Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells - PubMed (original) (raw)

Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells

Daphne Georlette et al. Genes Dev. 2007.

Abstract

Myb-MuvB (MMB)/dREAM is a nine-subunit complex first described in Drosophila as a repressor of transcription, dependent on E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent on DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNA interference and microarray expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupied 3538 chromosomal sites and was promoter-proximal to 32% of Drosophila genes. MMB contains multiple DNA-binding factors, and the data highlighted the combinatorial way by which the complex was targeted and utilized for regulation. Interestingly, only a subset of chromatin-bound complexes repressed genes normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression, whereas at other nonoverlapping sites, Myb was critical for repression. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants.

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Figures

Figure 1.

Cells depleted for MMB over a long period of time exhibit cell cycle progression defects and genome instability. (A) Accumulation of individual MMB complex members is dependent on multiple protein interactions. Shown is a schematic diagram of the accumulation of each protein after RNAi treatment. Arrow type depicts the amount that each protein was reduced in the absence of the indicated complex member, as follows: (solid) <25% of control RNAi levels; (large dash) 25%–50% of control RNAi levels; (small dash) 50%–75% of control RNAi levels. Asterisks denote proteins for which the immunoblot quantification was not performed (Lin-52 and DP). (B) Kc cells were subjected to three consecutive RNAi treatments (over 14 d) to deplete MMB (Myb, Mip120, and Mip130, see inset), as denoted by red asterisks. RNAi with a nonspecific RNA derived from a pBSK+ (named SK+) was used as control. Cells were diluted at the end of each RNAi treatment into fresh media containing a new aliquot of dsRNA (the reason for the drop in cell count at each RNAi point). Cell growth (expressed as the number of cells per milliliter, solid line) and protein expression (dashed line) were monitored at specific time points. Times at which the samples were collected for microarray expression, FACS, and metaphase spread analyses are indicated by black arrows. (C) MMB depletion results in the accumulation of abnormal chromosomes. Shown are representative metaphase spreads from Kc cells treated using RNAi for individual MMB members and stained with DAPI. Kc cells treated with SK–dsRNA were used as control. (Left panel) (WT) control metaphase spread exhibiting a normal tetraploid female karyotype; (ISCC) impaired sister chromatid cohesion; (F/CD) fragmentation/condensation defects; (P) polyploidy. (Right panel) Fifty metaphase spreads were counted after 14 d of RNAi treatment as described above (see inset for targeted protein) and analyzed for chromosomal defects (ISCC, F/CD, and P). The results are representative of three independent experiments. The most common abnormality was ISCC–F/CD for loss of Myb, Mip120, and Mip130, whereas those defects were less severe upon the loss of E2F2.

Figure 2.

MMB represses or activates gene expression. (Top) Venn diagram showing the classes of genes repressed by MMB. Three classes of genes were thoroughly analyzed: genes repressed by the two core proteins (Mip120 and Mip130), E2F2, RBF1, and RBF2 (RBFs) but not Myb (Class A); genes repressed by the two core proteins (Mip120 and Mip130) and Myb but not E2F2 (Class B); and genes repressed by only Mip120 and Mip130 without either E2F2 or Myb (Class C). Each of these classes were further subdivided according to the requirement of the additional MMB members Mip40, Lin-52, both RBFs, and L(3)MBT (see pie charts on the right side of the figure). Numbers in parentheses refer to the number of genes found in each class. (Bottom) Venn diagram representing the classes of genes activated by MMB. The genes activated by Mip120, Mip130, and Myb belong to Class D. This class was further subdivided according to the requirement of the additional MMB members Mip40 and Lin-52 (see pie chart on the right side of the figure). A detailed list of all the genes regulated by MMB (A–R in the Venn diagrams) is given in Supplementary Table 1.

Figure 3.

The MMB complex is involved in both transcriptional repression and activation. RNAi was used to specifically eliminate members of the MMB complex as indicated on the top. Total RNA was isolated from Kc cells treated with dsRNA for 4 d and analyzed by Northern blot for the abundance of the MMB-regulated transcripts using probes specific for the genes indicated on the right (Classes A to D). (nsRNA) Nonspecific RNAi (SK control). GADPH was used as a loading control (bottom).

Figure 4.

All five MMB members are colocalized throughout the Drosophila genome. DNA segments bound by MMB members were isolated using ChIP and identified using high-resolution tiling microarrays containing >3 million probe pairs of 25 nt in length spanning the entire nonrepeat portion of the Drosophila genome (see Materials and Methods for details). (A) Illustration of the colocalization of the MMB members on a 2.5-Mb portion of chromosome X, as viewed in the Affymetrix Integrated Genome Browser (IGB, Affymetrix). Each vertical line shows the enrichment for the binding of the selected MMB member (Lin-52, pink; Mip130, red; Mip120, green; Myb, blue; and E2F2, yellow). The indicated annotations (white) are from RefSeq. Annotations above the chromosome X coordinates are transcribed left to right, and annotations below the coordinates are transcribed right to left. (B) Distribution of the distance between bound probes and the closest TSS from Flybase (14,395 genes) for Myb (black), Mip120 (red), Mip130 (green), E2F2 (blue), and Lin-52 (turquoise). The null distribution of the distance between all probes and the closest TSS are shown as a black dotted line. (C) Chart showing the percentage of regions bound by all MMB members (Myb, Mip120, Mip130, E2F2, and Lin-52), as well as by subclasses of MMB members (one to four MMB members). A detailed list of these different categories can be found in Supplementary Table 2. The class where all members are bound is subdivided into four subclasses based on the value of the lrpeak; i.e., the smoothed [log2 ratio (ChIP sample/input)] (inset of the pie chart): lrpeak of all members >2 (i), lrpeak of all members >2 except Myb (2 > lrpeak > 0.5) (ii), lrpeak of all members >2 except E2F2 (2 > lrpeak > 0.5) (iii), and other possibilities with (2 > lrpeak Myb and E2F2 > 0.5) (iv).

Figure 5.

MMB is primarily targeted to DNA as a complex. The main population of the regions called “bound” based on our statistics criteria are enriched for the entire MMB complex; i.e., Myb, Mip120, Mip130, E2F2, and Lin-52 (3538 sites, Category A). This class was subdivided into four subclasses based on the value of the lrpeak; i.e., the smoothed [log2 ratio (ChIP sample/input)]: 997 sites where lrpeak of all members >2 (A1), 501 sites where lrpeak of all members >2 except Myb (2 > lrpeak > 0.5) (A2), 429 sites where lrpeak of all members >2 except E2F2 (2 > lrpeak > 0.5) (A3), and 1611 remaining sites (other) where 2 > lrpeak Myb and E2F2 > 0.5 (A4). The number of sites bound by MMB subcomplexes was much smaller compared with that of sites bound by the entire complex (Categories B–E). This behavior was independent of the _q_-value used in the statistical analysis (see Supplementary Table 2 for details).

Figure 6.

DNA binding by individual MMB subunits varies by the class of genes regulated by MMB. (Top) Shown are the locations of individual MMB subunits (Myb, Mip120, Mip130, E2F2, and Lin-52) for four representative MMB-regulated genes [the Class A gene CG13084 (A); the Class B gene fs(1)N (B); the Class C gene CG6737 (C), and the Class D gene squ (CG4711) (D)], as viewed in the Affymetrix IGB following ChIP–chip analysis. The white signals show the location of enrichment for the MMB member indicated on the left. (A–D, bottom) ChIPs were performed using antibodies against members of the MMB complex, and the isolated DNA was analyzed by qPCR for the presence of promoter fragments derived from each class of the regulated genes. The relative fold enrichment for each ChIP was calculated by determining the ratio of intensities of the experimental regions to the actin promoter. For each promoter, the mock ChIP (NS) was normalized to a value of 1 (see Materials and Methods for details). The results are representative of three independent experiments.

Figure 7.

E2F2 and Myb behave as mutually exclusive targeting factors for MMB. (A) A single MMB complex regulates gene expression in Drosophila Kc cells. Graphical representations of the genomic region surrounding the Class A qtc (left) and Class B CG31100 (right) loci are on the top. The location of the qtc and CG3100 qPCR products, labeled a–g, are shown as boxes on the top. Black arrows indicate the start and direction of transcription. Histograms displaying the qPCR results following ChIP for the proteins indicated in each inset box are summarized at the bottom. The relative fold enrichment for each ChIP was calculated by determining the ratio of intensities of the experimental regions to the actin promoter following qPCR. The mock ChIP data (NS) was normalized to a value of 1. Shown are the results and standard deviations for three independent experiments for each region. For both genes, the qPCR data were in agreement with the MMB location determined by ChIP–chip. (B) E2F2 and Myb are mutually exclusive for targeting MMB. ChIP was performed on chromatin isolated from E2F2- and Myb-depleted Kc cells following RNAi treatment using the antibodies against MMB members indicated on the bottom of each panel. qPCR was performed for the Class A qtc (left) and Class B CG31100 (right) promoters for the PCR products labeled “d” in each case. The relative fold enrichment for each ChIP was calculated by determining the ratio of intensities of the experimental regions to the actin promoter following qPCR. The mock ChIP data (NS) was normalized to a value of 1. Shown are the results and standard deviations for three independent experiments. MMB association (as measured by Mip120 and Mip130 binding) was decreased at the qtc promoter when E2F2 was removed (left) and at the CG31100 promoter when Myb was removed (right), demonstrating that E2F2 and Myb are indeed responsible for MMB targeting at Class A and Class B gene promoters, respectively.

Figure 8.

Analysis of promoter regions bound by MMB for the enrichment of E2F and Myb DNA-binding sites. (A) The MEME program identified conserved E2F sites at the Class A promoters (left) and Myb sites at the Class B promoters (right). Motifs were constructed using WebLogo (

http://weblogo.berkeley.edu

). The height of a letter represents the relative frequency of occurrence. Exact locations of the regulatory motifs of the analyzed regions are given in Supplementary Table 4. (B) Analysis of the overrepresentation of E2F (TTSSSSS) and Myb (YAACKG) consensus binding sites among the promoters of genes bound by MMB. Flybase genes (14,395) were scanned for both consensus sites within 1 kb upstream of their TSS, and the average occurrence of the studied motif was used as a population mean (3.5 and 1.7 for E2F and Myb sites, respectively). A one-sample _t_-test was run to ask whether the true average in the sample was higher than the population mean. Promoters of Class A genes are significantly enriched for E2F sites (left), whereas promoters of Class B and D genes are significantly enriched for Myb sites (right). Occurrence of E2F site is also higher at the promoters strongly bound by MMB, but Myb—while a Myb site—is overrepresented at the promoters strongly bound by MMB but E2F2 (see Materials and Methods for details). Data are given in Supplementary Table 4.

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Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells - PubMed (original) (raw)