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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01302

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Highly specific interactions between bHLH transcription factors and chromatin during retina development

¹ University of Geneva, Biochemistry Department, 30 quai Ernest-Ansermet, 1211 Geneva, Switzerland
² New York University School of Medicine, Department of Pathology, 550 First Avenue, New York, NY 10016, USA
³ University of Lausanne, Institute of Research in Ophthalmology and the Eye Hospital Jules Gonin, 15 avenue de France, 1004 Lausanne, Switzerland

View larger version (11K): [in a new window]   Fig. 1. Schematic representation of chromatin immunoprecipitation experiments. (A) Outline of ChIP experiment in retina and optic tectum. Dissected tissues were homogenized in the presence of formaldehyde. Crosslinked chromatin was prepared using a standard procedure, sonicated and incubated with appropriate antibodies. Specific DNA fragments in immunoprecipitates were quantified by real-time PCR. (B) Retinal ganglion cells (RGC) isolated using panning with anti-THY1 antibody. Dissociated retinal cells expressing THY1 were retained on dishes coated with anti-THY1 antibody and fixed with formaldehyde. Fixed cells were processed for ChIP as in A. (C) Schematic representation of upstream regions of the analyzed genes. The black squares are E-boxes and arrows indicate the primers used for amplification.

View larger version (24K): [in a new window]   Fig. 2. In vivo occupancy of neuronal-specific promoters by ATH5 and NGN2 as a function of developmental stage. Antibodies directed against ATH5 (A,C) and NGN2 (B) were used to immunoprecipitate crosslinked chromatin fragments prepared from E3 to E12 neuroretinas and optic tecta. Immunoprecipitates were analyzed for the abundance of ATH5 (A,B), ß3 (B,C), NeuroM (A) regulatory sequences and ß3 ORF (C) by real-time PCR. Data are normalized relative to ATH5 promoter occupancy by ATH5 protein (A,C) in E6 retina (IP efficiency: 0.08%), and for NGN2 protein (B) in E3 retina (IP efficiency: 0.08%). NR, neuroretina; OT, optic tectum. *P=0.02, Student's t-test.

View larger version (31K): [in a new window]   Fig. 3. ATH5-expressing cells in the developing chick retina. (A,B) Serial transverse sections at stage E2.5 were hybridized with an ATH5-specific antisense riboprobe. ATH5 is expressed in the central retina (gray) in a domain corresponding to 30% of the surface of the retina. (C) Cell counting reveals that 88% of the cells in the central domain express ATH5, representing 26% of the total retinal cell population. The dissociated cells of E6 retinas were hybridized with an ATH5-specific antisense riboprobe and the ration of labeled to unlabeled cells was determined.

View larger version (20K): [in a new window]   Fig. 4. Histone H3 K4 methylation on the ATH5 and ß3 promoters during retina and optic tectum development. Chromatin from E3-E18 retinas and optic tecta were immunoprecipitated with an antibody specific for the dimethylated K4 of histone H3. ATH5 and ß3 promoters sequences in the precipitates were quantified by real-time PCR. Methylation levels are shown relative to E6 retina in A for the ATH5 promoter (IP efficiency, 0.7%) and to E18 retina in B for the ß3 promoter (IP efficiency, 1.1%). *P=0.02, Student's t-test.

View larger version (19K): [in a new window]   Fig. 5. Methylation of histone H3 at NeuroM and NeuroD promoters as a function of retina and optic tectum developmental stage. Quantitative ChIP experiments were performed with an antibody specific for the dimethylated K4 of histone H3. Results are normalized relative to the value observed for E18 retina [NeuroM promoter (A), IP efficiency 1.2%; NeuroD promoter (B), IP efficiency 0.2%].

View larger version (19K): [in a new window]   Fig. 6. Histone H3 methylation monitored in isolated RGCs. (A) THY1 immunostaining of ganglion cells purified with anti-THY1 antibody. (B-E) Chromatin from purified RGCs was immunoprecipitated with an antibody directed against the dimethylated K4 of histone H3. Enrichments of ATH5 (B), ß3 (C), NeuroM (D) and NeuroD (E) promoter sequences are normalized relative to the value observed for E6 (B) or E18 (C-E) in the corresponding whole retina experiment.