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Required enhancer-matrin-3 network interactions for a homeodomain transcription program.

Skowronska-Krawczyk D, Ma Q, Schwartz M, Scully K, Li W, Liu Z, Taylor H, Tollkuhn J, Ohgi KA, Notani D, Kohwi Y, Kohwi-Shigematsu T, Rosenfeld MG - Nature (2014)

Bottom Line: Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program.The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation.These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

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Co-localization of Pit1 and matrin-3 on enhancersa, ChIP-Seq analysis of Pit1 and H3K4me2 genome-wide co-localization in GC rat pituitary somatotrope cell line. b, Most DNA sites co-bound by Pit1 and H3K4me2 lie outside gene proximal promoters. c, HA-tagged Pit1 immunoprecipitated from 293-T cells. Co-purifying factors identified with mass spectrometry. d, Co-immunoprecipitation of Pit1 followed by Western blot analysis confirmed Pit1:endogenous matrin 3 and hnRNPU interactions in GC cells. e, Most matrin-3/H3K4me2 sites in GC cells lie outside gene proximal promoters. f, Heat map of ChIP-Seq data on non-promoter genome-wide association of Pit1, H3K4me2, H3K4me1, H3K27Ac, matrin-3 in GC cells centered on Pit1 sites and categorized as transcription termination sites (TTS), intergenic, intronic sites. g, Meta-analysis of matrin-3 ChIP-seq data h, Examples of immuno-FISH experiments showing matrin3 spots (red) colocalized with GH locus spots (green) in GH-expressing GC cells. Chart represents count of percentage of signals exhibiting co-localization, n≥200, +/− SD.
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Figure 1: Co-localization of Pit1 and matrin-3 on enhancersa, ChIP-Seq analysis of Pit1 and H3K4me2 genome-wide co-localization in GC rat pituitary somatotrope cell line. b, Most DNA sites co-bound by Pit1 and H3K4me2 lie outside gene proximal promoters. c, HA-tagged Pit1 immunoprecipitated from 293-T cells. Co-purifying factors identified with mass spectrometry. d, Co-immunoprecipitation of Pit1 followed by Western blot analysis confirmed Pit1:endogenous matrin 3 and hnRNPU interactions in GC cells. e, Most matrin-3/H3K4me2 sites in GC cells lie outside gene proximal promoters. f, Heat map of ChIP-Seq data on non-promoter genome-wide association of Pit1, H3K4me2, H3K4me1, H3K27Ac, matrin-3 in GC cells centered on Pit1 sites and categorized as transcription termination sites (TTS), intergenic, intronic sites. g, Meta-analysis of matrin-3 ChIP-seq data h, Examples of immuno-FISH experiments showing matrin3 spots (red) colocalized with GH locus spots (green) in GH-expressing GC cells. Chart represents count of percentage of signals exhibiting co-localization, n≥200, +/− SD.

Mentions: During pituitary development, the POU-homeodomain transcription factor, Pit1, is necessary for differentiation of thyrotrope, lactotrope, and somatotrope cell types in both mice and humans10,11. To further understand the molecular basis for Pit1-mediated gene activation, we mapped the genomic localization of Pit1 by ChIP-seq using specific Pit-1 antibody (Fig. S1a, b) in a growth hormone (GH)-expressing rat pituitary cell line (GC). Of 14,466 Pit1 binding sites identified, >80% overlapped with H3K4me2 histone marks but not with transcription start sites, indicative of enhancer elements5 (Fig. 1a, b, Supplementary Table 1).


Required enhancer-matrin-3 network interactions for a homeodomain transcription program.

Skowronska-Krawczyk D, Ma Q, Schwartz M, Scully K, Li W, Liu Z, Taylor H, Tollkuhn J, Ohgi KA, Notani D, Kohwi Y, Kohwi-Shigematsu T, Rosenfeld MG - Nature (2014)

Co-localization of Pit1 and matrin-3 on enhancersa, ChIP-Seq analysis of Pit1 and H3K4me2 genome-wide co-localization in GC rat pituitary somatotrope cell line. b, Most DNA sites co-bound by Pit1 and H3K4me2 lie outside gene proximal promoters. c, HA-tagged Pit1 immunoprecipitated from 293-T cells. Co-purifying factors identified with mass spectrometry. d, Co-immunoprecipitation of Pit1 followed by Western blot analysis confirmed Pit1:endogenous matrin 3 and hnRNPU interactions in GC cells. e, Most matrin-3/H3K4me2 sites in GC cells lie outside gene proximal promoters. f, Heat map of ChIP-Seq data on non-promoter genome-wide association of Pit1, H3K4me2, H3K4me1, H3K27Ac, matrin-3 in GC cells centered on Pit1 sites and categorized as transcription termination sites (TTS), intergenic, intronic sites. g, Meta-analysis of matrin-3 ChIP-seq data h, Examples of immuno-FISH experiments showing matrin3 spots (red) colocalized with GH locus spots (green) in GH-expressing GC cells. Chart represents count of percentage of signals exhibiting co-localization, n≥200, +/− SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358797&req=5

Figure 1: Co-localization of Pit1 and matrin-3 on enhancersa, ChIP-Seq analysis of Pit1 and H3K4me2 genome-wide co-localization in GC rat pituitary somatotrope cell line. b, Most DNA sites co-bound by Pit1 and H3K4me2 lie outside gene proximal promoters. c, HA-tagged Pit1 immunoprecipitated from 293-T cells. Co-purifying factors identified with mass spectrometry. d, Co-immunoprecipitation of Pit1 followed by Western blot analysis confirmed Pit1:endogenous matrin 3 and hnRNPU interactions in GC cells. e, Most matrin-3/H3K4me2 sites in GC cells lie outside gene proximal promoters. f, Heat map of ChIP-Seq data on non-promoter genome-wide association of Pit1, H3K4me2, H3K4me1, H3K27Ac, matrin-3 in GC cells centered on Pit1 sites and categorized as transcription termination sites (TTS), intergenic, intronic sites. g, Meta-analysis of matrin-3 ChIP-seq data h, Examples of immuno-FISH experiments showing matrin3 spots (red) colocalized with GH locus spots (green) in GH-expressing GC cells. Chart represents count of percentage of signals exhibiting co-localization, n≥200, +/− SD.
Mentions: During pituitary development, the POU-homeodomain transcription factor, Pit1, is necessary for differentiation of thyrotrope, lactotrope, and somatotrope cell types in both mice and humans10,11. To further understand the molecular basis for Pit1-mediated gene activation, we mapped the genomic localization of Pit1 by ChIP-seq using specific Pit-1 antibody (Fig. S1a, b) in a growth hormone (GH)-expressing rat pituitary cell line (GC). Of 14,466 Pit1 binding sites identified, >80% overlapped with H3K4me2 histone marks but not with transcription start sites, indicative of enhancer elements5 (Fig. 1a, b, Supplementary Table 1).

Bottom Line: Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program.The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation.These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

Show MeSH
Related in: MedlinePlus