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. 2016 Jul 19;7(29):45398-45413.
doi: 10.18632/oncotarget.9982.

Deregulation of polycomb repressor complex 1 modifier AUTS2 in T-cell leukemia

Stefan Nagel  1 , Claudia Pommerenke  1 , Corinna Meyer  1 , Maren Kaufmann  1 , Hans G Drexler  1 , Roderick A F MacLeod  1
Affiliations

Deregulation of polycomb repressor complex 1 modifier AUTS2 in T-cell leukemia

Stefan Nagel et al. Oncotarget. .

Abstract

Recently, we identified deregulated expression of the B-cell specific transcription factor MEF2C in T-cell acute lymphoid leukemia (T-ALL). Here, we performed sequence analysis of a regulatory upstream section of MEF2C in T-ALL cell lines which, however, proved devoid of mutations. Unexpectedly, we found strong conservation between the regulatory upstream region of MEF2C (located at chromosomal band 5q14) and an intergenic stretch at 7q11 located between STAG3L4 and AUTS2, covering nearly 20 kb. While the non-coding gene STAG3L4 was inconspicuously expressed, AUTS2 was aberrantly upregulated in 6% of T-ALL patients (public dataset GSE42038) and in 3/24 T-ALL cell lines, two of which represented very immature differentiation stages. AUTS2 expression was higher in normal B-cells than in T-cells, indicating lineage-specific activity in lymphopoiesis. While excluding chromosomal aberrations, examinations of AUTS2 transcriptional regulation in T-ALL cells revealed activation by IL7-IL7R-STAT5-signalling and MEF2C. AUTS2 protein has been shown to interact with polycomb repressor complex 1 subtype 5 (PRC1.5), transforming this particular complex into an activator. Accordingly, expression profiling and functional analyses demonstrated that AUTS2 activated while PCGF5 repressed transcription of NKL homeobox gene MSX1 in T-ALL cells. Forced expression and pharmacological inhibition of EZH2 in addition to H3K27me3 analysis indicated that PRC2 repressed MSX1 as well. Taken together, we found that AUTS2 and MEF2C, despite lying on different chromosomes, share strikingly similar regulatory upstream regions and aberrant expression in T-ALL subsets. Our data implicate chromatin complexes PRC1/AUTS2 and PRC2 in a gene network in T-ALL regulating early lymphoid differentiation.

Keywords: IL-7; IL7R; MEF2C; PRC1; PRC2.

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Conflict of interest statement

The authors declare they have no known conflicts of interest in this work.

Figures

Figure 1
Figure 1. MEF2C expression and genomic regions at 5q14 and 7q11
(A) Expression analysis of MEF2C in T-ALL patients (public dataset GSE42038) indicated aberrant overexpression in 19% samples (left). RQ-PCR analysis of MEF2C in T-ALL cell lines demonstrated aberrant expression in 4 cell lines (middle). RQ-PCR analysis of MEF2C in primary cells showed high expression levels in B-cells, silencing in T-cells, and aberrant expression in T-ALL cells (right). (B–D) The 5q14 and 7q11 homologous sequence (boxed region) is located in the upstream region of MEF2C (B), in the downstream region of STAG3L4 (C) or in the upstream region of AUTS2 (D). This region contains two blocks of conserved TF binding sites which are named A and B (data were obtained from UCSC Genome Bioinformatics). The conserved STAT5 binding site is indicated in red.
Figure 2
Figure 2. Expression analyses of STAG3L4 and AUTS2
(A) RQ-PCR analysis of STAG3L4 in T-ALL cell lines (left). Expression analysis of STAG3L4 in T-ALL patient samples (public dataset GSE42038) (right). (B) RQ-PCR analysis of AUTS2 in T-ALL cell lines indicated overexpression in 3 cell lines (left). Expression analysis of AUTS2 in T-ALL patients (GSE42038) indicated aberrant overexpression in 6% of the samples (right). (C) Western blot analysis of AUTS2 in selected T-ALL cell lines shows elevated expression in LOUCY and PER-117. Tubulin A served as loading control. (D) RQ-PCR analysis of AUTS2 in primary cells showed high expression levels in B-cells, reduced levels in T-cells and overexpression in LOUCY and PER-117.
Figure 3
Figure 3. Gain at 7q11, IL7-STAT5 and MEF2C activate AUTS2 expression
(A) Genomic profiling of T-ALL cell lines JURKAT, LOUCY and PER-117 shows copy number states of chromosome 7, indicating whole chromosome triploidy in PER-117 (above, left). Quantitative PCR analysis of AUTS2 indicated 3 copies in PER-117 (Above, right). FISH analysis of AUTS2 (orange) and chromosome 7 (green) in DND-41, LOUCY and PER-117 indicates absence of chromosomal rearrangements at AUTS2 (below). (B) RQ-PCR analysis (above) after siRNA-mediated knockdown of STAT5, treatment with IL7, and forced overexpression of STAT5 in LOUCY and JURKAT cells. Western blot analyses (below) confirmed knockdown, increased phosphorylation and overexpression of STAT5. Tubulin A and SMAD1 served as loading controls. (C) Sequencing results of a IL7R gene section in DND-41 cells show wild type configuration of one allele (above) and an insertion in the other allele (below). This insertion encodes 4 amino acid residues comprising cysteine which mediates aberrant receptor activation. (D) SiRNA-mediated knockdown of IL7R in DND-41 resulted in reduced expression of AUTS2. (E) SiRNA-mediated knockdown of MEF2C in LOUCY resulted in reduced expression of AUTS2. (F) Reporter gene assay analyzing 3 potential MEF2C binding sites in the upstream region of AUTS2 demonstrated direct binding and regulation of AUTS2 by MEF2C.
Figure 4
Figure 4. AUTS2 and PCGF5 regulate MSX1 expression
(A) SiRNA-mediated knockdown of AUTS2 in LOUCY resulted in reduced expression of MSX1 but not of HOXA10 or MEF2C (left). SiRNA-mediated knockdown of AUTS2 in PER-117 resulted in reduced expression of MSX1 (middle). SiRNA-mediated knockdown of AUTS2 in JURKAT showed no change in MSX1 expression levels (middle). Forced expression of AUTS2 in JURKAT resulted in MSX1 upregulation while HOXA10 levels remained unaffected (right). (B) RQ-PCR analysis of PCGF5 in selected T-ALL cell lines (left). PER-117 showed the lowest PCGF5 expression level. RQ-PCR analysis of PCGF5 in primary hematopoietic cells showed elevated expression levels in B- and T-cells and reduced levels in T-ALL cell lines (right). (C) SiRNA-mediated knockdown of PCGF5 in LOUCY (left) and JURKAT (middle) resulted in enhanced expression of MSX1 but not of HOXA10. SiRNA-mediated knockdown of PCGF5 in PER-117 (middle) left MSX1 expression unperturbed. Forced expression of PCGF5 in PER-117 resulted in reduced expression levels of MSX1 (right).
Figure 5
Figure 5. PRC1, PRC2 and RUNX1 regulate MSX1
(A) RQ-PCR analysis of MSX1 in JURKAT and LOUCY after treatment with HDAC-inhibitor TSA resulted in enhanced expression levels (left). RQ-PCR analysis of MSX1 in LOUCY after treatment with EP300-inhibitor ICBP112 resulted in reduced expression levels (middle). (B) RQ-PCR analysis of MSX1 in JURKAT and LOUCY after treatment with EZH2-inhibitor DZNep resulted in enhanced expression levels in JURKAT but showed no change in EZH2-negative LOUCY (left). Forced expression of EZH2 in LOUCY resulted in reduced expression of MSX1 (left). ChIP analysis of H3K27me3 and H2A119ub1 in JURKAT and LOUCY demonstrated H3K27-trimethylation at MSX1 in JURKAT but not in LOUCY (right). (C) SiRNA-mediated knockdown of RUNX1 in JURKAT and LOUCY resulted in increased expression of MSX1 (left). In LOUCY and PER-117 forced expression of RUNX1 mediated decreased MSX1 expression (right). (D) Expression analysis of AUTS2 (left) and MSX1 (right) in T-ALL patients (public dataset GSE42038) demonstrated that the overlap of patients with maximal RNA expression of both AUTS2 and MSX1 was statistically significant (p = 0.0053, hypergeometric test, n = 20 draws of N = 79 patients), supporting the observed activating impact of AUTS2 on MSX1 expression.
Figure 6
Figure 6. Gene regulatory network comprising AUTS2 and MSX1
This figure summarizes the results obtained in this study. IL7-STAT5-signalling is located upstream of MEF2C and AUTS2. AUTS2 interacts with PCGF5/PRC1.5 turning the repressive impact of this complex into an activatory, resulting in elevated expression of MSX1. PRC2 mediates repressive H3K27-trimethylation and AUTS2 activatory histone-acetylation. MSX1 is regulated by PRC1.5 and PRC2 while HOXA10 is regulated just by PRC2. Both, HOXA10 and MSX1 are involved in lymphoid differentiation.
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