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Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation.

Tummala P, Mali RS, Guzman E, Zhang X, Mitton KP - Mol. Vis. (2010)

Bottom Line: Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation.Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI).Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309-4401, USA.

ABSTRACT

Purpose: During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites.

Methods: High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10-12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT-PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d).

Results: ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region.

Conclusions: High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

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Determination of Pol-II active-region/gene associations and changes to gene activation state for specific genes. RNA-Polymerase-II (Pol-II) ChIP-on-chip data could be examined on any continuous scale from entire chromosomes to single genes. Determination of Pol-II active regions, the association of active regions to genes, and the calculation of specific metrics for each region, required the sequential use of TAS and the TransPath program. A: First, TAS generated interval tracks where Pol-II signal levels were above an intermediate threshold. This is illustrated for a region surrounding Pde6B, a key marker of photoreceptor terminal maturation. Second, interval track data were processed using TransPath to determine active regions. Active regions were comprised of one or more interval tracks in close proximity. Two active regions are illustrated in the P25 neural retina. B: Higher resolution view of the active region surrounding the Pde6b TSS. Signals for individual tiling probes (35 bp spacing) are visible. For each active region, TransPath calculated specific metrics, such as the Pol-II peak signal ratio (P25/P2), as illustrated by the vertical black bars indicating signal maxima from the P25 and P2 time point samples. Genes in view: Phosophodiesterase 6b (Pde6b), Polycomb group ring-finger 3 (Pcgf3), Complexin 1 (Cplx1).
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f1: Determination of Pol-II active-region/gene associations and changes to gene activation state for specific genes. RNA-Polymerase-II (Pol-II) ChIP-on-chip data could be examined on any continuous scale from entire chromosomes to single genes. Determination of Pol-II active regions, the association of active regions to genes, and the calculation of specific metrics for each region, required the sequential use of TAS and the TransPath program. A: First, TAS generated interval tracks where Pol-II signal levels were above an intermediate threshold. This is illustrated for a region surrounding Pde6B, a key marker of photoreceptor terminal maturation. Second, interval track data were processed using TransPath to determine active regions. Active regions were comprised of one or more interval tracks in close proximity. Two active regions are illustrated in the P25 neural retina. B: Higher resolution view of the active region surrounding the Pde6b TSS. Signals for individual tiling probes (35 bp spacing) are visible. For each active region, TransPath calculated specific metrics, such as the Pol-II peak signal ratio (P25/P2), as illustrated by the vertical black bars indicating signal maxima from the P25 and P2 time point samples. Genes in view: Phosophodiesterase 6b (Pde6b), Polycomb group ring-finger 3 (Pcgf3), Complexin 1 (Cplx1).

Mentions: Post inspection of the ChIP-on-chip data at internal markers of terminal maturation or any genes of interest was accomplished with the IGB program. Rho and Pde6b (Phosphodiesterase-6b) provided internal controls as genes previously shown to be inactive at P2 and active at P25 [8,9]. The TransPath program was used to correlate active regions with specific genes and then calculate metrics, such as the Pol-II peak signal ratio (P25/P2). This process is illustrated in Figure 1 for Pde6b. Pde6b (chromosome 5) encodes phosphodiesterase-6b, a rod-specific subunit of phosphodiesterase that is part of the phototransduction pathway. Mutations of human PDE6B cause retinal degeneration through the gradual loss of rod photoreceptors [10].


Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation.

Tummala P, Mali RS, Guzman E, Zhang X, Mitton KP - Mol. Vis. (2010)

Determination of Pol-II active-region/gene associations and changes to gene activation state for specific genes. RNA-Polymerase-II (Pol-II) ChIP-on-chip data could be examined on any continuous scale from entire chromosomes to single genes. Determination of Pol-II active regions, the association of active regions to genes, and the calculation of specific metrics for each region, required the sequential use of TAS and the TransPath program. A: First, TAS generated interval tracks where Pol-II signal levels were above an intermediate threshold. This is illustrated for a region surrounding Pde6B, a key marker of photoreceptor terminal maturation. Second, interval track data were processed using TransPath to determine active regions. Active regions were comprised of one or more interval tracks in close proximity. Two active regions are illustrated in the P25 neural retina. B: Higher resolution view of the active region surrounding the Pde6b TSS. Signals for individual tiling probes (35 bp spacing) are visible. For each active region, TransPath calculated specific metrics, such as the Pol-II peak signal ratio (P25/P2), as illustrated by the vertical black bars indicating signal maxima from the P25 and P2 time point samples. Genes in view: Phosophodiesterase 6b (Pde6b), Polycomb group ring-finger 3 (Pcgf3), Complexin 1 (Cplx1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2822553&req=5

f1: Determination of Pol-II active-region/gene associations and changes to gene activation state for specific genes. RNA-Polymerase-II (Pol-II) ChIP-on-chip data could be examined on any continuous scale from entire chromosomes to single genes. Determination of Pol-II active regions, the association of active regions to genes, and the calculation of specific metrics for each region, required the sequential use of TAS and the TransPath program. A: First, TAS generated interval tracks where Pol-II signal levels were above an intermediate threshold. This is illustrated for a region surrounding Pde6B, a key marker of photoreceptor terminal maturation. Second, interval track data were processed using TransPath to determine active regions. Active regions were comprised of one or more interval tracks in close proximity. Two active regions are illustrated in the P25 neural retina. B: Higher resolution view of the active region surrounding the Pde6b TSS. Signals for individual tiling probes (35 bp spacing) are visible. For each active region, TransPath calculated specific metrics, such as the Pol-II peak signal ratio (P25/P2), as illustrated by the vertical black bars indicating signal maxima from the P25 and P2 time point samples. Genes in view: Phosophodiesterase 6b (Pde6b), Polycomb group ring-finger 3 (Pcgf3), Complexin 1 (Cplx1).
Mentions: Post inspection of the ChIP-on-chip data at internal markers of terminal maturation or any genes of interest was accomplished with the IGB program. Rho and Pde6b (Phosphodiesterase-6b) provided internal controls as genes previously shown to be inactive at P2 and active at P25 [8,9]. The TransPath program was used to correlate active regions with specific genes and then calculate metrics, such as the Pol-II peak signal ratio (P25/P2). This process is illustrated in Figure 1 for Pde6b. Pde6b (chromosome 5) encodes phosphodiesterase-6b, a rod-specific subunit of phosphodiesterase that is part of the phototransduction pathway. Mutations of human PDE6B cause retinal degeneration through the gradual loss of rod photoreceptors [10].

Bottom Line: Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation.Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI).Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309-4401, USA.

ABSTRACT

Purpose: During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites.

Methods: High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10-12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT-PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d).

Results: ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region.

Conclusions: High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

Show MeSH
Related in: MedlinePlus