Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jun 17:9:151.
doi: 10.1186/1476-4598-9-151.

Polycomb repressor complex 2 regulates HOXA9 and HOXA10, activating ID2 in NK/T-cell lines

Affiliations

Polycomb repressor complex 2 regulates HOXA9 and HOXA10, activating ID2 in NK/T-cell lines

Stefan Nagel et al. Mol Cancer. .

Abstract

Background: NK- and T-cells are closely related lymphocytes, originating from the same early progenitor cells during hematopoiesis. In these differentiation processes deregulation of developmental genes may contribute to leukemogenesis. Here, we compared expression profiles of NK- and T-cell lines for identification of aberrantly expressed genes in T-cell acute lymphoblastic leukemia (T-ALL) which physiologically regulate the differentiation program of the NK-cell lineage.

Results: This analysis showed high expression levels of HOXA9, HOXA10 and ID2 in NK-cell lines in addition to T-cell line LOUCY, suggesting leukemic deregulation therein. Overexpression experiments, chromatin immuno-precipitation and promoter analysis demonstrated that HOXA9 and HOXA10 directly activated expression of ID2. Concomitantly elevated expression levels of HOXA9 and HOXA10 together with ID2 in cell lines containing MLL translocations confirmed this form of regulation in both ALL and acute myeloid leukemia. Overexpression of HOXA9, HOXA10 or ID2 resulted in repressed expression of apoptosis factor BIM. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including components of polycomb repressor complex 2 (PRC2), indicated lacking expression of EZH2 in LOUCY and exclusive expression of HOP in NK-cell lines. Subsequent treatment of T-cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively. Moreover, siRNA-mediated knockdown of EZH2 in JURKAT enhanced HOXA10 expression, confirming HOXA10-repression by EZH2. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression.

Conclusions: Our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 which therefore contributes to T-cell leukemogenesis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Gene expression analysis by profiling. (A) Expression profiling data were analyzed by cluster analysis. Of note, all cells were ordered according to their origin. LOUCY was the most varied T-cell line. (B) Expression profiling data were transformed into a heat map, demonstrating 200 top/down expressed genes in NK/T-cell lines and CD34+ HSCs. Red indicates high, green low and black medium expression levels. Selected top/down expressed genes are shown on the right, including HOXA9, HOXA10, ID2 and RUNX2 highlighted in red.
Figure 2
Figure 2
Gene expression analysis by real-time PCR. Quantitative RT-PCR analysis of candidate genes HOXA9, HOXA10, ID2 and RUNX2 in selected T- and NK-cell lines. Of note, high expression levels of all four genes were confirmed in NK-92, YT and LOUCY. Expression levels of YT were set to 1.
Figure 3
Figure 3
Expression analysis in HELA cells. HELA cells were transfected with expression constructs for HOXA9 and HOXA10 in addition to a vector control. Subsequent RQ-PCR analysis of ID2 and RUNX2 demonstrated elevated ID2 transcript levels while that of RUNX2 remained unchanged.
Figure 4
Figure 4
Expression analysis in T-ALL cells. (A) JURKAT cells were lentivirally transduced for overexpression of HOXA9, HOXA10 and HOP, respectively. Subsequent RQ-PCR analysis of ID2 and RUNX2 demonstrated elevated ID2 transcription in HOXA9/HOXA10 overexpressing cells and reduced levels in HOP overexpressing cells. Expression levels of RUNX2 showed no alterations. (B) Western blot analysis demonstrated elevated expression of ID2 protein in JURKAT cells overexpressing HOXA10. STAT5 protein analysis served as loading control. (C) ChIP-PCR analysis in LOUCY (HOXA10-positive) and PEER (HOXA10-negative), using HOXA10 antibody demonstrated direct binding of HOXA10 to the ID2 promoter region at -2167 bp. (D) HELA cells were cotransfected with an ID2 promoter-construct and an expression-construct for HOXA10 or vector control. Reportergene activity was subsequently quantified by real-time PCR, indicating an activatory input by HOXA10.
Figure 5
Figure 5
Expression analysis in ALL cell lines. RQ-PCR analysis of HOXA9, HOXA10 and ID2 demonstrated higher expression levels in ALL cell lines carrying MLL-translocations. The highest expression levels of each gene were set to 1.
Figure 6
Figure 6
Expression analysis in AML cell lines. RQ-PCR analysis of HOXA9, HOXA10 and ID2 demonstrated higher expression levels in ALL cell lines carrying MLL-translocations. The highest expression levels of each gene were set to 1.
Figure 7
Figure 7
Target gene analysis of ID2. (A) RQ-PCR analysis of BIM demonstrated reduced expression levels in JURKAT cells overexpressing HOXA9, HOXA10 or ID2. (B) MTT assay of transduced JURKAT cells after treatment with etoposide indicated reduced apoptosis by HOXA10 overexpression.
Figure 8
Figure 8
Gene expression analysis of chromatin genes. Profiling data of selected chromatin genes were transformed into a heat map, demonstrating reduced expression levels of EZH2 in LOUCY and high expression levels of HOP in NK-cell lines NK-92 and YT. Red indicates high, green low and black medium expression levels.
Figure 9
Figure 9
Expression analysis in NK/T-cells. (A) RQ-PCR analysis of candidate gene EZH2 in selected T- and NK-cell lines. Of note, reduced EZH2 expression was confirmed in LOUCY. (B) RQ-PCR analysis of candidate gene HOP in selected T- and NK-cell lines. Of note, high expression levels were confirmed in NK-cell lines NK-92 and YT. (C) Western blot analysis of EZH2 protein expression in selected NK/T-cell lines. ERK1/2 protein expression served as loading control. Of note, in LOUCY no EZH2 protein was detectable.
Figure 10
Figure 10
DZNep treatment of T-ALL cells. (A) Western blot analysis of EZH2 protein expression in JURKAT and LOUCY cells treated with DZNep. ERK1/2 protein expression served as loading control. Treatment with DZNep reduced concentration dependent the protein levels of EZH2 in JURKAT cells. (B) RQ-PCR analysis of HOXA9 and HOXA10 in LOUCY and JURKAT cells treated with DZNep. Of note, LOUCY showed unaltered expression levels in contrast to JURKAT, demonstrating elevated HOXA10 expression after treatment.
Figure 11
Figure 11
Suppression of EZH2 in JURKAT. (A) RQ-PCR analysis of HOXA10 after siRNA-mediated knockdown of EZH2 demonstrated enhanced gene expression. (B) RQ-PCR analysis of HOXA10, ID2 and RUNX2 in JURKAT cells treated with DZNep. Of note, concomitantly with HOXA10, expression levels of ID2 rose after treatment with DZNep in contrast to RUNX2 expression.
Figure 12
Figure 12
Expression analysis of HOXA10. RQ-PCR analysis of HOXA10 in selected NK/T-cell lines after rapamycin treatment. Of note, reduced HOXA10 expression was detected in JURKAT and YT but not in LOUCY.
Figure 13
Figure 13
Effect of HOP in T- and NK-cells. (A) RQ-PCR analysis in HOP transduced JURKAT cells demonstrated downregulation of HOXA10 and ID2 expression (top). RQ-PCR analysis in siRNA-treated NK-92 and YT cells demonstrated upregulation of HOXA10 expression (below), confirming repressive activity of HOP and EZH2. (B) RQ-PCR analysis in transduced JURKAT cells treated with DZNep, demonstrating enhanced HOXA10 expression in HOP overexpressing cells.
Figure 14
Figure 14
Regulation of EZH2 expression via E2F/TFDP1. (A) Western blot analysis of selected NK/T-cell lines demonstrated the presence of E2F1 protein in all cells including LOUCY. (B) RT-PCR analysis of TFDP1 in selected NK/T-cell lines demonstrated reduced expression level in LOUCY. Expression of TEL served as positive control, NTC: no template control. (C) RQ-PCR analysis of LOUCY cells which were lentivirally transduced with an expression construct for TFDP1 demonstrated increased expression levels of EZH2 and decreased levels of HOXA10 and ID2 as compared to vector control cells.
Figure 15
Figure 15
Network of HOXA-regulation. The diagram depicts major chromatin complexes, including PRC1, PRC2, MLL and SET with selected components or mutated fusion proteins which regulate the transcription of HOXA genes.

Similar articles

Cited by

References

    1. Huntington ND, Vosshenrich CA, Di Santo JP. Developmental pathways that generate natural-killer-cell diversity in mice and humans. Nat Rev Immunol. 2007;7:703–714. doi: 10.1038/nri2154. - DOI - PubMed
    1. Anderson MK. At the crossroads: diverse roles of early thymocyte transcriptional regulators. Immunol Rev. 2006;209:191–211. doi: 10.1111/j.0105-2896.2006.00352.x. - DOI - PubMed
    1. Rothenberg EV, Moore JE, Yui MA. Launching the T-cell-lineage developmental programme. Nat Rev Immunol. 2008;8:9–21. doi: 10.1038/nri2232. - DOI - PMC - PubMed
    1. Rothenberg EV, Dionne CJ. Lineage plasticity and commitment in T-cell development. Immunol Rev. 2002;187:96–115. doi: 10.1034/j.1600-065X.2002.18709.x. - DOI - PubMed
    1. O'Neil J, Shank J, Cusson N, Murre C, Kelliher M. TAL1/SCL induces leukemia by inhibiting the transcriptional activity of E47/HEB. Cancer Cell. 2004;5:587–596. doi: 10.1016/j.ccr.2004.05.023. - DOI - PubMed

Publication types

MeSH terms