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Comparative Study
. 2005 Oct 11;102(41):14765-70.
doi: 10.1073/pnas.0503630102. Epub 2005 Sep 30.

MLL associates specifically with a subset of transcriptionally active target genes

Thomas A Milne  1 , Yali Dou, Mary Ellen Martin, Hugh W Brock, Robert G Roeder, Jay L Hess
Affiliations
Comparative Study

MLL associates specifically with a subset of transcriptionally active target genes

Thomas A Milne et al. Proc Natl Acad Sci U S A. .

Abstract

MLL (mixed-lineage leukemia) is a histone H3 Lys-4 specific methyltransferase that is a positive regulator of Hox expression. MLL rearrangements and amplification are common in acute lymphoid and myeloid leukemias and myelodysplastic disorders and are associated with abnormal up-regulation of Hox gene expression. Although MLL is expressed throughout hematopoiesis, Hox gene expression is sharply down-regulated during differentiation, suggesting that either the activity of MLL or its association with target promoters must be regulated. Here we show that MLL associates with actively transcribed genes but does not remain bound after transcriptional down-regulation. Surprisingly, MLL is associated not only with promoter regions but also is distributed across the entire coding regions of genes. MLL interacts with RNA polymerase II (pol II) and colocalizes with RNA pol II at a subset of actively transcribed target in vivo. Loss of function Mll results in defects in RNA pol II distribution. Together the results suggest that an intimate association between MLL and RNA pol II occurs at MLL target genes in vivo that is required for normal initiation and/or transcriptional elongation.

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Figures

Fig. 1.
Fig. 1.
Expression of MLL target genes in hematopoietic cells. (A) Schematic of the Hoxa9 locus showing three major Hoxa9 transcripts, one upstream transcript (a9a) and two downstream transcripts (a9b and a9T). The a9b and a9T transcripts originate from different start sites (data not shown). The positions of all Taqman primer/probe sets (1-11) used for Hoxa9 ChIP experiments are indicated by arrowheads above the schematic. The homeodomain (HD) is noted as a hatched box and is contained in an exon that is common to all three transcripts. Dashed lines indicate multiple transcription start sites. Open boxes on the line represent exons, black bars beneath the line are CpG-rich regions, light gray boxes beneath the line are AT-rich regions, and medium gray bars indicate regions of highest homology between mouse and human. Distances (in kb) are indicated below the schematic. (B) Meis1, Hoxa7, and all three Hoxa9 isoforms are expressed in MLL-ENL (4) and MLL-AF9 (5) transformed lines and are minimally expressed in neutrophils. Mll is expressed, but Hoxc8 is silent, in all three cell types.
Fig. 2.
Fig. 2.
Assessment of MLL binding to target loci in hematopoietic cells. (A) Schematic of the Meis1, Hoxa1, and Hoxa7 loci. Gray bars on the line represent exons; arrows and arrowheads indicate location of probe/primer sets used for quantitative PCR, and black bars represent CpG-rich regions. (B) Legend for C-E. Blue and pink represent the myeloblast cell lines MLL-ENL and MLL-AF9, respectively, and yellow indicates neutrophils. (C) ChIP using Abs specific for MLLC shows that MLL binds directly to the coding region of Meis1 and the promoter region of Hoxa7 in myeloblast cell lines but not in neutrophils. The Hoxa1 and Gapdh loci are not MLL binding targets. Error bars are shown for all, but in some cases they are too small to be visible. (D) Western blot showing MLLC expression (top band, arrowhead) in neutrophils. The lower band is a nonspecific band reported by Hsieh et al. (26). (E) Western blot showing MLLC expression (top band, arrowhead) in MLL-AF9 cells. (F) ChIP showing distribution of MLLC across the Hoxa9 and Hoxc8 loci in myeloblastic cell lines and neutrophils. MLLC binds to Hoxa9 upstream and coding regions, but not to Hoxc8, which is not expressed in the myeloblastic lines. No binding of MLLC is seen in neutrophils to either the silenced Hoxa9 or Hoxc8 loci. The white line marks the ATG start site. (G) ChIP using Abs specific for the RNA pol II CTD shows that the distribution of RNA pol II closely corresponds to MLL localization.
Fig. 3.
Fig. 3.
Mll association with the Hoxa9 locus in fibroblasts. (A) Quantitative PCR shows that Hoxa9b and Hoxa9T are expressed at higher levels in Mll+/+ fibroblasts relative to Mll-/- fibroblasts. Reexpression of MLL in Mll-/- cells (F-MLL#6 and F-MLL#16) up-regulates transcription of the Hoxa9a and a9T transcripts. The upstream a9a transcript, which is expressed in hematopoietic cells, is not expressed in any of these fibroblast cells. (B) Legend for C-E. Blue and pink represent Mll+/+ and Mll-/- fibroblast cells, respectively, and yellow represents the FMLL#16 line. The white line in C-E marks the ATG start of the Hoxa9. (C) MLLC binding. ChIP using Abs specific for the C-terminal (MLLC) proteolytic fragment of MLL shows highest levels of binding in the Hoxa9 coding and promoter region in Mll expressing cells and no binding at the transcriptionally inactive upstream region. Similar results were obtained in ChIP experiments by using Abs against MLLN. (D) MLLN binding. ChIP for the RNA pol II CTD shows that RNA pol II is distributed across the coding region of Hoxa9 in Mll+/+ cells and F-MLL#16 cells corresponding to peaks of Mll binding. A small but reproducible peak of RNA pol II is seen centered on the downstream Hoxa9 promoter (at ≈5 Kb) in Mll-/- cells. Stable reexpression of MLL results in redistribution of pol II in the downstream Hoxa9 coding region. Schematic indicates position of two alternative start sites. (E) RNA pol II binding.
Fig. 4.
Fig. 4.
MLL binds to RNA pol II and affects histone modifications and transcription elongation. (A) Interaction of MLL1-N and -C (indicated on the right) with either GST alone or RNA pol II C-terminal tail GST fusion (GST-CTD, indicated at the top). Consistent with previous experiments (30), an interaction of MLL2 and menin with the GST-CTD also was seen. In, input. (B) Immunoprecipitations performed with the Mll1-C Ab coprecipitates menin as well as the phospho-Ser-5 form of RNA pol II. Immunoprecipitations were performed in MLL-AF9 nuclear extracts. Control IPs with rabbit IgG using the same extracts also are shown. (C and D) ChIP using Abs specific for either Ser-2 (C) or Ser-5 (D) phosphorylated RNA pol II at the Hoxa9 TATA box, first exon and homeodomain. Taqman primer/probe sets used are shown in H. ChIP shows that Ser-2 is increased in the coding region (exon, HD) relative to the promoter (TATA), whereas Ser-5 is instead increased at the promoter (TATA) in Mll+/+ (blue) and F-MLL#16 (yellow) cells. Conversely, in Mll-/- cells (pink), Ser-2 and -5 are concentrated in small peaks at the promoter, suggesting a defect in transcription elongation. (E-H) ChIP for various histone modifications shows high levels of each mark at the promoter and in the coding regions of the Hoxa9 locus in Mll+/+ cells (blue). Each mark is drastically reduced in Mll-/- cells (pink) and is partially restored by MLL reexpression (yellow). Distances across the Hoxa9 locus (in kb) are shown across the bottom of each image. Positions of Taqman primer/probe sets are shown at the bottom of G and H. (E) Histone H3 dimethyl Lys-4 ChIP. (F) Histone H3 trimethyl Lys-4 ChIP. (G) Histone H3 acetyl Lys-9 ChIP. (H) Histone H3 dimethyl Lys-79 ChIP.
Fig. 5.
Fig. 5.
Mll and pol II association with other target genes in fibroblasts. (A) MLLC binding. ChIP using Abs specific for MLLC shows MLL binding at the Hoxa7, Meis1, FoxC1, and FoxC2 loci but not at Hoxa1, Pbx1, Pbx3, Mll or Gapdh. Positions of Taqman primer/probe sets used for quantification are shown in Figs. 2 and 8. (B) Menin binding. Menin binds to the same target genes as Mll. (C) RNA pol II binding. RNA pol II shows an Mll and menin-dependent increase in binding for the Hoxa7, Meis1, FoxC1, and FoxC2 target loci, but pol II binding is independent of Mll and menin at the Hoxa1, Pbx1, Pbx3, Mll, and Gapdh genes.
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