Genome-wide analysis of menin binding provides insights into MEN1 tumorigenesis

Abstract

Multiple endocrine neoplasia type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression but that the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin-targeted genes several hundred-fold beyond that previously described and provide potential insights to the endocrine tumor bias observed in MEN1 patients.

Figure 1. Strategy Used to Analyze Data from Tiled Microarrays

(A and B) Scatterplots of single-array intensities of oligonucleotides obtained from chromatin immunoprecipitation with menin antibodies (B) and a control with no antibody (A). Successful experiments are identified as those that show enrichment of multiple probes in the Cy5 channel (chromatin immoprecipitated DNA) over the Cy3 channel (total genomic DNA). (C) Histogram of mean intensity ratios (log2 scale) from menin chromatin immunoprecipitations from three biological replicates. The distinct tail at the right-hand end corresponds to DNA fragments enriched by menin-ChIP. Genomic sites enriched for factor binding are identified using a computer program called ACME. ACME first sorts probes by their genomic location, and then slides a window of user-defined size along tiled regions. ACME then tests whether each window contains a higher than expected number of oligonucletide probes above a user-defined threshold, and then assigns a p-value to each probe on the array. For all arrays used in this study, the window was set to 1,000 bp and the threshold at 90% (indicated by the red bar). (D) Plots showing data before and after processing by ACME. Points in black represent the mean intensity ratio of oligonucleotide probes (right y axis). Points in red indicate corresponding significance values for each data point reported by ACME (left y axis). True-positive signals are represented by multiple probes that cluster within a given window. Single probes that yield high intensity ratios most likely represent noise and are automatically filtered out by the windowing/threshold analysis.

Figure 2. General Pattern of Menin Occupancy in HeLa S3 Cells

(A) Tiled oligonucleotides corresponding to a random 3 MB on Chromosome 7. Four positive signals indicative of menin occupancy are denoted by the arrows and are easily distinguishable from background noise. (B) Expanded view of one positive signal from (A) reveals multiple oligos with significant p-values clustered solely at the transcriptional start site of SNX13 (Sorting Nexin 13). (C) Pattern of menin occupancy at each of four homeobox clusters.

Figure 3. Occupancy of Menin, MLL, Rbbp5 Frequently Overlaps with Trimethylation of Lys4 at Histone H3

(A) Compared to occupancy at the HOX A cluster in HeLa cells, factor occupancy in HepG2 cells and pancreatic islets is nearly absent. (B) Overlap of factors at one representative locus, ASB3 (Ankyrin repeat- and Socs Box-containing protein 3). (C) Venn diagram showing the overlap of menin-bound promoters in HeLa S3, HepG2, and pancreatic islets. Promoters included in the tally had a confidence threshold of p < 0.0001.

Figure 4. Menin Sites Can Be Bound in the Presence or Absence of HMT Complex Members

(A) Comparison of promoters bound by menin and Rbbp5 showing the broad range in signal intensity for each factor. The −log10 p-value for each site is plotted on the x and y axes. Vertical and horizontal lines represent confidence thresholds at p < 0.0001. Points in the lower left quadrant represent promoters that are bound by neither menin nor Rbbp5. Points in the upper right quadrant represent promoters occupied by both menin and Rbbp5. Plots comparing menin to MLL and H3 K4 appeared similar (unpublished data). (B and C) Heat maps illustrating the overlap of factor-occupied promoters. Promoters bound by either menin (B) or MLL (C) at the p < 0.0001 confidence threshold in HeLa S3 cells were plotted with corresponding p-values for each indicated factor. The heat maps reveal subsets of genes that are bound by all factors (blue brackets), in addition to subsets that are bound by menin or MLL but not the other factors (green brackets). (D) Real-time PCR validation of randomly selected promoters determined by ChIP-chip to be bound by menin and not Rbbp5 (left), and vice versa (right). Each pair of bars corresponds to a unique promoter region.

Figure 5. Enrichment of Selected Genomic Regions

Real-time PCR testing of genomic regions from menin (A) and H3 K4 (B) chromatin immunoprecipitations. Median values are indicated by the red bars. Enrichment values for menin are less than 1 because C_t values for the menin ChIP-PCR were higher than that for total genomic DNA (see Materials and Methods). (C) Number of promoters bound by each factor in HeLa S3 cells at various p-values. Compared to a negative control experiment in which no antibodies were used in the ChIP (protein G), significant enrichment of promoters was detected for each factor analyzed.

Figure 6. Factor Occupancy Correlates with High Gene Expression but Absence of Menin Does Not Generally Affect Gene Expression Levels

(A) Overall expression of genes for which we had both expression and binding data in HeLa S3 cells was plotted (lane 1, “All expression”) and compared to the expression of genes whose promoter regions were bound (p < 0.0001) by each of the factors indicated at the top. Asterisks denote significance (p < 0.0001) as determined by two-tailed t-test analyses between each set of factor-bound genes compared to all genes. Box plots comparing ChIP-chip and expression data from HepG2 and pancreatic islets revealed similar correlations between expression and factor occupancy (unpublished data). (B) Overall expression of genes in islets isolated from wild-type control mice (lane 1, “All Expression”) compared to those normally bound by menin in wild-type islets (lane 2) and those normally bound by menin in islets from 15-wk (lane 3) and 25-wk (lane 4) mice that are conditionally null for Men1 (genotype: RIP-cre; Men1 ^−/−).

Figure 7. Hb9 Expression Is Elevated in Islets from Conventional and Conditional Men1 Knockout Mice

(A) Pancreatic sections from an 18-mo conventional Men1 knockout mouse (Men1^{+/ −} ) were analyzed for Hb9 expression by immunohistochemistry. Brown stain corresponds to positive Hb9 signal. Compared to normal-size islets (left), an atypical hyperplastic islet (middle) and tumor (right) show increased Hb9 expression. All pictures were taken from different regions of the same section, all at the same exposure. Top panels: ×100 magnification, bottom panels: ×200 to ×400. (B) Real-time PCR analysis of Men1 and Hlxb9 expression in Men1^+/+ and Men1 ^−/− islets from conditional animals. Blue bars indicate expression relative to Gapdh; red bars, expression relative to beta-actin; +/+, average expression of five control mice (three RIP-Cre; Men1^+/+, two Men1^+/+); 15–25 wk Men1 ^−/−, average of one pool of conditional islets at 15 wk (five) and three individual preps from 25-wk Men1 ^−/− islets (RIP-cre; Men1^loxP/loxP); and tumor, average expression of five tumors from conditional Men1 knockout mice (RIP-cre; Men1^loxP/loxP). Asterisks denote statistical significance (p < 0.02) for both blue and red bars as determined by two-tailed t-tests.

Figure 8. Hypothetical Model of Menin Function

See text.