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Novel cis-regulatory modules control expression of the Hairy and Enhancer of Split-1 (HES1) transcription factor in myoblasts.

Jeziorska DM, Koentges G, Vance KW - J. Biol. Chem. (2011)

Bottom Line: We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions.Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus.This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Systems Analysis, School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.

ABSTRACT
The expression profile of a gene is controlled by DNA sequences called cis-regulatory modules (CRMs). CRMs can function over large genomic distances and can be located many kilobases away from their target promoters. hes1 is a key developmental gene that is overexpressed in certain cancers and is a primary target of NOTCH signaling. Despite this, analysis of hes1 transcriptional control has been limited solely to its promoter. Here, we identify seven conserved DNA sequence blocks, representing the hes1 promoter and six novel CRMs, within 57 kb upstream of the mouse hes1 gene. We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions. We validate RBP-Jκ and TEAD family occupancy in cells in culture and test the response of each of these CRMs to active NOTCH. We show that two regions, CRM5 and CRM7, function as enhancers, and four can repress transcription. A pair of RBP-Jκ motifs arranged in a tail-tail configuration in CRM5 and the M-CAT motif in CRM7 are necessary for enhancer function. Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus. This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

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Related in: MedlinePlus

Enhancer associated co-activators occupy hes1 CRM5 and CRM7 in C2C12 cells. ChIP assays were performed using antibodies against p300 (A), NICD (B), and YAP or a nonspecific control (anti-GFP) (C). Input consists of DNA extracted from sonicated nuclei. Precipitated DNA was PCR-amplified using primers spanning the indicated regions. Results are expressed as specific enrichment over input. To do this, the signal for each PCR quantified, divided by the input and the background value of an IgG isotope control, was then subtracted.
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Figure 5: Enhancer associated co-activators occupy hes1 CRM5 and CRM7 in C2C12 cells. ChIP assays were performed using antibodies against p300 (A), NICD (B), and YAP or a nonspecific control (anti-GFP) (C). Input consists of DNA extracted from sonicated nuclei. Precipitated DNA was PCR-amplified using primers spanning the indicated regions. Results are expressed as specific enrichment over input. To do this, the signal for each PCR quantified, divided by the input and the background value of an IgG isotope control, was then subtracted.

Mentions: The p300 histone acetyltransferase can be found in complex with both the RBP-Jκ and TEAD2 transcription factors (44, 45). Importantly, p300 occupancy has also been shown to mark transcriptional enhancers (46). We therefore tested for p300 binding at hes1 CRM3–5 and -7, as these regions regulated the activity of the hes1 promoter in our reporter assays (Fig. 2A). In addition, we used p300 occupancy at CRM1 as a positive control. The results in Fig. 5A demonstrate that p300 is enriched on CRM1 as well as both enhancer CRMs, CRM5 and CRM7, compared with a nonspecific control. Furthermore, CRM3 and -4, which functioned to repress the hes1 promoter in our reporter assays, showed markedly reduced levels of p300. These data are consistent with the finding that p300 occupancy can predict enhancer activity in a tissue-specific manner (47) and correlate with the transcriptional activity of these CRMs as measured using reporter assays (Fig. 2).


Novel cis-regulatory modules control expression of the Hairy and Enhancer of Split-1 (HES1) transcription factor in myoblasts.

Jeziorska DM, Koentges G, Vance KW - J. Biol. Chem. (2011)

Enhancer associated co-activators occupy hes1 CRM5 and CRM7 in C2C12 cells. ChIP assays were performed using antibodies against p300 (A), NICD (B), and YAP or a nonspecific control (anti-GFP) (C). Input consists of DNA extracted from sonicated nuclei. Precipitated DNA was PCR-amplified using primers spanning the indicated regions. Results are expressed as specific enrichment over input. To do this, the signal for each PCR quantified, divided by the input and the background value of an IgG isotope control, was then subtracted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285341&req=5

Figure 5: Enhancer associated co-activators occupy hes1 CRM5 and CRM7 in C2C12 cells. ChIP assays were performed using antibodies against p300 (A), NICD (B), and YAP or a nonspecific control (anti-GFP) (C). Input consists of DNA extracted from sonicated nuclei. Precipitated DNA was PCR-amplified using primers spanning the indicated regions. Results are expressed as specific enrichment over input. To do this, the signal for each PCR quantified, divided by the input and the background value of an IgG isotope control, was then subtracted.
Mentions: The p300 histone acetyltransferase can be found in complex with both the RBP-Jκ and TEAD2 transcription factors (44, 45). Importantly, p300 occupancy has also been shown to mark transcriptional enhancers (46). We therefore tested for p300 binding at hes1 CRM3–5 and -7, as these regions regulated the activity of the hes1 promoter in our reporter assays (Fig. 2A). In addition, we used p300 occupancy at CRM1 as a positive control. The results in Fig. 5A demonstrate that p300 is enriched on CRM1 as well as both enhancer CRMs, CRM5 and CRM7, compared with a nonspecific control. Furthermore, CRM3 and -4, which functioned to repress the hes1 promoter in our reporter assays, showed markedly reduced levels of p300. These data are consistent with the finding that p300 occupancy can predict enhancer activity in a tissue-specific manner (47) and correlate with the transcriptional activity of these CRMs as measured using reporter assays (Fig. 2).

Bottom Line: We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions.Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus.This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Systems Analysis, School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.

ABSTRACT
The expression profile of a gene is controlled by DNA sequences called cis-regulatory modules (CRMs). CRMs can function over large genomic distances and can be located many kilobases away from their target promoters. hes1 is a key developmental gene that is overexpressed in certain cancers and is a primary target of NOTCH signaling. Despite this, analysis of hes1 transcriptional control has been limited solely to its promoter. Here, we identify seven conserved DNA sequence blocks, representing the hes1 promoter and six novel CRMs, within 57 kb upstream of the mouse hes1 gene. We identify 12 binding sites for the RBP-Jκ NOTCH effector and a single M-CAT motif within these regions. We validate RBP-Jκ and TEAD family occupancy in cells in culture and test the response of each of these CRMs to active NOTCH. We show that two regions, CRM5 and CRM7, function as enhancers, and four can repress transcription. A pair of RBP-Jκ motifs arranged in a tail-tail configuration in CRM5 and the M-CAT motif in CRM7 are necessary for enhancer function. Furthermore, these enhancers are occupied by transcriptional co-activators and loop onto the hes1 promoter within the endogenous hes1 locus. This work demonstrates the power of combining computational genomics and experimental methodologies to identify novel CRMs and characterize their function.

Show MeSH
Related in: MedlinePlus