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Research paper thumbnail of Phosphotryptic Peptide Analysis of Human Progesterone Receptors

Journal of Biological Chemistry, 1989

Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding fo... more Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding forms of 2120 kDa (B-receptors) and 94 kDa (A-receptors). Both are phosphorylated in hormone-untreated T47D,, breast cancer cells. Hormone treatment leads to receptor transformation and an increased phosphorylation state: the "P-labeling intensity is 3-5 times higher after progestin treatment and 8-10 times higher after RU 486 treatment. Only serine residues are phosphorylated. To determine whether there are unique phosphorylation sites in transformed nuclear PR, we analyzed the phosphopeptides of untransformed and transformed Aand B-receptors by tryptic cleavage and reverse-phase high pressure liquid chromatography. Untransformed Aand B-receptors share at least five common phosphopeptides, and a sixth is unique to B. Following transformation by either R5020 or RU 486, A-receptors generate at least six and B-receptors seven phosphopeptides. Compared with untransformed PR, there are at least two different phosphopeptides in transformed nuclear PR. Cyanogen bromide cleavage of transformed nuclear A-receptors, which lack the proximal 165 amino-terminal residues of the 933 amino acid B-receptors, produces two large fragments of 243 and 19 kDa. These fragments contain all of the 32P label and comprise amino acids 165-595. Cleavage of transformed B-receptors also produces peptides of 4 3 and 19 kDa plus an additional 36-kDa fragment corresponding to residues 1-165. No 32P-labeled low molecular mass peptides are detected. Thus, all the hormone-dependent phosphoserine residues produced in nuclei are located in the first 595 amino acids of human PR, representing the amino terminus and 28 residues of the DNA-binding domain.

Research paper thumbnail of Synthesis of Human Progesterone Receptors in T47D Cells

Journal of Biological Chemistry, 1989

Human progesterone receptors (PR) are structurally complex. At basal states there are two forms: ... more Human progesterone receptors (PR) are structurally complex. At basal states there are two forms: A-receptors of ~9 4 kDa and B-receptors which are triplets of ~1 1 4 , 117, and 120 kDa. All the proteins bind hormone and are phosphorylated. By using PR-rich T47D,, human breast cancer cells, pulse-labeling with [36S]methionine, and receptor immunopurification with anti-PR monoclonal antibodies, we show that PR are synthesized as single B-proteins of 114 kDa and single A-proteins of 94 kDa. The mature B-triplets form 6-10 h later by post-translational phosphorylation at sites restricted to the B-proteins. This slow maturation is not required for PR activation to hormone binding states, however, since Aand B-receptors that are less than 15 min old respond to progestins by undergoing transformation and nuclear binding accompanied by a rapid secondary phosphorylation common to both proteins. These studies explain the complex structure of the mature human B-receptors and the transformed Aand B-receptors, and address issues dealing with Aand B-proreceptor synthesis and receptor activation rates.

Research paper thumbnail of Human Progesterone Receptor Transformation and Nuclear Down-Regulation are Independent of Phosphorylation

Molecular Endocrinology, 1988

We have studied the phosphorylation of progesterone receptors (PR) in T47D CO human breast cancer... more We have studied the phosphorylation of progesterone receptors (PR) in T47D CO human breast cancer cells using a monoclonal antibody directed against human PR called AB-52. This antibody recognizes both the A-(M, ~ 94,000) and B-(M r ~ 120,000) hormone binding proteins of PR, and was used to immunoprecipitate phosphorylated receptors isolated from cells incubated in vivo with [ 32 P]orthophosphate. The specific activity, or phosphorylation levels, relative to protein levels was quantified by combined immunoblotting and autoradiography followed by densitometry. We find that immunopurified untransformed hormone-free receptors, which have a characteristic triplet B, singlet A structure, are phosphoproteins with similar levels of phosphate incorporation in all protein bands. If PR are first transformed to the nuclear binding form by treatment of cells with progesterone, and then labeled with [ 32 P]orthophosphate, the receptor proteins are additionally phosphorylated. These chromatin-bound hormone occupied receptors incorporate five to 10 times more labeled phosphate per total receptor protein than do PR from untreated cells during the same [ 32 P]incubation time. The second round of phosphorylation may also account for mobility shifts of transformed A-and B-receptors observed in sodium dodecyl sulfate-polyacrylamide gels. Both untransformed and transformed species of A-and Breceptors are phosphorylated only on serine residues, and neither the extent of phosphorylation, nor the phosphoamino acids, are affected by treatment of the cells with epidermal growth factor or insulin. We previously reported that after hormone binding and transformation of receptors to the tight chromatin binding state, PR undergo processing, or nuclear down-regulation. AB-52 was used to compare PR protein and phosphorylation levels when cells were treated for 0.5-48 h with progesterone or the synthetic progestin R5020.

Research paper thumbnail of Distinct roles for P-CREB and LEF-1 in TCR alpha enhancer assembly and activation on chromatin templates in vitro

Genes & Development, 1997

The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studi... more The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Here we have reconstituted regulated TCR alpha enhancer activity in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB. When added in combination, these factors activate the TCR alpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhanced transcription 10-fold in vitro, and this effect was abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhanced the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant contai...

Research paper thumbnail of NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro

Genes & Development, 1996

NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T ce... more NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T cells. In this report we examined the ability of different subunits of NF-kappa B to enhance HIV-1 transcription in vitro with chromatin templates. We find that the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by re...

Research paper thumbnail of Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro

Genes & Development, 1995

Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that... more Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. In this paper we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. First, we show that mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nucleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and that repression can be counteracted efficiently by preincubation of ...

Research paper thumbnail of Histone Acetyltransferases Regulate HIV-1 Enhancer Activity In Vitro

Genes & …, 1997

Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are ... more Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are potent inducers of HIV-1 transcription in latently infected T-cell lines. Activation of the integrated HIV-1 promoter is accompanied by the loss or rearrangement of a positioned nucleosome (nuc-1) near the viral RNA start site. Here we show that TSA strongly induces HIV-1 transcription on chromatin in vitro, concomitant with an enhancer factor-assisted increase in the level of acetylated histone H4. TSA treatment, however, did not detectably alter enhancer factor binding or the positioning of nuc-1 on the majority of the chromatin templates indicating that protein acetylation and chromatin remodeling may be limiting steps that occur only on transcriptionally competent templates, or that remodeling of nuc-1 requires additional factors. To assess the number of active chromatin templates in vitro, transcription was limited to a single round with low levels of the detergent Sarkosyl. Remarkably, HIV-1 transcription on chromatin was found to arise from a small number of active templates that can each support nearly 100 rounds of transcription, and TSA increased the number of active templates in each round. In contrast, transcription on naked DNA was limited to only a few rounds and was not responsive to TSA. We conclude that HIV-1 enhancer complexes greatly facilitate transcription reinitiation on chromatin in vitro, and act at a limiting step to promote the acetylation of histones or other transcription factors required for HIV-1 enhancer activity.

Research paper thumbnail of Phosphotryptic Peptide Analysis of Human Progesterone Receptors

Journal of Biological Chemistry, 1989

Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding fo... more Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding forms of 2120 kDa (B-receptors) and 94 kDa (A-receptors). Both are phosphorylated in hormone-untreated T47D,, breast cancer cells. Hormone treatment leads to receptor transformation and an increased phosphorylation state: the "P-labeling intensity is 3-5 times higher after progestin treatment and 8-10 times higher after RU 486 treatment. Only serine residues are phosphorylated. To determine whether there are unique phosphorylation sites in transformed nuclear PR, we analyzed the phosphopeptides of untransformed and transformed Aand B-receptors by tryptic cleavage and reverse-phase high pressure liquid chromatography. Untransformed Aand B-receptors share at least five common phosphopeptides, and a sixth is unique to B. Following transformation by either R5020 or RU 486, A-receptors generate at least six and B-receptors seven phosphopeptides. Compared with untransformed PR, there are at least two different phosphopeptides in transformed nuclear PR. Cyanogen bromide cleavage of transformed nuclear A-receptors, which lack the proximal 165 amino-terminal residues of the 933 amino acid B-receptors, produces two large fragments of 243 and 19 kDa. These fragments contain all of the 32P label and comprise amino acids 165-595. Cleavage of transformed B-receptors also produces peptides of 4 3 and 19 kDa plus an additional 36-kDa fragment corresponding to residues 1-165. No 32P-labeled low molecular mass peptides are detected. Thus, all the hormone-dependent phosphoserine residues produced in nuclei are located in the first 595 amino acids of human PR, representing the amino terminus and 28 residues of the DNA-binding domain.

Research paper thumbnail of Synthesis of Human Progesterone Receptors in T47D Cells

Journal of Biological Chemistry, 1989

Human progesterone receptors (PR) are structurally complex. At basal states there are two forms: ... more Human progesterone receptors (PR) are structurally complex. At basal states there are two forms: A-receptors of ~9 4 kDa and B-receptors which are triplets of ~1 1 4 , 117, and 120 kDa. All the proteins bind hormone and are phosphorylated. By using PR-rich T47D,, human breast cancer cells, pulse-labeling with [36S]methionine, and receptor immunopurification with anti-PR monoclonal antibodies, we show that PR are synthesized as single B-proteins of 114 kDa and single A-proteins of 94 kDa. The mature B-triplets form 6-10 h later by post-translational phosphorylation at sites restricted to the B-proteins. This slow maturation is not required for PR activation to hormone binding states, however, since Aand B-receptors that are less than 15 min old respond to progestins by undergoing transformation and nuclear binding accompanied by a rapid secondary phosphorylation common to both proteins. These studies explain the complex structure of the mature human B-receptors and the transformed Aand B-receptors, and address issues dealing with Aand B-proreceptor synthesis and receptor activation rates.

Research paper thumbnail of Human Progesterone Receptor Transformation and Nuclear Down-Regulation are Independent of Phosphorylation

Molecular Endocrinology, 1988

We have studied the phosphorylation of progesterone receptors (PR) in T47D CO human breast cancer... more We have studied the phosphorylation of progesterone receptors (PR) in T47D CO human breast cancer cells using a monoclonal antibody directed against human PR called AB-52. This antibody recognizes both the A-(M, ~ 94,000) and B-(M r ~ 120,000) hormone binding proteins of PR, and was used to immunoprecipitate phosphorylated receptors isolated from cells incubated in vivo with [ 32 P]orthophosphate. The specific activity, or phosphorylation levels, relative to protein levels was quantified by combined immunoblotting and autoradiography followed by densitometry. We find that immunopurified untransformed hormone-free receptors, which have a characteristic triplet B, singlet A structure, are phosphoproteins with similar levels of phosphate incorporation in all protein bands. If PR are first transformed to the nuclear binding form by treatment of cells with progesterone, and then labeled with [ 32 P]orthophosphate, the receptor proteins are additionally phosphorylated. These chromatin-bound hormone occupied receptors incorporate five to 10 times more labeled phosphate per total receptor protein than do PR from untreated cells during the same [ 32 P]incubation time. The second round of phosphorylation may also account for mobility shifts of transformed A-and B-receptors observed in sodium dodecyl sulfate-polyacrylamide gels. Both untransformed and transformed species of A-and Breceptors are phosphorylated only on serine residues, and neither the extent of phosphorylation, nor the phosphoamino acids, are affected by treatment of the cells with epidermal growth factor or insulin. We previously reported that after hormone binding and transformation of receptors to the tight chromatin binding state, PR undergo processing, or nuclear down-regulation. AB-52 was used to compare PR protein and phosphorylation levels when cells were treated for 0.5-48 h with progesterone or the synthetic progestin R5020.

Research paper thumbnail of Distinct roles for P-CREB and LEF-1 in TCR alpha enhancer assembly and activation on chromatin templates in vitro

Genes & Development, 1997

The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studi... more The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Here we have reconstituted regulated TCR alpha enhancer activity in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB. When added in combination, these factors activate the TCR alpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhanced transcription 10-fold in vitro, and this effect was abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhanced the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant contai...

Research paper thumbnail of NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro

Genes & Development, 1996

NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T ce... more NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T cells. In this report we examined the ability of different subunits of NF-kappa B to enhance HIV-1 transcription in vitro with chromatin templates. We find that the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by re...

Research paper thumbnail of Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro

Genes & Development, 1995

Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that... more Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. In this paper we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. First, we show that mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nucleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and that repression can be counteracted efficiently by preincubation of ...

Research paper thumbnail of Histone Acetyltransferases Regulate HIV-1 Enhancer Activity In Vitro

Genes & …, 1997

Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are ... more Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are potent inducers of HIV-1 transcription in latently infected T-cell lines. Activation of the integrated HIV-1 promoter is accompanied by the loss or rearrangement of a positioned nucleosome (nuc-1) near the viral RNA start site. Here we show that TSA strongly induces HIV-1 transcription on chromatin in vitro, concomitant with an enhancer factor-assisted increase in the level of acetylated histone H4. TSA treatment, however, did not detectably alter enhancer factor binding or the positioning of nuc-1 on the majority of the chromatin templates indicating that protein acetylation and chromatin remodeling may be limiting steps that occur only on transcriptionally competent templates, or that remodeling of nuc-1 requires additional factors. To assess the number of active chromatin templates in vitro, transcription was limited to a single round with low levels of the detergent Sarkosyl. Remarkably, HIV-1 transcription on chromatin was found to arise from a small number of active templates that can each support nearly 100 rounds of transcription, and TSA increased the number of active templates in each round. In contrast, transcription on naked DNA was limited to only a few rounds and was not responsive to TSA. We conclude that HIV-1 enhancer complexes greatly facilitate transcription reinitiation on chromatin in vitro, and act at a limiting step to promote the acetylation of histones or other transcription factors required for HIV-1 enhancer activity.