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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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Naturally occurring R271W mutation in human Pit1 affects ability of Pit1 to interact with matrin-3 rich networka, Western blot analysis showing defective interaction of Pit1R271W mutant with components of nuclear matrin-3 enriched network as well as β-catenin and Satb1. b, Biochemical extraction shows that the amount of R271W mutant protein decreased in the LIS-resistant fraction and associates instead with the looped-out DNA fraction. c, Schematic representation of Pit1 protein with added SAF domain. red “x” – location of amino-acid 271 d, Analysis of effect of overexpression of different forms of Pit1 protein in absence of endogenous Pit1; top panels: ChIP-qPCR analysis of matrin-3 association,; bottom panels: RT-qPCR analysis of transcriptional effect on selected targets. Experiments were repeated 3 or 4 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01, ***p<0.001) e, Analysis of effect of ex vivo overexpression of R271W mutant with and without SAF domain in e17.5 snell mutant pituitary glands. GH expression is rescued only when the R271W-SAF protein is overexpressed. Bottom: quantification of percentage of HA-positive GH expressing cells. Two pituitaries were used per condition; p-value calculated using χ2 test. f, Model: Association of Pit1-bound regulatory element with matrin-3 rich network and loss of this interaction based on failure of R271W mutant to interact with β-catenin and SATB1 causing loss of gene activation as in CPHD.
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Figure 4: Naturally occurring R271W mutation in human Pit1 affects ability of Pit1 to interact with matrin-3 rich networka, Western blot analysis showing defective interaction of Pit1R271W mutant with components of nuclear matrin-3 enriched network as well as β-catenin and Satb1. b, Biochemical extraction shows that the amount of R271W mutant protein decreased in the LIS-resistant fraction and associates instead with the looped-out DNA fraction. c, Schematic representation of Pit1 protein with added SAF domain. red “x” – location of amino-acid 271 d, Analysis of effect of overexpression of different forms of Pit1 protein in absence of endogenous Pit1; top panels: ChIP-qPCR analysis of matrin-3 association,; bottom panels: RT-qPCR analysis of transcriptional effect on selected targets. Experiments were repeated 3 or 4 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01, ***p<0.001) e, Analysis of effect of ex vivo overexpression of R271W mutant with and without SAF domain in e17.5 snell mutant pituitary glands. GH expression is rescued only when the R271W-SAF protein is overexpressed. Bottom: quantification of percentage of HA-positive GH expressing cells. Two pituitaries were used per condition; p-value calculated using χ2 test. f, Model: Association of Pit1-bound regulatory element with matrin-3 rich network and loss of this interaction based on failure of R271W mutant to interact with β-catenin and SATB1 causing loss of gene activation as in CPHD.

Mentions: Naturally occurring mutations in Pit1 underlie number of combined pituitary hormone deficiency (CPHD) cases in humans, with the R271 residue being the most frequently mutated9. An amino acid 271 maps to the last turn of helix3 in the homeodomain and is located outside the DNA binding surface. While the R271W mutation in Pit1 confers dominant-negative properties (Fig. S4a)9,, it does not affect protein stability or binding to its cognate DNA sites (Fig. S4b–d)9. We therefore investigated whether the R271W mutation affects the composition of the Pit1 complex. Co-IP of recombinant HA-tagged Pit1 proteins followed by Western blot analysis revealed that the R271W mutation disrupted the ability of Pit1 to interact with matrin3 and hnRNPU, apparently due to its inability to bind β-catenin and Satb1 (Fig. 4a). Additionally, biochemical fractionation indicated that the R271W mutant is largely lost from the insoluble fraction (Fig. 4b), although both, WT and R271W proteins were expressed at similar levels and found entirely localized to the nucleus (Fig. S4e). Instead R271W was detected in the ‘looped-out DNA’ fraction (Fig. 4b), suggesting that it had lost its ability to interact with the LIS resistant fraction.


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)

Naturally occurring R271W mutation in human Pit1 affects ability of Pit1 to interact with matrin-3 rich networka, Western blot analysis showing defective interaction of Pit1R271W mutant with components of nuclear matrin-3 enriched network as well as β-catenin and Satb1. b, Biochemical extraction shows that the amount of R271W mutant protein decreased in the LIS-resistant fraction and associates instead with the looped-out DNA fraction. c, Schematic representation of Pit1 protein with added SAF domain. red “x” – location of amino-acid 271 d, Analysis of effect of overexpression of different forms of Pit1 protein in absence of endogenous Pit1; top panels: ChIP-qPCR analysis of matrin-3 association,; bottom panels: RT-qPCR analysis of transcriptional effect on selected targets. Experiments were repeated 3 or 4 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01, ***p<0.001) e, Analysis of effect of ex vivo overexpression of R271W mutant with and without SAF domain in e17.5 snell mutant pituitary glands. GH expression is rescued only when the R271W-SAF protein is overexpressed. Bottom: quantification of percentage of HA-positive GH expressing cells. Two pituitaries were used per condition; p-value calculated using χ2 test. f, Model: Association of Pit1-bound regulatory element with matrin-3 rich network and loss of this interaction based on failure of R271W mutant to interact with β-catenin and SATB1 causing loss of gene activation as in CPHD.
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Related In: Results  -  Collection

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Figure 4: Naturally occurring R271W mutation in human Pit1 affects ability of Pit1 to interact with matrin-3 rich networka, Western blot analysis showing defective interaction of Pit1R271W mutant with components of nuclear matrin-3 enriched network as well as β-catenin and Satb1. b, Biochemical extraction shows that the amount of R271W mutant protein decreased in the LIS-resistant fraction and associates instead with the looped-out DNA fraction. c, Schematic representation of Pit1 protein with added SAF domain. red “x” – location of amino-acid 271 d, Analysis of effect of overexpression of different forms of Pit1 protein in absence of endogenous Pit1; top panels: ChIP-qPCR analysis of matrin-3 association,; bottom panels: RT-qPCR analysis of transcriptional effect on selected targets. Experiments were repeated 3 or 4 times, and p-values calculated using student’s two tailed t-test. (+/− SD; **p<0.01, ***p<0.001) e, Analysis of effect of ex vivo overexpression of R271W mutant with and without SAF domain in e17.5 snell mutant pituitary glands. GH expression is rescued only when the R271W-SAF protein is overexpressed. Bottom: quantification of percentage of HA-positive GH expressing cells. Two pituitaries were used per condition; p-value calculated using χ2 test. f, Model: Association of Pit1-bound regulatory element with matrin-3 rich network and loss of this interaction based on failure of R271W mutant to interact with β-catenin and SATB1 causing loss of gene activation as in CPHD.
Mentions: Naturally occurring mutations in Pit1 underlie number of combined pituitary hormone deficiency (CPHD) cases in humans, with the R271 residue being the most frequently mutated9. An amino acid 271 maps to the last turn of helix3 in the homeodomain and is located outside the DNA binding surface. While the R271W mutation in Pit1 confers dominant-negative properties (Fig. S4a)9,, it does not affect protein stability or binding to its cognate DNA sites (Fig. S4b–d)9. We therefore investigated whether the R271W mutation affects the composition of the Pit1 complex. Co-IP of recombinant HA-tagged Pit1 proteins followed by Western blot analysis revealed that the R271W mutation disrupted the ability of Pit1 to interact with matrin3 and hnRNPU, apparently due to its inability to bind β-catenin and Satb1 (Fig. 4a). Additionally, biochemical fractionation indicated that the R271W mutant is largely lost from the insoluble fraction (Fig. 4b), although both, WT and R271W proteins were expressed at similar levels and found entirely localized to the nucleus (Fig. S4e). Instead R271W was detected in the ‘looped-out DNA’ fraction (Fig. 4b), suggesting that it had lost its ability to interact with the LIS resistant fraction.

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