MLL protects CpG clusters from methylation within the Hoxa9 gene, maintaining transcript expression - PubMed (original) (raw)

MLL protects CpG clusters from methylation within the Hoxa9 gene, maintaining transcript expression

Frank E Erfurth et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Homeobox (HOX) genes play a definitive role in determination of cell fate during embryogenesis and hematopoiesis. MLL-related leukemia is coincident with increased expression of a subset of HOX genes, including HOXA9. MLL functions to maintain, rather than initiate, expression of its target genes. However, the mechanism of MLL maintenance of target gene expression is not understood. Here, we demonstrate that Mll binds to specific clusters of CpG residues within the Hoxa9 locus and regulates expression of multiple transcripts. The presence of Mll at these clusters provides protection from DNA methylation. shRNA knock-down of Mll reverses the methylation protection status at the previously protected CpG clusters; methylation at these CpG residues is similar to that observed in Mll null cells. Furthermore, reconstituting MLL expression in Mll null cells can reverse DNA methylation of the same CpG residues, demonstrating a dominant effect of MLL in protecting this specific region from DNA methylation. Intriguingly, an oncogenic MLL-AF4 fusion can also reverse DNA methylation, but only for a subset of these CpGs. This method of transcriptional regulation suggests a mechanism that explains the role of Mll in transcriptional maintenance, but it may extend to other CpG DNA binding proteins. Protection from methylation may be an important mechanism of epigenetic inheritance by regulating the function of both de novo and maintenance DNA methyltransferases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

MLL protects specific CpG residues in the Hoxa9 locus from methylation. (A) Schematic representation of the murine Hoxa9 region. Eight kilobases of the genomic region of Hoxa9 is represented. CpG Islands are numbered beneath. Upstream alternative first exon is labeled AB, the canonical Hoxa9 first exon is labeled CD and the canonical second exon, containing the homeodomain, is labeled II. Large asterisks indicate Mll binding as determined by ChIP. (B) MLL-dependent protection of CpG Island 1 from methylation. Direct bisulfite sequencing reveals a difference in methylation status in Mll+/+ MEF cells (circles) and _Mll_−/− MEF cells (X). Area under the curve analysis was performed on DNA sequence histograms and relative methylation percentage was determined for CpG residues. Each point on the graph represents a single CpG residue from the sequence below the graph. Beneath the graph are sequencing results from individual clones (10 clones each from Mll+/+ and _Mll_−/− MEFs) of PCR products from bisulfite treated template. Empty circles, unmethylated CpG residues; filled circles, methylated CpG residues. (C) Upon shRNA knockdown of Mll in Mll+/+ MEF cells, protection from methylation is lost. Triangles, shRNA Mll knockdown; circles, shRNA pSuper vector alone. Knockdowns were selected for 2 weeks. Four-week selection showed similar results (data not shown). (D) Upon add-back of MLL in _Mll_−/− MEF cells, protection from methylation returns. Filled circles, MLL add-back; x's, vector-only control. Data shown are from cells kept under selection for one week.

Fig. 2.

Fig. 2.

Mll-dependent expression of Hoxa9 transcripts and Mll binding to Hoxa9 AB region. (A) RNA from Mll+/+ and _Mll_−/− MEF cells was analyzed by real-time RT-PCR to detect expression of upstream AB and canonical Hoxa9 transcripts. Expression was normalized to gapdh from the same samples. (B) shRNA knockdown of Mll. RNA extracted from Mll+/+ MEF cells after infection with retrovirus carrying empty vector or shRNA directed against either Mll or CtBP (an unrelated gene) were analyzed by RT-PCR to detect expression of Mll (to confirm efficient knockdown), the upstream AB transcript, the canonical Hoxa9 transcript, or gapdh control with and without reverse transcription (± RT). Cells were kept under selection for 2 weeks. (C) ChIP analysis was performed by using a mixture of polyclonal anti-Mll antibodies, a commercial anti-Mll monoclonal antibody, or IgG control antibody. Enriched chromatin was amplified by using primers specific for the AB exon.

Fig. 3.

Fig. 3.

ITC measurement of the binding of the MLL CXXC domain to CpG1II oligonucleotide. Data shown is for addition of 10-μl aliquots of 550 μM CXXC domain to a 46 μM solution of CpG1II. The data were fit to a one-site binding model, giving _K_d = 7.8 ± 1.4 μM and n = 0.93 ± 0.04.

Fig. 4.

Fig. 4.

Add back of MLL-AF4 fusion reduces methylation of a portion of the protected CpG1 residues after 4 weeks and longer. _Mll_−/− MEF cells were transfected with either plasmid to express MLL-AF4 or empty vector. Cell populations were analyzed after 4 weeks of selection (squares, MLL-AF4; diamonds, vector control) or individual clones were selected, replated, and expanded (crosses and circles, two different representative MLL-AF4 add-back clones). DNA was subjected to bisulfite treatment and nested PCR and sequenced. Area under the curve analysis was performed on DNA sequencing histograms and relative methylation percentage was determined for CpG residues. Regions protected from DNA methylation by MLL-AF4 and by MLL (data from Fig. 1) are highlighted under the graph.

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