogenous retrovirus are conserved during primate evolution and possess enhancer activities in embryonic and hematopoietic cells.

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 beta-globin locus control region (beta-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. Functional studies utilizing the green fluorescent protein (GFP) gene as the reporter in transfection experiments show that the U3 region of the LTRs possesses strong enhancer activity in embryonic cells of widely different tissue origins and in adult cells of blood lineages. In both the genomic LTRs of embryonic placental cells and erythroid K562 cells and transfected LTRs of recombinant GFP plasmids in K562 cells, the U3 enhancer activates synthesis of RNAs that are initiated from a specific site 25 bases downstream of the AATAAA (TATA) motif in the U3 promoter. A second AATAAA motif in the R region does not serve as the TATA box or as the polyadenylation signal. The LTR-initiated RNAs extend through the R and U5 regions into the downstream genomic DNA. The results suggest that the ERV-9 LTR-initiated transcription process may modulate transcription of the associated gene loci in embryonic and hematopoietic cells.