"The 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".

Xiuping Yu ‡, Xingguo Zhu ‡ , Wenhu Pi, Jianhua Ling, Lan Ko, Yoshihiko Takeda and Dorothy Tuan

From the Department of Biochemistry and Molecular Biology and Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia, Augusta GA 30912

Address Correspondence to: Dorothy Tuan, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912, Tel: 706-721-0272; Fax: 706-721-6608;
email:   dtuanlo@mail.mcg.edu

‡ These authors contributed equally to the work.

    The solitary ERV-9 LTR located upstream of the HS5 site in the human b-globin Locus Control Region exhibits prominent enhancer activity in embryonic and erythroid cells. The LTR enhancer contains fourteen tandemly repeated subunits with recurrent CCAAT, GTGGGGA and GATA motifs. Here, we show that in erythroid K562 cells these DNA motifs bound 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-ep-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 e-globin promoter and silenced transcription of the GFP gene. The results indicate 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 stimulate the transcription of the downstream gene locus.

INTRODUCTION:

The human genome contains a large number of retrotransposons, the L1s, the Alus and the LTRs of human endogenous retroviruses, which comprise over 40% of the human chromosome DNA (1, 2). The retrotransposons have been suggested to be selfish DNAs that do not serve relevant host functions (3). However, the L1s and Alus contain respectively polymerase II and III promoters and the LTRs contain both enhancers and promoters (1) and can thus serve relevant host function by modulating transcription
of the cis-linked host genes (4-9).

In the human genome, there are approximately 50 copies of the ERV-9 endogenous retrovirus and an additional 3000-4000 copies of solitary ERV-9 LTRs (10, 11). The solitary LTRs contain the U3 enhancer and promoter region, the transcribed R region, whose 5’ end marks the initiation site of retroviral RNA
synthesis, and the U5 region (12), but no internal gag, pol and env genes. During primate evolution, the LTRs were self-replicated and stably integrated into various host chromosomal sites (13-15). The ERV-9 LTRs are associated with hematopoietic genes such as the b-like globin genes (15, 16), the selectin genes (GenBank AL022146) and the major histocompatibility genes (14). They are also associated with
embryonic genes such as the embryonic e-globin gene in the b-like globin gene cluster (15) and the
axin gene (16), which is expressed during embryogenesis (17) as well as in adult hematopoietic stem cells (18).

Compared with the LTRs of other families of endogenous retroviruses, the ERV-9 LTRs exhibit an unusual sequence feature: the U3 enhancer regions contain from 5 to 17 tandem repeats (15, 16, 19) comprising four closely related subunits 1, 2, 3 and 4 arranged in the order of (1-2-3-4)_n. The four subunits, each of 40 DNA
bases, contain combinations of three recurrent DNA sequences (Fig. 1A):

i ) ACACCAATCAGCA: The underlined CCAAT motif has been reported to bind the C/EBP family of hematopoietic transcription factors (20) and the ubiquitous transcription factor NF-Y. NF-Y is a trimeric complex composed of NF-YA, -YB and -YC, in which the YB and YC subunits bind to DNA through their histone fold domains and the YA subunit, recruited by this dimeric complex, binds through its specific DNA binding domain to the CCAAT motif (21, 22). Expression of NF-YA appears to be developmentally regulated as its level declines from proliferating progenitor cells to terminally differentiated cells during cellular differentiation (23, 24).

ii) TCTGTATCTAGCT: The underlined TATC (GATA) motif has been reported to bind the GATA family of transcription factors including GATA-1 and –2, which are expressed in hematopoietic progenitor cells and
play critical roles in hematopoiesis and globin gene transcription during erythropoiesis (25, 26).

iii) GGTGGGGACGTGGAGA: The underlined G-rich motifs bear high sequence identity to the DNA binding site of the myeloid transcription factor MZF1, a protein with 13 zinc fingers in which fingers 1-9 and 10-13 bind respectively to the 5’ and 3’ half sites AGTGGGGA and GAGGGGGAA in the target DNA (27-29). MZF1 is expressed in myeloid progenitor cells and has been reported to regulate myelopoiesis (30).

The human b-globin gene locus spans the embryonic e-, the fetal Gg- and Ag-, and the adult d- and b-globin genes arranged in the transcriptional order of 5’ e-Gg-Ag-d-b 3’ in the chromosome. The b-globin Locus Control Region (b-LCR), located 10-60 kb upstream of the globin genes and defined by DNase I
hypersensitive sites HS1, 2, 3, 4 and 5, plays a pivotal role in regulating transcriptional activation of the far downstream b-like globin genes (31-33). In an effort to define the 5’ border of the b-LCR, we previously cloned and sequenced the DNA further upstream of the HS5 site and discovered a solitary ERV-9 LTR located 1.5 kb upstream of the HS5 site in the 5’ boundary area of the human b-LCR (15). Subsequent studies showed that this 5’HS5 ERV-9 LTR possesses prominent enhancer activity in embryonic and
erythroid cell lines (16) and in oocytes and progenitor cells including the erythroid progenitor cells but not in differentiated somatic tissues in transgenic zebrafish and humans (34). Unlike the HS2 and HS3 enhancers of the b-LCR, whose enhancer activities are blocked by the HS5 site with reported insulator properties when HS5 is interposed between the enhancer and the cis-linked promoter (35, 36), the ERV-9 LTR enhancer is not blocked by the interposed HS5 site (37).

To further characterize the 5’ HS5 ERV-9 LTR to gain insight into its functional significance, here we identified the transcription factors that bound to and activated the LTR enhancer. Wildtype and mutant LTR-GFP plasmids were transfected into leukemic K562 cells that exhibit properties of erythroid progenitor cells and express the embryonic globin gene program (38) and the embryonic teratocarcinoma
PA-1 cells derived from primordial oocytes (39). The results showed that base substitutions in the CCAAT, GTGGGGA and GATA motifs reduced LTR enhancer activity, with the CCAAT mutation generating the largest reduction of up to 98%. Electrophoretic mobility shift assays (EMSAs) showed that these core motifs flanked by respective, consensus bases bound three transcription factors: ubiquitous NF-Y and
hematopoietic MZF1 and GATA-2. Furthermore, competitive EMSAs and western blots of proteins that bound to and were eluted from magnetic beads conjugated to the wildtype or mutant LTR enhancer probes showed that through protein/protein interactions, NF-Y bound at the CCAAT motif recruited MZF1 and GATA-2, but not Sp1 and GATA-1, and stabilize their binding to the neighboring GTGGGGA and GATA sites to assemble a novel LTR enhancer complex, NF-Y/MZF /GATA-2 . Chromatin immunoprecipitation (ChIP) assays confirmed that these transcription factors and the ERV-9 LTR were associated with one another in vivo. In the LTR-HS5-ep-GFP plasmid integrated into the genome of K562 cells, abolishing NF-Y binding to the LTR enhancer by mutating the CCAAT motif in the LTR enhancer closed down the chromatin structure of the e-globin promoter and silenced the transcription of the GFP gene. The results indicate that the robust LTR enhancer complex assembled by NF-Y could act over the intervening DNA to
remodel the chromatin structure and stimulate the transcription of a cis-linked gene locus in embryonic and erythroid progenitor cells.
 

Figure 1: Map of the ERV-9 LTR and construction and transfection of the wt and mutant (4-1)-P-GFP plasmids.

A. Map of the ERV-9 LTR in the human b-globin gene locus. Hatched box: the ERV-9 LTR. Solid boxes marked with vertical arrows: Hypersensitive sites HS5, 4. 3. 2 and 1 defining the b-LCR. Enlarged map of the LTR: U3, the region encompassing the LTR enhancer (E), containing 14 tendemly repeated subunits and the LTR promoter (P). Angled arrow at the 5’ border of R: initiation site of LTR transcription in the direction of the globin genes. Angled arrow above the globin genes: direction of transcription of the globin genes. Lower panel: DNA sequences of the LTR enhancer subunits 1-4 and of the LTR promoter. Underlined bases are potential binding sites for transcription factors; underlined AATAAA: the TATA box in the LTR promoter. Angled arrow: transcriptional initiation site marking the 5’ border of the R region.

B. Maps of the wt E-P-GFP and (4-1)-P-GFP plasmids and the mutant MI-IV-P-GFP plasmids.
4* and 1* : enhancer subunit 1 or 4 containing the respective mutations as indicated.

C. Enhancer activities of plasmids containing the wt and mutant (4-1) enhancers and the enhancerless P-GFP plasmid (GFP) determined by FACS analyses of the GFP levels expressed by the plasmids transiently transfected into K562 and PA-1 cells. The relative GFP level (Rel. GFP) was the ratio of the GFP level of the test plasmids to that of the (4-1)-P-GFP reference plasmid, which was set at 100 in both K562 and PA-1 cells, although the plasmid was 1.3x more active in PA-1 than in K562 cells. The values shown are means and standard deviations obtained from 3-4 independent determinations.

The solitary ERV-9 LTR located upstream of the b-globin LCR contains in the enhancer region fourteen tandem repeats of four closely related subunits 1, 2, 3 and 4 arranged in the order (1-2-3-4)₃-4-1. In this study, we showed that these enhancer subunits contain three recurrent sequences ACACCAATCAGCA,
GGTGGGGACGTGGAGA and AGCTAGATACAGAGT with the underlined core motifs and consensus flanking bases that bound both in vitro and in vivo the ubiquitous transcription factor NF-Y and the hematopoietic transcription factors MZF1 and GATA-2. As all three protein factors are expressed preferentially in progenitor cells (23, 25, 30), their binding to the LTR enhancer may provide a molecular basis for the earlier observation that the ERV-9 LTR enhancer is active in progenitor cells but not in
differentiated somatic cells in transgenic zebrafish and humans (34). Consistent with the finding that
the ERV-9 LTR enhancer is active also in the primordial oocytes of transgenic zebrafish and humans (34), the ERV-9 LTR enhancer bound to and was activated by NF-Y, MZF1 and a GATA-x factor in ovarian PA-1 cells derived from primordial oocytes (39).

In assembling the LTR enhancer complex, the protein/DNA and protein/protein interactions exhibited the following hierarchy of binding affinities: NF-Y/CCAAT > NF-Y/GATA-2; NF-Y/MZF1> MZF1/GATA-2 > MZF1/GTGGGGA; GATA-2/GATA. The strong affinity between NF-Y and the CCAAT motif, at the top of the binding hierarchy, is consistent with earlier reports that NF-YA in the trimeric complex through a specific DNA binding domain binds to the CCAAT motif with a kd of 10 ^-10 ~10 ^-11 , which is among the
highest for transcription factors (21, 22). In contrast, GATA-2/GATA and MZF1/GTGGGGA binding strengths were at the bottom of the binding hierarchy; hence, GATA-2 and MZF1 bound weakly to their cognate sites in the LTR enhancer and required NF-Y bound at the neighboring CCAAT site to recruit and stabilize their binding to the LTR enhancer. Our result is consistent with the finding that NF-Y is able to
recruit and stabilize binding of the sterol-regulatory-element-binding-protein (SREBP) to the neighboring sterol responsive element, which otherwise could not bind detectable amount of SREBP (52). Together, these findings indicate that NF-Y bound at the CCAAT motif could serve as a scaffold to recruit other transcription factors in assembling the LTR enhancer complex.

In erythroid K562 cells, MZF1 and GATA-2 appeared to be the preferred protein partners for NF-Y. Our results showed that NF-Y preferentially interacted with GATA-2 but not with GATA-1, even though GATA-2 is less abundant than GATA-1 in K562 cells (53). On the other hand, NF-Y and GATA-1 bound at the respective GATA and CCAAT sites in the promoter of the erythroid Gfi-1B gene have been reported to cooperatively activate the Gfi-1B promoter in K562 cells (54). However, this GATA site bound very weakly to GATA-1 but strongly to an un-identified protein (54). Since direct protein/protein interaction between NF-Y and GATA-1 was not shown in this study, the possibility cannot be excluded that the observed
cooperativity between NF-Y and GATA-1 was mediated indirectly through their interactions with an un-identified third protein and not by direct NF-Y/ GATA-1 interaction.

In K562 cells, NF-Y did not interact with Sp1. This finding appears to contradict earlier reports that NF-Y bound at the CCAAT motif cooperated with Sp1 bound at the neighboring Sp1 site to activate the resident promoters (22, 55-57). Those studies were, however, carried out with nuclear extracts of non-hematopoietic cells, which apparently did not express MZF1 and GATA-2. Indeed, in the EMSA carried out with the nuclear extract of Friend erythroid leukemia cells, NF-Y bound at the CCAAT motif in the ferritin heavy
chain promoter did not recruit Sp1 to the consensus Sp1 sites flanking the CCAAT motif (58). These findings indicate that in hematopoietic cells, NF-Y interacted preferentially with MZF1 and GATA-2 but not with Sp1. However, in non-hematopoietic cells that did not express MZF1 and GATA-2, NF-Y then interacted with the ubiquitously expressed Sp1.

The strong affinity between NF-Y and the CCAAT motif, at the top of the binding hierarchy, indicates that in the enhancer repeats of ERV-9 LTR, (1-2-3-4), the CCAAT motifs in subunits 1, 2 and 3 initially bound NF-Y, which then recruited MZF1 and GATA-2 to the neighboring GTGGGGA and GATA sites in subunits 2 and 4 to assemble a modular enhancer complex: [E1/NF-Y]•[E2/NF-Y/GATA-2]•[E3/NF-Y]•[E4/MZF1/GATA-2] in K562 cells. In PA-1 cells expressing no GATA-2, the modular enhancer complex appeared to be: [E1/NF-Y]•[E2/NF-Y]•[E3/NF-Y]•[E4/MZF1/GATAx].
 

NF-Y bound at the CCAAT motif in the promoter has been reported to be able to open up the chromatin structure of the resident promoters (50, 51). In the trimeric NF-Y complex, the NF-YB and -YC subunits bear both sequence and structure similarities to histone 2B and 2A and can compete with histone 2B and 2A for binding to the histones 3 and 4 tetramer core to disrupt formation of the nucleosomes (59, 60). It is thus possible that in the (4-1)-HS5-ep-GFP plasmid, the e-globin promoter, by itself unable to assemble the NF-Y
complex, required the NF-Y delivered from the LTR enhancer to disrupt the nucleosomes and open up its chromatin structure. Indeed, in the integrated (4-1)-HS5-ep-GFP plasmid, the e-globin promoter existed in an accessible chromatin structure and was able to activate transcription of the linked GFP gene to a high
level. The ability of the ERV-9 LTR enhancer to transmit its enhancer activity over the intervening HS5 site did not appear to be dependent on the e-globin promoter or special structural features of the (4-1)-HS5-ep-GFP plasmid, since the LTR enhancer coupled to the LTR promoter was able to act across the HS5 site and other intervening DNAs to activate transcription of the further downstream gene in several different plasmids (15, 16, 37). In contrast, in MI-HS5-ep-GFP, the mutated CCAAT motif in the LTR enhancer could not bind NF-Y to assemble an active LTR enhancer complex. In the absence of NF-Y
delivered from the LTR enhancer, the e-globin promoter existed in a closed chromatin structure and was unable to activate transcription of the GFP gene. The results indicate that NF-Y assembled a robust LTR enhancer complex, which could act over the intervening HS5 site to remodel the chromatin structure of the downstream e-globin promoter.

In the endogenous b-globin gene locus, the ERV-9 LTR spanning 14 enhancer subunits with a total of 10 NF-Y binding sites is located near the HS5 site in the 5’ boundary of the b-LCR. Whether the ERV-9 LTR enhancer complex containing multiple NF-Y’s can act across the b-LCR to remodel the chromatin structure of the e-globin promoter located 25 kb downstream of it is at the moment not known. However, it has been reported that in the mouse erythroleukemia MEL cells containing a human chromosome 11 spanning the b-globin gene locus, deletion of hypersensitive sites HS5, 4, 3 and 2 in the b-LCR, thus juxtaposing the ERV-9 LTR to the HS1 site, shut down transcription of the b-globin gene but did not close down the chromatin structure of the b-globin gene locus (61). This finding suggests the possibility that the multiple NF-Y’s assembled by the ERV-9 LTR enhancer are able to act over a long distance to remodel the chromatin structure of the b-globin gene locus. We are testing this possibility by experiments designed to study the effects of ERV-9 LTR deletion on chromatin structure and transcription of the downstream b-LCR and the globin genes in the 100 kb human b-globin gene locus during hematopoietic cell differentiation.

Acknowledgement:

We thank Dr. A. Phillips for helpful discussion of EMSA results and Dr. R. Markowitz for critical reading of the manuscript. This work was supported by NIH grant HL62308.

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