Biochbnica et Biophysica Acta, ! 129 (1991 ) 43-48 © 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4781/91/$03.50
43
BBAEXP 92312
Histones and DNA methylation in mammalian chromatin. II. Presence of non-inhibitory tightly-bound histones Paola Caiafa I, Anna Reale 2, Maria D'Erme ~, Paola Allegra ~, Raffaella Santoro and Roberto Strom 2 Departnu, nts of t Biochemical Sciences and 2 Human Biopathology, Unicersit3' of Rome "La Sapienza : Rome (Bah') and C, N.R. Centre fiJr Molecular Biolo~.. Ronw (Italy)
(Received 28 February 1991) (Revised manuscript received 19 July 1991)
Key words: lflstone: Tightly-hound chromatin protein: DNA Methylation
After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid.soluble tightly.bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct Hi-, H2A- , HZB-, H 3- and H4.1ike components, the 'digested matrix' being however characterized by the absence of tightly-bound H t. These tightly. bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H I components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H t has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.
Introduction Enzymic methylation of DNA cytosine moieties occurs, in eukaryotic cells, within the chromatin structure. The complexity of chromatin organization is therefore to be taken into consideration in studies aimed to the identification of proteic factors susceptible of modifying the activity of DNA methyltransferase. The problem can be tackled by trying to detect specific proteins which preferentially recognize, along DNA, peculiar highly methylated - or, conversely, fully unmethylated - sequences. Neither 'core' histones nor the so-called 'high-mobility group' (HMG) proteins have been found to exhibit any preferential binding to methylated DNA [1,2]. Zhang et al. [3] have however been able to purify a 'methylated DNA binding protein' (MDBP), present in all mammalian tissues [4], which binds preferentially to specific DNA sequences
Correspondence: P. Caiafa, Dipartimento di Scienze Biochimiche. Universith di Roma "La Sapienza', Piazzale Aldo Moro 5. 00185 Rome, Italy.
characterized by a high proportion of methylated pyrimidines [3,5,6]. More recently, Mechan et al. [7] have described a methyI-CpG binding protein (MeCP), widely present in mammalian cells, that binds to a variety of non-specific DNA sequences containing methylated CpG and apparently confers [8] specific in vivo protection to the methylated CpGs. As an alternative approach, in vitro studies on chromatin proteins have proven that relatively crude preparations of HMG proteins fail to affect the activity of mammalian DNA methyltransferase [9] Studies carried out by Wang et al. [10] on the role played by specific HMG components on the in vitro methylation of DNA have, however, shown that the HMG-14 protein, which according to some authors [11,12] is typically present in transcribing nucleosomes, is a potent inhibitor of DNA methyitransferase - 100% inhibition being obtained in the presence of a 2-fold excess of HMG-14 with respect to DNA. HMG-I and HMG-2 exert instead a significant inhibitory effect on enzymic DNA methylation only under suitable ionic strength conditions (40 mM KCi). Also, histone proteins appear, at least in vitro, to affect the process of enzymic DNA methylation [9].
More recent studies from this laboratory [13] have shown that, within the 'physiological' range of histones-to-DNA ratios, only H~ is responsible for this inhibition, whose magnitude depends moreover on the secondary structure of the histone(s). On the other hand, the so-called 'tightly-bound non,histone proteins' [14-17], which are not dissociated from chromatin even upon exposure to very high ionic strength (above 2 M NaCI) - and which are reportedly related to the functional state of chromatin [14,15,18-21] - appear to play, in vitro as well as in vivo, an important role in facilitating the enzymic methylation of chmmatin DNA [22,23]. h~ the present study we describe a particular subset of tightly.bound proteins which, though resisting extraction even at high ionic strength, are quite similar to 'typical' histones in terms o1" acid solubility, apparent molecular mass and specific immunoreactivity. These 'tightly-bound histones', at variance from 'typical' ones, do not cause any inhibitory effect on the in vitro enzymic DNA methylation. Purified preparations of
the tightly-bound H rlike component were even found to exert a significant activation of the DNA methylation process. Materials and Methods
Isolation of chromatin and of chromatin subfractions Whole chromatin was prepared from human placenta according to Rickwood and Birnie [24], chromatin-bound proteinases being irreversibly inhibited by addition of 1 mM phenylmethylsulfonylfluoride (PMSF), which was thereafter present throughout all preparation steps. After extensive dialysis against 10 mM Tris-HCi buffer (pH 7.4) containing 0.5 mM dithiothreitol (DTT), 5 mM Na-EDTA and 1 mM PMSF, repeated treatment for i h at 4 ° C with 0.6 M NaC! in a 10 mM Tris-HCI + 2() mM Na-EDTA buffer (pH 7.4), yielded a histone Hi-depicted chromatin. Complete removal of all the histones and of the other loosely-bound chromatin proteins, could be obtained by further exposure to 2 M NaCI, yielding a 'stripped
Chromatin dissociatedfor 30 rainby low-salt" treatment: 0,6 M NaCI+ 20 mM EDTAin 10 mM Tris-HCI(pH 8) buffer ('LS buffer"); centrifugedat 10,000q)m for 10 min on a 8S34 Sorvall rotor -...- twice--I
SUPERNATANT
I
PELL r I
extractionwith 10% (w/v) HCIO4 J
I HI"deleted chr°matin I
I m°sa'Y'~ur
43
BBAEXP 92312
Histones and DNA methylation in mammalian chromatin. II. Presence of non-inhibitory tightly-bound histones Paola Caiafa I, Anna Reale 2, Maria D'Erme ~, Paola Allegra ~, Raffaella Santoro and Roberto Strom 2 Departnu, nts of t Biochemical Sciences and 2 Human Biopathology, Unicersit3' of Rome "La Sapienza : Rome (Bah') and C, N.R. Centre fiJr Molecular Biolo~.. Ronw (Italy)
(Received 28 February 1991) (Revised manuscript received 19 July 1991)
Key words: lflstone: Tightly-hound chromatin protein: DNA Methylation
After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid.soluble tightly.bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct Hi-, H2A- , HZB-, H 3- and H4.1ike components, the 'digested matrix' being however characterized by the absence of tightly-bound H t. These tightly. bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H I components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H t has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.
Introduction Enzymic methylation of DNA cytosine moieties occurs, in eukaryotic cells, within the chromatin structure. The complexity of chromatin organization is therefore to be taken into consideration in studies aimed to the identification of proteic factors susceptible of modifying the activity of DNA methyltransferase. The problem can be tackled by trying to detect specific proteins which preferentially recognize, along DNA, peculiar highly methylated - or, conversely, fully unmethylated - sequences. Neither 'core' histones nor the so-called 'high-mobility group' (HMG) proteins have been found to exhibit any preferential binding to methylated DNA [1,2]. Zhang et al. [3] have however been able to purify a 'methylated DNA binding protein' (MDBP), present in all mammalian tissues [4], which binds preferentially to specific DNA sequences
Correspondence: P. Caiafa, Dipartimento di Scienze Biochimiche. Universith di Roma "La Sapienza', Piazzale Aldo Moro 5. 00185 Rome, Italy.
characterized by a high proportion of methylated pyrimidines [3,5,6]. More recently, Mechan et al. [7] have described a methyI-CpG binding protein (MeCP), widely present in mammalian cells, that binds to a variety of non-specific DNA sequences containing methylated CpG and apparently confers [8] specific in vivo protection to the methylated CpGs. As an alternative approach, in vitro studies on chromatin proteins have proven that relatively crude preparations of HMG proteins fail to affect the activity of mammalian DNA methyltransferase [9] Studies carried out by Wang et al. [10] on the role played by specific HMG components on the in vitro methylation of DNA have, however, shown that the HMG-14 protein, which according to some authors [11,12] is typically present in transcribing nucleosomes, is a potent inhibitor of DNA methyitransferase - 100% inhibition being obtained in the presence of a 2-fold excess of HMG-14 with respect to DNA. HMG-I and HMG-2 exert instead a significant inhibitory effect on enzymic DNA methylation only under suitable ionic strength conditions (40 mM KCi). Also, histone proteins appear, at least in vitro, to affect the process of enzymic DNA methylation [9].
More recent studies from this laboratory [13] have shown that, within the 'physiological' range of histones-to-DNA ratios, only H~ is responsible for this inhibition, whose magnitude depends moreover on the secondary structure of the histone(s). On the other hand, the so-called 'tightly-bound non,histone proteins' [14-17], which are not dissociated from chromatin even upon exposure to very high ionic strength (above 2 M NaCI) - and which are reportedly related to the functional state of chromatin [14,15,18-21] - appear to play, in vitro as well as in vivo, an important role in facilitating the enzymic methylation of chmmatin DNA [22,23]. h~ the present study we describe a particular subset of tightly.bound proteins which, though resisting extraction even at high ionic strength, are quite similar to 'typical' histones in terms o1" acid solubility, apparent molecular mass and specific immunoreactivity. These 'tightly-bound histones', at variance from 'typical' ones, do not cause any inhibitory effect on the in vitro enzymic DNA methylation. Purified preparations of
the tightly-bound H rlike component were even found to exert a significant activation of the DNA methylation process. Materials and Methods
Isolation of chromatin and of chromatin subfractions Whole chromatin was prepared from human placenta according to Rickwood and Birnie [24], chromatin-bound proteinases being irreversibly inhibited by addition of 1 mM phenylmethylsulfonylfluoride (PMSF), which was thereafter present throughout all preparation steps. After extensive dialysis against 10 mM Tris-HCi buffer (pH 7.4) containing 0.5 mM dithiothreitol (DTT), 5 mM Na-EDTA and 1 mM PMSF, repeated treatment for i h at 4 ° C with 0.6 M NaC! in a 10 mM Tris-HCI + 2() mM Na-EDTA buffer (pH 7.4), yielded a histone Hi-depicted chromatin. Complete removal of all the histones and of the other loosely-bound chromatin proteins, could be obtained by further exposure to 2 M NaCI, yielding a 'stripped
Chromatin dissociatedfor 30 rainby low-salt" treatment: 0,6 M NaCI+ 20 mM EDTAin 10 mM Tris-HCI(pH 8) buffer ('LS buffer"); centrifugedat 10,000q)m for 10 min on a 8S34 Sorvall rotor -...- twice--I
SUPERNATANT
I
PELL r I
extractionwith 10% (w/v) HCIO4 J
I HI"deleted chr°matin I
I m°sa'Y'~ur
