Materials and Methods

Antibodies
Affinity purified rabbit polyclonal anti-MLL1 antibody 173 and 170 were previously described (14). Control polyclonal rabbit IgG antibody was obtained from Upstate Biotechnologies. Monoclonal anti-Pol II antibody 8WG16 was directed against the highly conserved Pol II CTD. Histone H4 K4 trimethyl antibody was obtained from AbCam (ab6002). Histone H3 carboxy terminal antibody (ab1791) was obtained from AbCam.

Genome-Wide Location Analysis
The protocol described here was adapted from (40). Briefly, 2.0 x 108 U937 cells were fixed with 1% final concentration formaldehyde for 20 minutes at room temperature, harvested and rinsed with 1x PBS. The resultant cell pellet was sonicated, and DNA fragments were enriched by immunoprecipitation with a factor specific antibody. After reversal of the crosslinking, the enriched DNA was amplified using ligation-mediated PCR (LM-PCR), and then fluorescently labeled using high concentration Klenow polymerase and a dNTP-fluorophore. A sample of reference DNA not enriched by immunoprecipitation was subjected to LM-PCR and labeled with a different fluorophore. Both IP-enriched and unenriched pools of labeled DNA were hybridized to the Human 19K array. Each experiment was performed independently in duplicate.

Human 19K DNA Microarray
Design and quality control for the human 13K array has been described (41). Expansion of the 13K array (13,000 features) to 19K (19,000 features) was performed as following. We collated all genes predicted by Refseq, Ensembl, and MGC from build 34 of the human genome sequence. Primers were designed against genes that were included in at least 2 of the 3 databases. Proximal promoter features were produced representing 1) all genes mismapped in the original NCBI build 22 (April 2001), and 2) All genes predicted to encode transcriptional regulators as described in GO annotation. The promoter regions spanned 750bp upstream to 250bp downstream of the consensus transcription start sites. DNA representing regions –3775 to -2625, -2775 to –1625, -1775 to –625, -775 to +375, +225 to +1375, and +1225 to +2375 relative to the transcription start site of 275 diverse genes was also included. We identified the largest gaps between all predicted genes and designed 5 x 1 Kb probes to each of the largest 143 of these gaps (all over 1.6 Mb). Of these, 623 primer pairs were found. All sequences are at least 800Kb from the nearest gene. Binding data from these intergenic control probes were used to normalize the array data and to estimate the significance of binding ratios for all other probes.

Comparing Location and Expression Data
We used Affymetrix expression data from the Novartis v2 dataset (42). Data from replicate tissue samples were averaged and expression ratios were generated by dividing each value by the row-wise average. The data were converted to log base 2 and normalized so that the column and row-wise medians were 0. Data for genes represented by multiple probes were averaged giving 12847 unique genes. The genes were ordered with a 1-dimensional self-organizing map and then clustered using average-linkage hierarchical clustering (43). We isolated binding data for the 10223 probes confirmed to correspond to proximal promoters (3’ end no more than 500bp from the transcription start site). For genes represented by multiple probes, we selected the probe with the most significant p-value (9336 unique genes). This gave 6132 genes for which we had both binding and expression data. Binding events were scored for MLL1, Pol II and histone H3 trimethyl K4 at the p=0.001 confidence level.

ChIP Analysis
Chromatin obtained from 5 x 106 crosslinked U937 cells was used for each chromatin immunoprecipitation. ChIP analysis was adapted from (40) using rabbit IgG, Pol II, MLL1, and H3-K4 trimethyl antibodies described above. After reversal of cross links and DNA isolation, samples were subjected to PCR analysis using primers within the proximal promoter region (+1 to +250) of Meis1, HoxA11, and HoxB13. Primers against the upstream region (-750 to -500) of Actin-alpha were used as control.