Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 14 (1), R5

Tissue-specific Direct Targets of Caenorhabditis Elegans Rb/E2F Dictate Distinct Somatic and Germline Programs

Tissue-specific Direct Targets of Caenorhabditis Elegans Rb/E2F Dictate Distinct Somatic and Germline Programs

Michelle Kudron et al. Genome Biol.

Abstract

Background: The tumor suppressor Rb/E2F regulates gene expression to control differentiation in multiple tissues during development, although how it directs tissue-specific gene regulation in vivo is poorly understood.

Results: We determined the genome-wide binding profiles for Caenorhabditis elegans Rb/E2F-like components in the germline, in the intestine and broadly throughout the soma, and uncovered highly tissue-specific binding patterns and target genes. Chromatin association by LIN-35, the C. elegans ortholog of Rb, is impaired in the germline but robust in the soma, a characteristic that might govern differential effects on gene expression in the two cell types. In the intestine, LIN-35 and the heterochromatin protein HPL-2, the ortholog of Hp1, coordinately bind at many sites lacking E2F. Finally, selected direct target genes contribute to the soma-to-germline transformation of lin-35 mutants, including mes-4, a soma-specific target that promotes H3K36 methylation, and csr-1, a germline-specific target that functions in a 22G small RNA pathway.

Conclusions: In sum, identification of tissue-specific binding profiles and effector target genes reveals important insights into the mechanisms by which Rb/E2F controls distinct cell fates in vivo.

Figures

Figure 1
Figure 1
Genes with tissue-specific binding by LIN-35, EFL-1 and DPL-1 exhibit unique properties. (a) Single-gene examples of the typical binding profile for each tissue-specific dataset. The key for each factor and tissue is to the left of the tracks. One track is shown for each factor in sets corresponding to each tissue, with a control (input) sample for each tissue (black). Red, germline-specific promoter; blue, endogenous promoter; orange, intestine-specific promoter. (b) Graph showing the fraction of binding sites for each tissue-specific dataset not readily assignable to at least one nearby coding gene. (c) The fraction of candidate gene targets with germline-intrinsic or oogenesis-enriched expression based on [21] (about 0.105 of genes in the genome, marked by black line). Bars marked with an asterisk have significant over-representation (P < 1.8 × e-15 or lower; hypergeometric probability test). (d) The chromosomal distribution of candidate gene targets for each tissue-specific dataset. Statistically significant deviations from the expected value of 1 (marked with a black line) are indicated by an asterisk (P < 1.0e-05, Pearson's chi-square). (e) Gene Ontology analysis of candidate target genes in each tissue, with Gene Ontology category 'molecular process', and the extent of enrichment indicated by the bar. Up to ten categories, all with more than two-fold enrichment and a P-value < 0.05, are shown. Redundant categories were removed manually for each tissue. Full analysis available in Additional file 5.
Figure 2
Figure 2
LIN-35 exhibits reduced binding in the germline. (a) Example of binding across the entirety of chromosome I for each factor in the germline (indicated by the promoter, either Ppie-1, red, or Pmex-5, green), and for LIN-35 in the soma (Plin-35, blue) and the intestine (Pges-1, orange). Three individual genes are shown below, two of which are germline-specific and show little (left) or no (middle) LIN-35 binding, and one that is broadly bound and shows LIN-35 binding in somatic tissues and not in the germline. (b) Venn diagram comparing overlap of binding sites between Pmex-5:LIN-35:GFP and Ppie-1:EFL-1:GFP. (c) In situ hybridization of candidate germline-specific genes (par-3 and rme-2) in wild-type (wt) and lin-35 gonads. AS, antisense probe; S, sense probe. Distal tip marked by an asterisk. Scale bars = 50 μm.
Figure 3
Figure 3
Tissue-specific target genes in lin-35, efl-1, and dpl-1 mutants are differentially regulated. (a) Over-representation of tissue-specific candidate gene targets in different published microarray expression datasets. The expression datasets on the x-axis labeled E2F gonad and LIN gonad are from [11], while the 20°C data sets are from [20], and the 26°C data sets are from [10]. Statistically significant deviations are indicated by asterisks (P < 1.0e-05, hypergeometric probability test). (b) qRT-PCR results for selected germline-specific candidate target genes that were not considered regulated in the gonad microarray datasets. Fold difference of expression was compared between control (unc-4) and mutant (unc-4 dpl-1) RNA from dissected gonads, and normalized to hexokinase expression. Error bars indicate technical replicates. The dashed line indicates 1.5-fold difference. (c) qRT-PCR results for selected soma-specific (blue), intestine-specific (orange), and broadly bound (black) candidate target genes that did not show any regulation in the 20°C or 26°C L1 microarray datasets. The fold difference of expression of each gene was compared between wild-type (N2) and lin-35(n745) mutant L1s raised at 26°C, and normalized to actin (act-3) expression. Error bars indicate technical replicates. The dashed line indicates 1.5-fold difference.
Figure 4
Figure 4
mes-4 is a direct target of LIN-35/EFL-1/DPL-1 in somatic tissues. (a) Binding profile of LIN-35, EFL-1, DPL-1 and HPL-2 in multiple tissues at the mes-4 locus. (b) qRT-PCR verification of up-regulation of mes-4 transcript levels in lin-35 mutants relative to wild type. The error bar indicates standard error of biological replicates. (c) Binding profiles of EFL-1 at the mes-4 locus in the soma using the GFP antibody (blue) and in wild-type (magenta) and lin-35 (cyan) using the EFL-1 antibody. An input control for each is shown below in black.
Figure 5
Figure 5
22G small RNA pathways mediate the lin-35 larval arrest phenotype. (a) Binding profiles at loci encoding candidate small RNA pathway regulatory proteins. (b) qRT-PCR analysis of transcript levels of candidate small RNA pathway regulators in the dissected gonads of wild-type and dpl-1 mutants. Error bars indicate technical replicates. (c) qRT-PCR analysis of transcript levels of candidate small RNA pathway regulators in L1 larvae of wild-type and lin-35 mutants. Error bars indicate biological replicates. (d) Assay of the high temperature larval arrest phenotype of lin-35 mutants at 26°C upon RNAi of candidate small RNA regulators (listed on the x-axis) and empty vector control (L4440).
Figure 6
Figure 6
HPL-2 requires LIN-35 for recruitment to intestine-specific binding sites. (a) Single-gene examples of the typical binding profiles for intestine-specific targets. (b) Binding site width for each tissue-specific candidate target set. Error bars represent standard error.

Similar articles

See all similar articles

Cited by 29 PubMed Central articles

See all "Cited by" articles

References

    1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;14:646–674. doi: 10.1016/j.cell.2011.02.013. - DOI - PubMed
    1. Chen HZ, Tsai SY, Leone G. Emerging roles of E2Fs in cancer: an exit from cell cycle control. Nat Rev Cancer. 2009;14:785–797. doi: 10.1038/nrc2696. - DOI - PMC - PubMed
    1. Xu X, Bieda M, Jin VX, Rabinovich A, Oberley MJ, Green R, Farnham PJ. A comprehensive ChIP chip analysis of E2F1, E2F4, and E2F6 in normal and tumor cells reveals interchangeable roles of E2F family members. Genome Res. 2007;14:1550–1561. doi: 10.1101/gr.6783507. - DOI - PMC - PubMed
    1. Lee BK, Bhinge AA, Iyer VR. Wide-ranging functions of E2F4 in transcriptional activation and repression revealed by genome-wide analysis. Nucleic Acids Res. 2011;14:3558–3573. doi: 10.1093/nar/gkq1313. - DOI - PMC - PubMed
    1. Ceol CJ, Horvitz HR. dpl-1 DP and efl-1 E2F act with lin-35 Rb to antagonize Ras signaling in C. elegans vulval development. Mol Cell. 2001;14:461–473. doi: 10.1016/S1097-2765(01)00194-0. - DOI - PubMed

Publication types

MeSH terms

Feedback