Sam John | Symbiosis Institute of Telecom Management (original) (raw)

Papers by Sam John

ACM Sigcse Bulletin, 2003

Abstract The World Wide Web is increasingly becoming an integrated extension of users&amp... more Abstract The World Wide Web is increasingly becoming an integrated extension of users' computing environments, with content indexed and retrieved through Web browsers. Web browsers are increasingly being used as computer science curriculum delivery mechanism, for both ...

ABSTRACT Finding just the right example to answer a question can be difficult for CS1 students an... more ABSTRACT Finding just the right example to answer a question can be difficult for CS1 students and teachers. For this to work well there must be an intuitive interface coupled to an appropriate set of focused examples. The examples then provide the scaffolding to enable students' ...

... We describe a mobile service platform that authenticates users who send service requests from... more ... We describe a mobile service platform that authenticates users who send service requests from various mobile devices, transcodes video content based on user and device profiles, and authorizes the delivery of content from a media server to the proper ...

Wireless Networks, 2003

iMobile 1 is an enterprise mobile service platform that allows resource-limited mobile devices to... more iMobile 1 is an enterprise mobile service platform that allows resource-limited mobile devices to communicate with each other and to securely access corporate contents and services. The original iMobile architecture consists of devlets that provide protocol interfaces to different mobile devices and infolets that access and transcode information based on device profiles. iMobile Enterprise Edition (iMobile EE) is a redesign of the original iMobile architecture to address the security, scalability, and availability requirements of a large enterprise such as AT&T. iMobile EE incorporates gateways that interact with corporate authentication services, replicated iMobile servers with backend connections to corporate services, a reliable message queue that connects iMobile gateways and servers, and a comprehensive service profile database that governs operations of the mobile service platform. The iMobile EE architecture was also extended to provide personalized multimedia services, allowing mobile users to remotely control, record, and request video contents. iMobile EE aims to provide a scalable, secure, and modular software platform that makes enterprise services easily accessible to a growing list of mobile devices roaming among various wireless networks.

Methods, 1998

Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core h... more Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core histones to transcriptional activity of cellular chromatin. New insights into this field were gained on the identification of the first nuclear, type A histone acetyltransferase (HAT). The yeast transcriptional adaptor protein Gcn5 was identified as a nuclear HAT and thus provided a direct link between pathways of transcriptional activation and histone acetylation. However, while recombinant Gcn5 can efficiently acetylate free histone H3 and, to a lesser extent, H4 it is unable to acetylate nucleosomal histones. It is therefore very likely that additional proteins are required for Gcn5mediated acetylation of chromosomal histones. We have recently shown that Gcn5 is the catalytic subunit of two highmolecular-weight histone acetyltransferase complexes in yeast. In addition to the Gcn5-containing ADA and SAGA HAT complexes we have identified two other HAT complexes in yeast. These are called NuA3 and NuA4 for their predominant specificity to acetylate histones H3 and H4, respectively. Here we describe the identification and characterization of four native nuclear high-molecular-weight HAT complexes in Saccharomyces cerevisiae. These purified HATs can be used in a variety of functional assays to further address questions of how acetylation has an impact on transcriptional regulation.

Methods, 1997

ture, suggesting that the primary steps leading to An early step in a pathway leading to transcri... more ture, suggesting that the primary steps leading to An early step in a pathway leading to transcriptional initiatranscriptional initiation involve interactions betion involves the rearrangement of chromatin at gene regulatween activators and nucleosomes . tory sequences. To study this process, we have developed a

Bioessays, 1998

A hallmark feature of mitosis is the extinction of bulk cellular transcription. The mechanism by ... more A hallmark feature of mitosis is the extinction of bulk cellular transcription. The mechanism by which transcription is abrogated is likely linked to mitotic specific events such as chromosome condensation. Recent studies 1,2 that probe the structure of genes that can be reactivated rapidly after mitotic repression (early G1) suggest that there are structural distortions in the promoter regions of these genes. These distortions are absent in genes that are typically repressed or reactivated in later phases of the cell cycle (late G1, S, or G2). Such changes in the chromatin structure of these genes may create a transient window for transcription factor binding and rapid reactivation of genes in subsequent phases of the cell cycle.

Genes & Development, 1995

The translational positions of nucleosomes in the promoter region of the mouse mammary tumor viru... more The translational positions of nucleosomes in the promoter region of the mouse mammary tumor virus (MMTV) were defined at high resolution. Nucleosome boundaries were determined in primer extension assays using full-length single-stranded mononucleosomal DNA prepared from cells treated with formaldehyde, a reversible protein-DNA cross-linking agent.

Molecular Cell, 2008

The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function ar... more The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function are unknown. We have characterized glucocorticoid receptor (GR) binding events and chromatin structural transitions across GR-induced or -repressed genes. This analysis reveals that GR binding invariably occurs at nuclease-accessible sites (DHS). A remarkable diversity of mechanisms, however, render these sites available for GR binding. Accessibility of the GR binding sites is either constitutive or hormone inducible. Within each category, some DHS sites require the Brg1-containing Swi/Snf complex, but others are Brg1 independent, implicating a different remodeling complex. The H2A.Z histone variant is highly enriched at both inducible and constitutive DHS sites and is subject to exchange during hormone activation. The DHS profile is highly cell specific, implicating cell-selective organization of the chromatin landscape as a critical determinant of tissue-selective receptor function. Furthermore, the widespread requirement for chromatin remodeling supports the recent hypothesis that the rapid exchange of receptor proteins occurs during nucleosome reorganization.

Molecular Biology of The Cell, 2008

Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI... more Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI/SNF family of chromatin-remodeling complexes. These complexes are involved in essential processes such as cell cycle, growth, differentiation, and cancer. Using imaging approaches in a cell line that harbors tandem repeats of stably integrated copies of the steroid responsive MMTV-LTR (mouse mammary tumor virus-long terminal repeat), we show that BRG1 and BRM are recruited to the MMTV promoter in a hormone-dependent manner. The recruitment of BRG1 and BRM resulted in chromatin remodeling and decondensation of the MMTV repeat as demonstrated by an increase in the restriction enzyme accessibility and in the size of DNA fluorescence in situ hybridization (FISH) signals.

Journal of Cell Biology, 2007

Molecular Endocrinology, 2006

The glucocorticoid receptor (GR) dynamically interacts with response elements in the mouse mammar... more The glucocorticoid receptor (GR) dynamically interacts with response elements in the mouse mammary tumor virus (MMTV) promoter to regulate steroid-dependent transcription. In a clonal mammary carcinoma cell line containing a tandem array of MMTV promoter-reporter gene cassettes integrated at a single genomic locus, direct binding of a green fluorescent protein (GFP)-GR fusion protein to the MMTV regulatory elements can be observed in living cells. After ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA fluorescence in situ hybridization (FISH). High-resolution fluorescence images were acquired from large numbers of randomly selected cells. Images were analyzed with a novel automated computer algorithm, measuring the RNA FISH signal and the relative GFP-GR fluorescence intensity at the MMTV array for each cell. Although dexamethasone increased the mean RNA FISH signal approximately 10-fold, RU486 produced only about a 2-fold induction, as expected for this mixed antagonist. For all treatment conditions, the relative GFP-GR fluorescence at the array for the averaged cells paralleled the RNA FISH measurements, suggesting that image analysis accurately detected an increase in steady-state GR association with the MMTV array that was responsible for the increase in transcriptional activity. The antagonist-dependent decreases in GR association with the MMTV promoter were confirmed by chromatin immunoprecipitation experiments, supporting the image analysis results. A pronounced cell-to-cell variability was observed in RNA FISH signal and GR-MMTV association within treatment groups. We observed a nonlinear relationship between GR-MMTV association and RNA FISH in individual cells, indicating that differences in GR-MMTV interaction account for some, but not all, of the transcriptional heterogeneity between individual cells. In selected cell subpopulations with equal levels of GR-MMTV association, there was a decrease in RNA FISH signal with RU486 treatment compared with dexamethasone treatment. These results indicate that stochastic events occurring after GR-promoter association, such as the actions of chromatin remodeling complexes or other cofactors, change in a ligand-dependent manner and regulate heterogeneous transcription in individual cells.

Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I... more Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I-hypersensitive region (HSR) spanning the hormone responsive element (HRE) in the long terminal repeat. Restriction enzymes were used to characterize the accessibility of various sites within the HSR of mouse mammary tumor virus long terminal repeat-reporter constructions in four different cell lines. The glucocorticoid-dependent HSR was found to span minimally 187 bases, a stretch of DNA longer than that associated with histones in the core particle. Although the 5-most receptor binding site within the HRE is downstream of ؊190, hypersensitive sites were found further upstream to at least ؊295. The relationship in the accessibility between pairs of sites in the vicinity of the HSR was further examined in one cell line by a two-enzyme restriction access assay. In the uninduced state, the accessibilities at these sites were found to be independent of each other. In contrast, when stimulated with hormone, the accessibilities at these sites were observed to become linked. That is, once a distinct promoter was activated, all of the sites within the HSR of that molecule became accessible. The HSR formed along an invariant stretch of DNA sequence despite the multiplicity of nucleosome frames in the nucleosome B region, where the HRE is located. The results indicate that the macroscopic length of the HSR does not arise from core length-remodeling events in molecules containing Nuc-B in alternative positions.

Journal of Cell Science, 2009

Molecular Cell, 2006

Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 se... more Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 serves as a coactivator for the glucocorticoid receptor (GR). Furthermore, a subfraction of cellular HDAC1 is acetylated after association with the GR, and this acetylation event correlates with a decrease in promoter activity. HDAC1 in repressed chromatin is highly acetylated, while the deacetylase found on transcriptionally active chromatin manifests a low level of acetylation. Acetylation of purified HDAC1 inactivates its deacetylase activity, and mutation of the critical acetylation sites abrogates HDAC1 function in vivo. We propose that hormone activation of the receptor leads to progressive acetylation of HDAC1 in vivo, which in turn inhibits the deacetylase activity of the enzyme and prevents a deacetylation event that is required for promoter activation. These findings indicate that HDAC1 is required for the induction of some genes by the GR, and this activator function is dynamically modulated by acetylation.

Endocrinology, 2009

We have characterized the kinetic response of gene targets throughout the murine genome to transc... more We have characterized the kinetic response of gene targets throughout the murine genome to transcriptional modulation by the glucocorticoid receptor (GR). In contrast to a model in which multiple genes are either repressed or activated during the GR response, the vast majority of responsive genes are subject to complex regulation profiles, frequently with alternate activation and repression phases. We also observe that GR binding at response elements does not always correlate with the target gene response profile. Thus, the cellular response to GR stimulation involves a highly orchestrated series of regulatory actions and not simply a binary response to hormone.

Chromosome Research, 2006

Eucaryotic gene transcriptional switches utilize changes both in the activity and composition of ... more Eucaryotic gene transcriptional switches utilize changes both in the activity and composition of soluble transcription factor complexes, and epigenetic modifications to the chromatin template. Until recently, alternate states of promoter activity have been associated with the assembly of relatively stable multiprotein complexes on target genes, with transitions in the composition of these complexes occurring on the time scale of minutes or hours. The development of living cell techniques to characterize transcription factor function in real time has led to an alternate view of highly dynamic protein/template interactions. In addition, emerging evidence suggests that energy-dependent processes contribute significantly to the rapid movement of proteins in living cells, and to the exchange of sequence-specific DNA-binding proteins with regulatory elements. Potential mechanisms involved in the unexpectedly rapid flux of factor/template interactions are discussed in the context of a “return-to-template” model for transcription factor function.

Biochemistry, 1991

Activation of mouse mammary tumor virus transcription by the hormone-bound glucocorticoid recepto... more Activation of mouse mammary tumor virus transcription by the hormone-bound glucocorticoid receptor results in disruption of a nucleosome that is specifically positioned on the promoter. Limited treatment of cells with the histone deacetylase inhibitor sodium butyrate prevents receptor-dependent promoter activation and nucleosome disruption [Bresnick, E. H., John, S., Berard, D. S., LeFebvre, P., & Hager, G. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3977-3981]. On the basis of this observation, we undertook a series of experiments to compare the structure of normal and hyperacetylated mouse mammary tumor virus chromatin. Although butyrate prevents hormone-induced restriction enzyme cutting specifically in the B nucleosome region, chromatin containing hyperacetylated histones does not differ from normal chromatin in general sensitivity to restriction enzymes. Indirect end-labeling analysis of micrococcal nuclease digested chromatin reveals that nucleosomes are identically phased on the mouse mammary tumor virus long terminal repeat in normal and hyperacetylated chromatin. A synthetic DNA fragment spanning the B nucleosome region was reconstituted into a monosome by using core particles containing normal or hyperacetylated histones. Analysis of the structure of reconstituted monosomes by nondenaturing polyacrylamide gel electrophoresis, salt stability, thermal stability, restriction enzyme accessibility, and exonuclease III or DNase I footprinting reveals no effect of histone hyperacetylation on monosome structure. These observations suggest that histone hyperacetylation does not induce a major change in the structure of mouse mammary tumor virus chromatin, such as nucleosome unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochimica Et Biophysica Acta-gene Structure and Expression, 2004

Following a hormone signal, steroid/nuclear receptors bind regulatory elements in chromatin and i... more Following a hormone signal, steroid/nuclear receptors bind regulatory elements in chromatin and initiate the recruitment of a variety of multi-protein complexes to promoter sequences. These complexes ultimately lead to the recruitment of general transcription factors and the initiation of transcription. Traditional models suggest that these factors remain statically bound to each other and to chromatin until other signals are received to reduce transcription. Recent findings demonstrate that the processes and actions involved are much more complex than traditional models convey, and that the movement of receptors and coactivators is remarkably dynamic. Transcription factors are highly mobile in the nuclear environment, and interact only briefly with target sites in the nucleus. As a result of these transient interactions, promoters move through many states during activation and repression. Two general concepts emerge from current data: (1) Various transcription factors appear to follow "ordered recruitment" to promoters on a time scale of minutes to hours in response to a stimulus. During this response, the proteins that interact with chromatin may cycle on and off the promoter multiple times. (2) During these ordered recruitment cycles, the individual molecules that form functional complexes often exchange rapidly on a time scale of seconds. This rapid exchange of molecules within a formed complex occurs independently of long-term cycling on chromatin. Several processes are implicated in rapid nuclear dynamics, including potential roles for molecular chaperones, the proteasome degradation machinery and chromatin remodeling complexes.

ACM Sigcse Bulletin, 2003

Abstract The World Wide Web is increasingly becoming an integrated extension of users&amp... more Abstract The World Wide Web is increasingly becoming an integrated extension of users' computing environments, with content indexed and retrieved through Web browsers. Web browsers are increasingly being used as computer science curriculum delivery mechanism, for both ...

ABSTRACT Finding just the right example to answer a question can be difficult for CS1 students an... more ABSTRACT Finding just the right example to answer a question can be difficult for CS1 students and teachers. For this to work well there must be an intuitive interface coupled to an appropriate set of focused examples. The examples then provide the scaffolding to enable students' ...

... We describe a mobile service platform that authenticates users who send service requests from... more ... We describe a mobile service platform that authenticates users who send service requests from various mobile devices, transcodes video content based on user and device profiles, and authorizes the delivery of content from a media server to the proper ...

Wireless Networks, 2003

iMobile 1 is an enterprise mobile service platform that allows resource-limited mobile devices to... more iMobile 1 is an enterprise mobile service platform that allows resource-limited mobile devices to communicate with each other and to securely access corporate contents and services. The original iMobile architecture consists of devlets that provide protocol interfaces to different mobile devices and infolets that access and transcode information based on device profiles. iMobile Enterprise Edition (iMobile EE) is a redesign of the original iMobile architecture to address the security, scalability, and availability requirements of a large enterprise such as AT&T. iMobile EE incorporates gateways that interact with corporate authentication services, replicated iMobile servers with backend connections to corporate services, a reliable message queue that connects iMobile gateways and servers, and a comprehensive service profile database that governs operations of the mobile service platform. The iMobile EE architecture was also extended to provide personalized multimedia services, allowing mobile users to remotely control, record, and request video contents. iMobile EE aims to provide a scalable, secure, and modular software platform that makes enterprise services easily accessible to a growing list of mobile devices roaming among various wireless networks.

Methods, 1998

Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core h... more Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core histones to transcriptional activity of cellular chromatin. New insights into this field were gained on the identification of the first nuclear, type A histone acetyltransferase (HAT). The yeast transcriptional adaptor protein Gcn5 was identified as a nuclear HAT and thus provided a direct link between pathways of transcriptional activation and histone acetylation. However, while recombinant Gcn5 can efficiently acetylate free histone H3 and, to a lesser extent, H4 it is unable to acetylate nucleosomal histones. It is therefore very likely that additional proteins are required for Gcn5mediated acetylation of chromosomal histones. We have recently shown that Gcn5 is the catalytic subunit of two highmolecular-weight histone acetyltransferase complexes in yeast. In addition to the Gcn5-containing ADA and SAGA HAT complexes we have identified two other HAT complexes in yeast. These are called NuA3 and NuA4 for their predominant specificity to acetylate histones H3 and H4, respectively. Here we describe the identification and characterization of four native nuclear high-molecular-weight HAT complexes in Saccharomyces cerevisiae. These purified HATs can be used in a variety of functional assays to further address questions of how acetylation has an impact on transcriptional regulation.

Methods, 1997

ture, suggesting that the primary steps leading to An early step in a pathway leading to transcri... more ture, suggesting that the primary steps leading to An early step in a pathway leading to transcriptional initiatranscriptional initiation involve interactions betion involves the rearrangement of chromatin at gene regulatween activators and nucleosomes . tory sequences. To study this process, we have developed a

Bioessays, 1998

A hallmark feature of mitosis is the extinction of bulk cellular transcription. The mechanism by ... more A hallmark feature of mitosis is the extinction of bulk cellular transcription. The mechanism by which transcription is abrogated is likely linked to mitotic specific events such as chromosome condensation. Recent studies 1,2 that probe the structure of genes that can be reactivated rapidly after mitotic repression (early G1) suggest that there are structural distortions in the promoter regions of these genes. These distortions are absent in genes that are typically repressed or reactivated in later phases of the cell cycle (late G1, S, or G2). Such changes in the chromatin structure of these genes may create a transient window for transcription factor binding and rapid reactivation of genes in subsequent phases of the cell cycle.

Genes & Development, 1995

The translational positions of nucleosomes in the promoter region of the mouse mammary tumor viru... more The translational positions of nucleosomes in the promoter region of the mouse mammary tumor virus (MMTV) were defined at high resolution. Nucleosome boundaries were determined in primer extension assays using full-length single-stranded mononucleosomal DNA prepared from cells treated with formaldehyde, a reversible protein-DNA cross-linking agent.

Molecular Cell, 2008

The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function ar... more The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function are unknown. We have characterized glucocorticoid receptor (GR) binding events and chromatin structural transitions across GR-induced or -repressed genes. This analysis reveals that GR binding invariably occurs at nuclease-accessible sites (DHS). A remarkable diversity of mechanisms, however, render these sites available for GR binding. Accessibility of the GR binding sites is either constitutive or hormone inducible. Within each category, some DHS sites require the Brg1-containing Swi/Snf complex, but others are Brg1 independent, implicating a different remodeling complex. The H2A.Z histone variant is highly enriched at both inducible and constitutive DHS sites and is subject to exchange during hormone activation. The DHS profile is highly cell specific, implicating cell-selective organization of the chromatin landscape as a critical determinant of tissue-selective receptor function. Furthermore, the widespread requirement for chromatin remodeling supports the recent hypothesis that the rapid exchange of receptor proteins occurs during nucleosome reorganization.

Molecular Biology of The Cell, 2008

Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI... more Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI/SNF family of chromatin-remodeling complexes. These complexes are involved in essential processes such as cell cycle, growth, differentiation, and cancer. Using imaging approaches in a cell line that harbors tandem repeats of stably integrated copies of the steroid responsive MMTV-LTR (mouse mammary tumor virus-long terminal repeat), we show that BRG1 and BRM are recruited to the MMTV promoter in a hormone-dependent manner. The recruitment of BRG1 and BRM resulted in chromatin remodeling and decondensation of the MMTV repeat as demonstrated by an increase in the restriction enzyme accessibility and in the size of DNA fluorescence in situ hybridization (FISH) signals.

Journal of Cell Biology, 2007

Molecular Endocrinology, 2006

The glucocorticoid receptor (GR) dynamically interacts with response elements in the mouse mammar... more The glucocorticoid receptor (GR) dynamically interacts with response elements in the mouse mammary tumor virus (MMTV) promoter to regulate steroid-dependent transcription. In a clonal mammary carcinoma cell line containing a tandem array of MMTV promoter-reporter gene cassettes integrated at a single genomic locus, direct binding of a green fluorescent protein (GFP)-GR fusion protein to the MMTV regulatory elements can be observed in living cells. After ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA fluorescence in situ hybridization (FISH). High-resolution fluorescence images were acquired from large numbers of randomly selected cells. Images were analyzed with a novel automated computer algorithm, measuring the RNA FISH signal and the relative GFP-GR fluorescence intensity at the MMTV array for each cell. Although dexamethasone increased the mean RNA FISH signal approximately 10-fold, RU486 produced only about a 2-fold induction, as expected for this mixed antagonist. For all treatment conditions, the relative GFP-GR fluorescence at the array for the averaged cells paralleled the RNA FISH measurements, suggesting that image analysis accurately detected an increase in steady-state GR association with the MMTV array that was responsible for the increase in transcriptional activity. The antagonist-dependent decreases in GR association with the MMTV promoter were confirmed by chromatin immunoprecipitation experiments, supporting the image analysis results. A pronounced cell-to-cell variability was observed in RNA FISH signal and GR-MMTV association within treatment groups. We observed a nonlinear relationship between GR-MMTV association and RNA FISH in individual cells, indicating that differences in GR-MMTV interaction account for some, but not all, of the transcriptional heterogeneity between individual cells. In selected cell subpopulations with equal levels of GR-MMTV association, there was a decrease in RNA FISH signal with RU486 treatment compared with dexamethasone treatment. These results indicate that stochastic events occurring after GR-promoter association, such as the actions of chromatin remodeling complexes or other cofactors, change in a ligand-dependent manner and regulate heterogeneous transcription in individual cells.

Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I... more Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I-hypersensitive region (HSR) spanning the hormone responsive element (HRE) in the long terminal repeat. Restriction enzymes were used to characterize the accessibility of various sites within the HSR of mouse mammary tumor virus long terminal repeat-reporter constructions in four different cell lines. The glucocorticoid-dependent HSR was found to span minimally 187 bases, a stretch of DNA longer than that associated with histones in the core particle. Although the 5-most receptor binding site within the HRE is downstream of ؊190, hypersensitive sites were found further upstream to at least ؊295. The relationship in the accessibility between pairs of sites in the vicinity of the HSR was further examined in one cell line by a two-enzyme restriction access assay. In the uninduced state, the accessibilities at these sites were found to be independent of each other. In contrast, when stimulated with hormone, the accessibilities at these sites were observed to become linked. That is, once a distinct promoter was activated, all of the sites within the HSR of that molecule became accessible. The HSR formed along an invariant stretch of DNA sequence despite the multiplicity of nucleosome frames in the nucleosome B region, where the HRE is located. The results indicate that the macroscopic length of the HSR does not arise from core length-remodeling events in molecules containing Nuc-B in alternative positions.

Journal of Cell Science, 2009

Molecular Cell, 2006

Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 se... more Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 serves as a coactivator for the glucocorticoid receptor (GR). Furthermore, a subfraction of cellular HDAC1 is acetylated after association with the GR, and this acetylation event correlates with a decrease in promoter activity. HDAC1 in repressed chromatin is highly acetylated, while the deacetylase found on transcriptionally active chromatin manifests a low level of acetylation. Acetylation of purified HDAC1 inactivates its deacetylase activity, and mutation of the critical acetylation sites abrogates HDAC1 function in vivo. We propose that hormone activation of the receptor leads to progressive acetylation of HDAC1 in vivo, which in turn inhibits the deacetylase activity of the enzyme and prevents a deacetylation event that is required for promoter activation. These findings indicate that HDAC1 is required for the induction of some genes by the GR, and this activator function is dynamically modulated by acetylation.

Endocrinology, 2009

We have characterized the kinetic response of gene targets throughout the murine genome to transc... more We have characterized the kinetic response of gene targets throughout the murine genome to transcriptional modulation by the glucocorticoid receptor (GR). In contrast to a model in which multiple genes are either repressed or activated during the GR response, the vast majority of responsive genes are subject to complex regulation profiles, frequently with alternate activation and repression phases. We also observe that GR binding at response elements does not always correlate with the target gene response profile. Thus, the cellular response to GR stimulation involves a highly orchestrated series of regulatory actions and not simply a binary response to hormone.

Chromosome Research, 2006

Eucaryotic gene transcriptional switches utilize changes both in the activity and composition of ... more Eucaryotic gene transcriptional switches utilize changes both in the activity and composition of soluble transcription factor complexes, and epigenetic modifications to the chromatin template. Until recently, alternate states of promoter activity have been associated with the assembly of relatively stable multiprotein complexes on target genes, with transitions in the composition of these complexes occurring on the time scale of minutes or hours. The development of living cell techniques to characterize transcription factor function in real time has led to an alternate view of highly dynamic protein/template interactions. In addition, emerging evidence suggests that energy-dependent processes contribute significantly to the rapid movement of proteins in living cells, and to the exchange of sequence-specific DNA-binding proteins with regulatory elements. Potential mechanisms involved in the unexpectedly rapid flux of factor/template interactions are discussed in the context of a “return-to-template” model for transcription factor function.

Biochemistry, 1991

Activation of mouse mammary tumor virus transcription by the hormone-bound glucocorticoid recepto... more Activation of mouse mammary tumor virus transcription by the hormone-bound glucocorticoid receptor results in disruption of a nucleosome that is specifically positioned on the promoter. Limited treatment of cells with the histone deacetylase inhibitor sodium butyrate prevents receptor-dependent promoter activation and nucleosome disruption [Bresnick, E. H., John, S., Berard, D. S., LeFebvre, P., & Hager, G. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3977-3981]. On the basis of this observation, we undertook a series of experiments to compare the structure of normal and hyperacetylated mouse mammary tumor virus chromatin. Although butyrate prevents hormone-induced restriction enzyme cutting specifically in the B nucleosome region, chromatin containing hyperacetylated histones does not differ from normal chromatin in general sensitivity to restriction enzymes. Indirect end-labeling analysis of micrococcal nuclease digested chromatin reveals that nucleosomes are identically phased on the mouse mammary tumor virus long terminal repeat in normal and hyperacetylated chromatin. A synthetic DNA fragment spanning the B nucleosome region was reconstituted into a monosome by using core particles containing normal or hyperacetylated histones. Analysis of the structure of reconstituted monosomes by nondenaturing polyacrylamide gel electrophoresis, salt stability, thermal stability, restriction enzyme accessibility, and exonuclease III or DNase I footprinting reveals no effect of histone hyperacetylation on monosome structure. These observations suggest that histone hyperacetylation does not induce a major change in the structure of mouse mammary tumor virus chromatin, such as nucleosome unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochimica Et Biophysica Acta-gene Structure and Expression, 2004

Following a hormone signal, steroid/nuclear receptors bind regulatory elements in chromatin and i... more Following a hormone signal, steroid/nuclear receptors bind regulatory elements in chromatin and initiate the recruitment of a variety of multi-protein complexes to promoter sequences. These complexes ultimately lead to the recruitment of general transcription factors and the initiation of transcription. Traditional models suggest that these factors remain statically bound to each other and to chromatin until other signals are received to reduce transcription. Recent findings demonstrate that the processes and actions involved are much more complex than traditional models convey, and that the movement of receptors and coactivators is remarkably dynamic. Transcription factors are highly mobile in the nuclear environment, and interact only briefly with target sites in the nucleus. As a result of these transient interactions, promoters move through many states during activation and repression. Two general concepts emerge from current data: (1) Various transcription factors appear to follow "ordered recruitment" to promoters on a time scale of minutes to hours in response to a stimulus. During this response, the proteins that interact with chromatin may cycle on and off the promoter multiple times. (2) During these ordered recruitment cycles, the individual molecules that form functional complexes often exchange rapidly on a time scale of seconds. This rapid exchange of molecules within a formed complex occurs independently of long-term cycling on chromatin. Several processes are implicated in rapid nuclear dynamics, including potential roles for molecular chaperones, the proteasome degradation machinery and chromatin remodeling complexes.