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Delineating MEIS1 cis-regulatory elements active in hematopoietic cells

MEIS1 (Myeloid ecotropic viral integration site 1) is a homeodomain transcription factor that cooperates with Hox family proteins in both normal and leukemic hematopoiesis.1 MEIS1 is highly expressed in hematopoietic stem cells (HSCs), and its level decreases during differentiation but with a relatively higher expression in the megakaryocytic lineage.2, 3 MEIS1 expression is often elevated in leukemia,4 and it is one of the leukemic stem cell (LSC)-related signature genes whose higher expression is correlated with worse prognosis.5 Previous work, including our own, has shown that MEIS1 acts as an important driver for leukemogenesis.1 Elevated expression of MEIS1 with concurrent high expression of HOX can induce rapid-onset AML in murine models.6 Decreased MEIS1 expression via short hairpin RNA-mediated knockdown has been shown to significantly reduce LSC potential.7, 8 Together, these findings point to the importance of tightly regulated expression of MEIS1 for normal hematopoiesis and reveal how its deregulated expression can be a major factor in leukemogenesis.

A major challenge, however, to understanding the regulation of MEIS1 is the large genomic region it spans (around 140 kb in both mouse and human with 98% non-coding region), where many potential cis-regulatory elements could function together to regulate its expression. Our previous and recent studies have provided some insights into the nature of the MEIS1 promoter region with an essential role in the regulation of MEIS1 expression9 (Supplementary Figure 1). Two recent studies have further identified the presence of multiple additional cis-regulatory elements within the MEIS1 locus through analysis of evolutionarily conserved regions from zebrafish to human.10, 11 Although such evolutionary-based approaches have helped to identify some MEIS1 cis-regulatory elements, including several regions that could be potentially involved in hematopoiesis, it leaves open the possibility of missing important regulatory regions that are not so highly conserved from fish to human.

To identify cis-regulatory elements that are especially important in the leukemia-associated MEIS1 upregulation, we took a different approach and searched for additional candidate MEIS1 enhancers on the basis of their chromatin status, namely, open and active chromatin characterized by DNase I hypersensitivity and high histone H3 acetylation. We selected five human leukemic cell lines, plus 293 cells, which served as an example of MEIS1-expressing non-hematopoietic cells, to cover a wide range of MEIS1 expression levels spanning over three orders of magnitude. We grouped them into three categories on the basis of their relative MEIS1 expression: high expressers MEIS1Hi (KG1 and U937); medium expressers MEIS1Med (K562 and 293); and low/non-expressers MEIS1Lo (Jurkat and HL60; Figure 1a). From our DNase I hypersensitivity site (DHS) mapping (Figure 1b), we identified a total of 31 significant DHSs within the MEIS1 region, with the majority of these DHSs common to at least two cell lines. Significantly fewer DHSs were detected at the MEIS1 locus in the MEIS1Lo cell lines, suggesting a less open chromatin structure. We noticed many common DHSs, especially within the MEIS1Med or MEIS1Hi cell line groups, such as at +134 and +140 kb in the MEIS1Med (K562 and 293; Supplementary Figure 2) and at +8.8 and +10.6 kb in the MEIS1Hi cell lines (U937 and KG1; Supplementary Figure 3). We examined the level of histone H3K9/14 acetylation, which is typically found at active enhancers12 within the MEIS1 locus with a focus on these DHSs through chromatin immunoprecipitation (ChIP)-real time PCR (Figure 1c). The MEIS1Lo cell lines displayed much weaker histone H3 acetylation over the MEIS1 locus, consistent with their less opened chromatin. In contrast, we identified three open and active chromatin regions that may function as enhancers in MEIS1-expressing cell lines. For both the MEIS1Hi and MEIS1Med cell lines, a high histone H3 acetylation level was apparent in the distal promoter region at −2 kb DHS (defined here as E1). Interestingly, the +8 to 12 kb region at the beginning of intron 6, especially around +10.6 kb DHS (defined here as E2), was significantly enriched with histone H3 acetylation in the MEIS1Hi cell lines. In addition, the MEIS1Med cell lines had pronounced histone H3 acetylation at the 3′ end region around +140 kb DHS (defined here as E3). As E1 and E2 are within and E3 is close to CpG islands, we further demonstrated that DNA within these three regions was hypomethylated when they were enriched with histone H3 acetylation, which correlated well with their active chromatin status (Supplementary Figure 4).

Figure 1
figure1

Identification of MEIS1 candidate cis-regulatory elements. (a) Human cell lines used in the current study display variable expression levels of MEIS1 in the quantitative real-time PCR (RT-PCR) assay. Data are normalized with GAPDH as internal control and displayed as the relative levels to the MEIS1 expression level in K562 cells. On the basis of the MEIS1 expression level, the cell lines are categorized into MEIS1Lo, MEIS1Med and MEIS1Hi cell lines. (b) Summary of the DNase I hypersensitivity mapping assay across the MEIS1 locus. ‘1’ indicates that the corresponding band of a DNase I hypersensitive site (DHS) on Southern blot was readily detected by eye. (c) Summary of the enrichment of histone H3 acetylation (K9/14) across the MEIS1 locus. Data are normalized to the enrichment of histone H3 acetylation (K9/14) at the promoter region of GAPDH. The candidate enhancer regions of E1, E2 and E3 are highlighted.

We focused our interest on these three candidate enhancer regions. When we analyzed the available epigenome data within the MEIS1 locus from normal blood populations, we found that all these regions were epigenetically active in the more stem cell-enriched CD34+ population, but silenced in the more mature peripheral blood mononuclear cell population, in which the MEIS1 level is diminished (Figure 2a and Supplementary Figure 5). This suggests an involvement of these enhancer candidates in normal hematopoiesis. We demonstrated that these regions contain enhancer function through a luciferase assay (Figure 2b). The addition of a 300–350 bp fragment from E1, E2 or E3 to the MEIS1 promoter significantly increased its activity in both MEIS1Hi U937 and MEIS1Med K562, while they failed to enhance the MEIS1 promoter activity in the MEIS1Lo Jurkat cell line. As active enhancer elements typically increase the expression of a particular gene through their close physical interactions with the gene promoter via looping within chromatin,13 we performed a chromosome conformation capture (3C) assay in three representative MEIS1Hi, MEIS1Med and MEIS1Lo cell lines (U937, K562 and HL60, respectively) to detect the frequency of association between the candidate enhancers and the MEIS1 promoter regions (Figure 2c). As expected, given their close proximity, the frequency of interaction between the fragments containing E1 and the MEIS1 promoter was high in all cell lines tested with a slight increase in the MEIS1Hi U937 cell line. Most interestingly, the interactions between the fragments containing E2 and the MEIS1 promoter were significantly stronger in U937, whereas relatively stronger interactions between the 3′ end of MEIS1 including the E3 and the MEIS1 promoter in the MEIS1Med K562 cell line compared with other cell lines were observed. These frequencies of interaction are essentially proportional to the local levels of DNase I sensitivity and histone acetylation. As two of the three candidate enhancer regions (E2 and E3) also contain conserved binding sites of CTCF (Supplementary Figure 6), a well-known factor involved in chromatin association,14 we suspected CTCF may be involved in bringing these candidate enhancers to the proximal region of promoter. Through CTCF ChIP assay, we detected a strong CTCF association at the upstream −6 kb DHS in all the cell lines and also found strong CTCF binding at +140 kb E3 exclusively in the two MEIS1Med cell lines. This suggests that CTCF may be involved in establishing the loop between the 5′ and 3′ ends of the MEIS1 locus when E3 is epigenetically active and, in doing so, it bringing E3 adjacent to the MEIS1 promoter. We did not, however, detect very strong CTCF association at E2 except for a very slight enrichment (approximately two- to threefold) in the two MEIS1Hi cell lines (Figure 2d). This suggests that other mechanisms are involved in bringing the epigenetically active E2 adjacent to the promoter.

Figure 2
figure2

Functional validation of candidate enhancer regions. (a) A snapshot of MEIS1 locus at http://epigenomegateway.wustl.edu/browser/, highlighting the representative epigenetic data from mobilized CD34+cells and peripheral blood mononuclear cells (PBMCs). The three enhancer regions are marked by arrows. In CD34+ cells, they are enriched with active chromatin markers, such as DNase I Hypersensivity, H3K4 monomethylation and trimethylation, H3K27 acetylation and so on, whereas in PBMCs, these regions are strongly repressed with significantly enriched H3K9 and H3K27 trimethylation. (b) Enhancer activities measured through the dual luciferase assay. The enhancer activity is presented as fold change compared with the vector carrying MEIS1 promoter alone. ‘C’ is a size as well as CTCF binding control (more detail in the Supplementary Information). The three enhancers display enhancer activities in MEIS1Hi (U937) and MEIS1Med (K562) cell lines. (c) The interactions between the promoter region of MEIS1 and the candidate enhancer regions are evaluated through the chromosome conformation capture (3C) assay. The HindIII digestion sites are highlighted by shaded gray boxes except the fragment including MEIS1 promoter region, which is used as the anchor region, highlighted in black. (d) CTCF enrichment at the predicted CTCF binding sites in the MEIS1 locus. Data are normalized to the CTCF enrichment at the GAPDH promoter region.

In summary, we have expanded on our previous work to further elucidate mechanisms involved in the regulation of MEIS1 in the context of leukemic settings. We selected the candidate regions on the basis of their epigenetic status in different leukemic conditions and identified three candidate enhancers, namely, E1 at −2 kb, E2 at +10.6 kb and E3 at +140 kb. Compared with previous studies on the basis of conservation between zebrafish to human, our approach was complementary in finding the less conserved cis-regulatory elements. Although E3 is evolutionarily highly conserved and was included in the study by Royo et al.10 (HHc2:067156), in which it demonstrated enhancer activity in the G0 zebrafish, the corresponding regions of E1 and E2 are missing in zebrafish genome and have not been characterized previously (Supplementary Figure 7). The E1 is, however, conserved between mouse and human. Although E2 is less conserved, a predicted 18 bp CTCF binding site within E2 is conserved between mouse and human (Supplementary Figure 6).

On the basis of the evidence of these three regions maintaining active chromatin status in HSC-enriched CD34+ cells, but adopting features of silenced chromatin in peripheral blood mononuclear cells, we suggest these elements can also function in normal hematopoiesis. Under different leukemic conditions, these three regions demonstrated cell-type-specific enhancer function, as suggested by their variable chromatin status. Although the −2 kb E1 enhancer displayed an active chromatin status in all the MEIS1-expressing cells, an active E2 region was more associated with MEIS1Hi cell lines and an active E3 region was more associated with MEIS1Med cell lines. In addition, the closer association between the distant downstream enhancer E2 or E3 and the MEIS1 promoter, as demonstrated in the 3C assay, suggests that they function as enhancers in vivo when they are in the active chromatin status. This apparent differential enhancer usage in the various leukemic cell lines may be related to the heterogeneous nature of leukemia.15 Together, these findings suggest that a further exploration of the regulation of these newly-recognized enhancers, particularly E2, will provide important insights into the mechanisms underlying the dysregulation of MEIS1 expression in leukemia.

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Acknowledgements

We sincerely thank other lab members in Dr Keith Humphries’ laboratory for helpful discussion. This work is supported by the Terry Fox Foundation Program Project Grant (TFF-122869) and CIHR (RMF-92093).

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Correspondence to K R Humphries.

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Xiang, P., Wei, W., Lo, C. et al. Delineating MEIS1 cis-regulatory elements active in hematopoietic cells. Leukemia 28, 433–436 (2014). https://doi.org/10.1038/leu.2013.287

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