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Journal of Biological …, 2009
Numerous post-translational modifications have been identified in histones. Most of these occur w... more Numerous post-translational modifications have been identified in histones. Most of these occur within the histone tails, but a few have been identified within the histone core sequences. Histone core post-translational modifications have the potential to directly modulate nucleosome structure and consequently DNA accessibility. Here, we identify threonine 45 of histone H3 (H3T45) as a site of phosphorylation in vivo. We find that phosphorylation of H3T45 (H3T45ph) increases dramatically in apoptotic cells, around the time of DNA nicking. To further explore this connection, we analyzed human neutrophil cells because they are short-lived cells that undergo apoptosis in vivo. Freshly isolated neutrophils contain very little H3T45ph, whereas cells cultured for 20 h possess significant amounts; the kinetics of H3T45ph induction closely parallel those of caspase-3 activation. Cytokine inhibition of neutrophil apoptosis leads to reduced levels of H3T45ph. We identify protein kinase C-␦ as the kinase responsible for H3T45ph in vitro and in vivo. Given the nucleosomal position of H3T45, we postulate that H3T45ph induces structural change within the nucleosome to facilitate DNA nicking and/or fragmentation.
Nucleic Acids Research, 1993
The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here,... more The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here, we show that the DNA-PK modifies c-Jun in vitro and that serine residue 249 (Ser-249) is required for phosphorylation to occur. This residue corresponds to one of three sites of c-Jun that are phosphorylated in vivo and which negatively regulate c-Jun DNA binding in vitro. However, we find that phosphorylation of c-Jun by the DNA-PK does not interfere with DNA binding, indicating that phosphorylation at other sites is required for this effect. Mutagenesis of the phosphorylated region of c-Jun reveals that the primary amino acid sequence recognised by the DNA-PK consists of the sequence Ser-GIn, and that adjacent acidic residues potentiate kinase activity. Furthermore, when this site is placed within the context of a second protein, it confers DNA-PK directed phosphorylation upon that protein. Our findings will facilitate identification of DNA-PK phosphorylation sites in other transcription factors.
Cell Research, 2011
Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to e... more Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place.
Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory... more Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.
Current Opinion in Genetics & Development, 2004
Nature, 2002
Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone... more Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.
Cell, 2002
and against the reversible nature of methylation and consider ways in which this modification may... more and against the reversible nature of methylation and consider ways in which this modification may be dis-University of Cambridge Tennis Court Road placed from the histones if methylation were to be dynamically controlled. Cambridge CB2 1QR United Kingdom Methylation of Histones The major methylation sites within histone tails are the Methylation of histones mediates transcriptional sibasic amino acid side chains of lysine and arginine resilencing at heterochromatin sites and affects regulated dues (Kouzarides, 2002; Zhang and Reinberg, 2001). The transcription at euchromatic loci. So is the methyl positions of many of these methylated residues within group a permanent mark on histones, or can it be the histone N-terminal tails have now been mapped (Figremoved by an active process necessary for regulated ure 1A), and the histone methyltransferases (HMTs) pergene expression?
Nature, 2005
Histones package DNA, and post-translational modifications of histones can regulate access to DNA... more Histones package DNA, and post-translational modifications of histones can regulate access to DNA. Until recently, histone methylation-unlike all other histone modifications-was considered a permanent mark. The discovery of enzymes that reverse the methylation of lysines and arginines challenges our current thinking on the unique nature of histone methylation, and substantially increases the complexity of histone modification pathways.
Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2006
Journal of Biological …, 2005
Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas meth... more Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas methylation of K9/H3 is tightly associated with gene inactivity. These are well characterized sites of methylation within histones, but there are numerous other, less characterized, sites of modification. In Saccharomyces cerevisiae, methylation of K36/H3 has been linked to active genes, but little is known about this methylation in higher eukaryotes. Here we analyzed for the first time the levels and spatial distribution of di- and tri-methyl (di- and tri-Me) K36/H3 in metazoan genes. We analyzed chicken genes that are developmentally regulated, constitutively active, or inactive. We found that active genes contain high levels of these modifications compared with inactive genes. Furthermore, in actively transcribed regions the levels of di- and tri-Me K36/H3 peak toward the 3' end of the gene. This is in striking contrast to the distributions of di- and tri-Me K4/H3, which peak early in actively transcribed regions. Thus, di/tri-Me K4/H3 and di/tri-Me K36/H3 are both useful markers of active genes, but their genic distribution indicates differing roles. Our data suggest that the unique spatial distribution of di- and tri-Me K36/H3 plays a role in transcriptional termination and/or early RNA processing.
Molecular Cell, 2010
Histone-modifying enzymes require recruitment to specific genomic loci in order to elicit their e... more Histone-modifying enzymes require recruitment to specific genomic loci in order to elicit their effects. In this issue, Kleine-Kohlbrecher et al. reveal how a histone demethylase is targeted to specific genes, thereby providing mechanistic insight into recruitment and regulation.
Immunity, 1999
Zhu et al., 1998). Due to the common binding with Institute of Pathology immunophilins and inhibi... more Zhu et al., 1998). Due to the common binding with Institute of Pathology immunophilins and inhibition of calcineurin, the immu- † Institute of Medical Radiation Research nosuppressants cyclosporin A and FK506 are potent and Cell Biology inhibitors of nuclear translocation and activity of all four University of Wuerzburg NF-AT factors (Rao et al., 1997). D-97080 Wuerzburg In addition to nuclear translocation, calcineurin also Germany appears to control DNA binding of NF-AT factors. Whereas ‡ Wellcome/CRC Institute and Department of Pathology hyperphosphorylation was shown to inhibit DNA binding University of Cambridge of NF-ATp, dephosphorylation by calcineurin (or other Cambridge CB2 1QR phosphatases) strongly stimulated the binding of NF-United Kingdom ATp to DNA (Park et al. , 1995). A further level at which the activity of NF-AT factors might be controlled is the level of transactivation. Inducible transactivation do-Summary mains (TADs) have been identified in numerous eukaryotic transcription factors whose activity is rapidly in-NF-ATc, an inducibly expressed transcription factor, duced upon cellular stimulation. This has been shown controls gene expression in T lymphocytes and cardioin detail for the C-terminal TAD of inducible transcription myocytes. We show here that the transcriptional cofactor Elk-1/TCF-1, which contains multiple Ser/ThrPro activators CBP/p300 bind to and control the activity phosphorylation motifs. Within seconds after cellular of the inducible N-terminal transactivation domain of stimulation, extracellular signals are transmitted through
Journal of Biological …, 2009
Numerous post-translational modifications have been identified in histones. Most of these occur w... more Numerous post-translational modifications have been identified in histones. Most of these occur within the histone tails, but a few have been identified within the histone core sequences. Histone core post-translational modifications have the potential to directly modulate nucleosome structure and consequently DNA accessibility. Here, we identify threonine 45 of histone H3 (H3T45) as a site of phosphorylation in vivo. We find that phosphorylation of H3T45 (H3T45ph) increases dramatically in apoptotic cells, around the time of DNA nicking. To further explore this connection, we analyzed human neutrophil cells because they are short-lived cells that undergo apoptosis in vivo. Freshly isolated neutrophils contain very little H3T45ph, whereas cells cultured for 20 h possess significant amounts; the kinetics of H3T45ph induction closely parallel those of caspase-3 activation. Cytokine inhibition of neutrophil apoptosis leads to reduced levels of H3T45ph. We identify protein kinase C-␦ as the kinase responsible for H3T45ph in vitro and in vivo. Given the nucleosomal position of H3T45, we postulate that H3T45ph induces structural change within the nucleosome to facilitate DNA nicking and/or fragmentation.
Nucleic Acids Research, 1993
The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here,... more The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here, we show that the DNA-PK modifies c-Jun in vitro and that serine residue 249 (Ser-249) is required for phosphorylation to occur. This residue corresponds to one of three sites of c-Jun that are phosphorylated in vivo and which negatively regulate c-Jun DNA binding in vitro. However, we find that phosphorylation of c-Jun by the DNA-PK does not interfere with DNA binding, indicating that phosphorylation at other sites is required for this effect. Mutagenesis of the phosphorylated region of c-Jun reveals that the primary amino acid sequence recognised by the DNA-PK consists of the sequence Ser-GIn, and that adjacent acidic residues potentiate kinase activity. Furthermore, when this site is placed within the context of a second protein, it confers DNA-PK directed phosphorylation upon that protein. Our findings will facilitate identification of DNA-PK phosphorylation sites in other transcription factors.
Cell Research, 2011
Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to e... more Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place.
Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory... more Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.
Current Opinion in Genetics & Development, 2004
Nature, 2002
Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone... more Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.
Cell, 2002
and against the reversible nature of methylation and consider ways in which this modification may... more and against the reversible nature of methylation and consider ways in which this modification may be dis-University of Cambridge Tennis Court Road placed from the histones if methylation were to be dynamically controlled. Cambridge CB2 1QR United Kingdom Methylation of Histones The major methylation sites within histone tails are the Methylation of histones mediates transcriptional sibasic amino acid side chains of lysine and arginine resilencing at heterochromatin sites and affects regulated dues (Kouzarides, 2002; Zhang and Reinberg, 2001). The transcription at euchromatic loci. So is the methyl positions of many of these methylated residues within group a permanent mark on histones, or can it be the histone N-terminal tails have now been mapped (Figremoved by an active process necessary for regulated ure 1A), and the histone methyltransferases (HMTs) pergene expression?
Nature, 2005
Histones package DNA, and post-translational modifications of histones can regulate access to DNA... more Histones package DNA, and post-translational modifications of histones can regulate access to DNA. Until recently, histone methylation-unlike all other histone modifications-was considered a permanent mark. The discovery of enzymes that reverse the methylation of lysines and arginines challenges our current thinking on the unique nature of histone methylation, and substantially increases the complexity of histone modification pathways.
Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2006
Journal of Biological …, 2005
Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas meth... more Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas methylation of K9/H3 is tightly associated with gene inactivity. These are well characterized sites of methylation within histones, but there are numerous other, less characterized, sites of modification. In Saccharomyces cerevisiae, methylation of K36/H3 has been linked to active genes, but little is known about this methylation in higher eukaryotes. Here we analyzed for the first time the levels and spatial distribution of di- and tri-methyl (di- and tri-Me) K36/H3 in metazoan genes. We analyzed chicken genes that are developmentally regulated, constitutively active, or inactive. We found that active genes contain high levels of these modifications compared with inactive genes. Furthermore, in actively transcribed regions the levels of di- and tri-Me K36/H3 peak toward the 3' end of the gene. This is in striking contrast to the distributions of di- and tri-Me K4/H3, which peak early in actively transcribed regions. Thus, di/tri-Me K4/H3 and di/tri-Me K36/H3 are both useful markers of active genes, but their genic distribution indicates differing roles. Our data suggest that the unique spatial distribution of di- and tri-Me K36/H3 plays a role in transcriptional termination and/or early RNA processing.
Molecular Cell, 2010
Histone-modifying enzymes require recruitment to specific genomic loci in order to elicit their e... more Histone-modifying enzymes require recruitment to specific genomic loci in order to elicit their effects. In this issue, Kleine-Kohlbrecher et al. reveal how a histone demethylase is targeted to specific genes, thereby providing mechanistic insight into recruitment and regulation.
Immunity, 1999
Zhu et al., 1998). Due to the common binding with Institute of Pathology immunophilins and inhibi... more Zhu et al., 1998). Due to the common binding with Institute of Pathology immunophilins and inhibition of calcineurin, the immu- † Institute of Medical Radiation Research nosuppressants cyclosporin A and FK506 are potent and Cell Biology inhibitors of nuclear translocation and activity of all four University of Wuerzburg NF-AT factors (Rao et al., 1997). D-97080 Wuerzburg In addition to nuclear translocation, calcineurin also Germany appears to control DNA binding of NF-AT factors. Whereas ‡ Wellcome/CRC Institute and Department of Pathology hyperphosphorylation was shown to inhibit DNA binding University of Cambridge of NF-ATp, dephosphorylation by calcineurin (or other Cambridge CB2 1QR phosphatases) strongly stimulated the binding of NF-United Kingdom ATp to DNA (Park et al. , 1995). A further level at which the activity of NF-AT factors might be controlled is the level of transactivation. Inducible transactivation do-Summary mains (TADs) have been identified in numerous eukaryotic transcription factors whose activity is rapidly in-NF-ATc, an inducibly expressed transcription factor, duced upon cellular stimulation. This has been shown controls gene expression in T lymphocytes and cardioin detail for the C-terminal TAD of inducible transcription myocytes. We show here that the transcriptional cofactor Elk-1/TCF-1, which contains multiple Ser/ThrPro activators CBP/p300 bind to and control the activity phosphorylation motifs. Within seconds after cellular of the inducible N-terminal transactivation domain of stimulation, extracellular signals are transmitted through