CELL BIOCHEMISTRY AND FUNCTION VOL. 9:
119-123 (1991)
Chromatin Phospholipid Changes During Rat Liver Development ELlSABETTA ALBIT, MARIAPIA VIOLA MAGNIT, REMO LAZZARINIt AND PETER B. GAHANf tlstituto di Patologia Generale, Universita di Perugia, Italy $Biosphere Sciences Division, King's College London, U.K.
The chromatin extracted from rat hepatocytes of different ages has been shown to contain a phospholipid fraction representing 0.47-0.59 per cent of total chromatin in newborn animals and 0-22 per cent in 45-day-old animals. No such age-related differences are observed in the nuclei. The phospholipidcomposition of the nuclei at different ages shows a higher level of sphingomyelin and a lower level of phosphatidylserine in newborn than in adult animals. Chromatin phospholipids have a completely different composition from that of nuclei with respect to age, particularly in newborn rats, where there is a decrease in phosphatidylcholine and an increase in phosphatidylserine. KEY
WORDS--Chromatin; phospholipids; hepatocytes; development,
INTRODUCTION The presence of a phospholipid fraction in chromatin has been demonstrated by both histochemical and biochemical t e c h i q ~ e s . 'This ~ ~ fraction, on extraction from chromatin isolated from rat hepatocytes, has been shown to have a phospholipid and fatty acid composition which differs from that of interphase n ~ c l e i .Its ~ synthesis and turnover also differ from the phospholipids of nuclear chromatin and m i c r o ~ o r n e s .Whilst ~ ~ ~ the synthesis of nuclear membrane phospholipids does not vary in relation to the S phase, chromatin phospholipid synthesis does increase in relation to DNA synthesis.'.' During the first few days after birth, hepatocytes undergo a biochemical maturation accompanied by an increase in the amount of DNA/diploid nucleus as a consequence of a de nouo synthesis of a DNA aliquot characterized by a different AT content.'-1° Synthesis of chromatin phospholipids also increases during cell duplication following
Addressee lor correspondence: Peter B. Gahan, King's College London, Carnpden Hill Road. Kensington, London W8 7AH, U.K. 0263-6484/9 1!020119-05 $05.00 Q') 1991 by John Wiley & Sons, Ltd.
hepatectomy and is correlated with DNA synthesk6 However, the rates of synthesis of the component phospholipids vary: phosphatidylserine and phosphatidylcholine increasing greatly at the beginning of S phase and others, such as phosphatidylethanolamine, doing so at the end of S phase (unpublished data), indicating the possibility of a different role for each phospholipid. It may be that the changes in phospholipids could be related to two possible, but not mutually exclusive, events. Either the duplication of some DNA strands might be regulated by specific linkages to different phospholipids, or the higher levels of newly synthesized phospholipids may be involved in transcriptional control of the newly synthesized DNA through an on/off switching controlled by phosphatidylserine In view and phosphatidylcholine, respectively.' of the indication of the possible involvement of the chromatin-associated phospholipid with RNA synthesis,' ' - 1 3 the hepatocytes from newborn rats could provide a suitable model on which to investigate further the role(s) of these phospholipids. As a first stage, we report the analysis of nuclear and chromatin phospholipids, extracted from hepatocytes isolated at 0 and 6 days. These results are compared with those from hepatocytes of 45-dayold rats.
'-'
120 MATERIALS AND METHODS
E. ALBl ET A L .
Biochemical Determinations
The lipids were extracted with chloroformmethanol16 and analysed chromatographically Sprague Dawley rats of both sexes, either at birth on thin layer silica gels in a bidimensional (100 animals per experiment) or 6 days (90 animals system using chloroform: methano1:ammonia per experiment) or 45 days (6 animals per experi- (65:25:4v/v) for the first run. The gels were ment) old, were killed and the pooled livers kept dried and exposed for 10min to concentrated in liquid nitrogen. A small portion of the liver HCl vapour and dried again. The second run was was used for the isolation of microsomes,6 the made with ch1oroform:methanol:acetone:acetic acid:distilled water (70: 15:30:15:7.5 v/v). The remainder for nuclei and chromatin preparations. lipids were detected with iodine vapour and scraped into test tubes for inorganic phosphorus d e t e r m i n a t i ~ n .The ~ ~ ~exposure to HCl vapour Nuclei Isolarion between the two runs allows a better separation of The nuclei were prepared according to the meth- phosphatidylinositol and phosphatidylserine. The concentrations of DNA,” RNA’* and protein” od of Bresnick et a l l 4 Briefly, the liver was homogenized in a cold were determined in both isolated nuclei and chrosolution of 2.2 M sucrose containing 3.3 mM CaCI, matin. The purity of the single preparations was and 1 mM phenylmethylsulfonyfluoride (PMFS) determined as previously described.I3 and centrifuged at 50 000 g for 60 min. The sediment is formed by an homogeneous population of RESULTS hepatocyte nuclei without contamination from other kinds of nuclei present in the liver. The Nuclei and Chromatin Composition (Table l a separated nuclei were washed twice in the physio- and b ) logical solution as previously de~cribed.~ No great differences in nuclear composition between 0-6 and 45 days were observed, only the RNA changing with age, increasing in 6-day-old Chromatin Extraction and decreasing in 45-day-old rats. The results reThe nuclei were gently suspended for 5 min in ported for 45-day-old animals are similar to those 1 mM PMFS, pH 7.4 containing 0.3 per cent Triton reported previ~usly.~ X- 100, 0.25 M sucrose, 10 mM Tris and centrifuged The isolated chromatin contained more DNA for 5 min at 5000 g. The swelling of the nuclei and and less protein than the nuclei. Nevertheless, the extraction of chromatin was done according to the amount of DNA differed with respect to age, being method of Shaw and Huang.‘’ The nuclei were higher in the newborn than in adult rats. The RNA washed four times with hypotonic solution increased significantly during the first six days of (0.075 M NaClO.024 M EDTA, 1 mM PMHS pH 8) life in relation to the cell maturation process.20 and with progressively less concentrated Tris sol- However, the most striking variation occurred in utions (from 5 0 m to ~ 0 . 4 ~ ) All . solutions con- the level of phospholipids which at 0-6 days was tained 1 mM PMFS and all procedures were double that observed in 45-day-old rats (Table 1). performed at 0-4°C. Note that the composition reported for 45 days Finally, the material was resuspended in 1 mM remains unchanged with age.21 This cannot be PMFS (100 ml for about 20 g of liver), brought to attributed to a faulty preparation of the chromatin pH 8 with ammonium hydroxide and stirred over- as then a similar increase in protein levels should night at 14°C. The solution was then centrifuged at have been observed. 90 000 g. The sediment containing chromatin was resuspended in 10 mM Tris pH 8. The preparation Phospholipid Composition (Table 2 ) was controlled: (i) by light microscopy after methyThe analysis of the composition of phospholipids lene blue staining in order to verify the swelling of the nuclei and the separation of chromatin; (ii) by from the microsomal fraction showed no significant the biochemical marker previously described (glu- changes with age for phosphatidylcholine, phosco~e-6-phosphatase).~ phatidylserine and phosphatidylethanolamine, Animals
121
CHROMATIN PHOSPHOLIPIDS IN DEVELOPMENT
Table la. Composition of isolated hepatocyte nuclei and chromatin at different ages.
DNA 0 days 6 days 45 dys RNA 0 days 6 days 45 days Proteins 0 days 6 days 45 days Phospholipids 0 days 6 days 45 days
Days DNA RNA Prot. Phosp.
Nuclei
Chromatin
%
%
18.70 f 1.76 (5) 17.77 & 2.09 (3) 23.74 f 0.65 (3)
+
38.01 f 3.20 (5) 34.02 f 3.49 (3) 30.30 f 2.56 (3)
+
3.43 1.05 ( 5 ) 5.25 k 1.03 (3) 3.39 f 0.30 (3)
5.58 1.40 (5) 8.81 f 0.54 (3) 4.50 0.34 (3)
76.10 2.36 (5) 73.53 3.72 (3) 71.33 f 2.30 (3)
55.77 f 3.70 (5) 57.31 f 2.69 (3) 64.80 rt 2.57 (3)
+
1-73f 0.34 ( 5 ) 1.33 f 0.26 (3) 1.93 0.32 (3)
+
+
0.47 +_ 0.015 (5) 0.59 & 0.10 (3) 0.22 & 0.06 (3)
Nuclei Chromatin N./Chr. 016 6/45 0145 016 6/45 0145 0 6 45 S S NS NS S S* S S NS S NS NS S S S NS S S* S NS S S S* S S NS NS S* S* S S* NS NS NS S S
Table 1b.
RNA 0 days 6 days 45 dys Proteins 0 days 6 days 45 days Phospholipids 0 days 6 days 45 days
Days RNA Prot. Phosp.
016 NS NS NS
Nuclei
Chromatin
04
%
0.1838 & 0.0513 (5) 0.2990 & 0,0392 (3) 0.1467 0.0134 (3)
0.1466 rt 0.0338 ( 5 ) 0.2630 0.0308 (3) 0.1507 k 0.0137 (3)
4.1028 k 0.4283 ( 5 ) 4-1870 f 0.5648(3) 2.9457 f 0.1 308 (3)
1.4796 f 0.0338 (5) 1.6810 & 0.2379 (3) 2.0713 0.2338 (3)
0.0924 & 0.0 144 ( 5 ) 0.0803 f 0.0133 (3) 0.0813 +0.0111 (3)
0.0120 k 0~0011( 5 ) 0.0176 OC037 (3) 0.0086 k 0,0022 (3)
Nuclei 6/45 S NS NS
0145 NS S NS
+
+
016 S NS S
Chromatin 6/45 0145 S NS S S S S
The values are expressed as mg of RNA or proteins or phospholipids per mg DNA. In parenthesis the number of experiments and at foot of table significance values, S: p < 0.05 and S: p < 0.001.
~
The v'iluec expressed as "6 of different components, reprcbent the average + S D In parenthesis the number ofexperlments and at foot of table significance values. S. p < 0 05 a n d S * p
119-123 (1991)
Chromatin Phospholipid Changes During Rat Liver Development ELlSABETTA ALBIT, MARIAPIA VIOLA MAGNIT, REMO LAZZARINIt AND PETER B. GAHANf tlstituto di Patologia Generale, Universita di Perugia, Italy $Biosphere Sciences Division, King's College London, U.K.
The chromatin extracted from rat hepatocytes of different ages has been shown to contain a phospholipid fraction representing 0.47-0.59 per cent of total chromatin in newborn animals and 0-22 per cent in 45-day-old animals. No such age-related differences are observed in the nuclei. The phospholipidcomposition of the nuclei at different ages shows a higher level of sphingomyelin and a lower level of phosphatidylserine in newborn than in adult animals. Chromatin phospholipids have a completely different composition from that of nuclei with respect to age, particularly in newborn rats, where there is a decrease in phosphatidylcholine and an increase in phosphatidylserine. KEY
WORDS--Chromatin; phospholipids; hepatocytes; development,
INTRODUCTION The presence of a phospholipid fraction in chromatin has been demonstrated by both histochemical and biochemical t e c h i q ~ e s . 'This ~ ~ fraction, on extraction from chromatin isolated from rat hepatocytes, has been shown to have a phospholipid and fatty acid composition which differs from that of interphase n ~ c l e i .Its ~ synthesis and turnover also differ from the phospholipids of nuclear chromatin and m i c r o ~ o r n e s .Whilst ~ ~ ~ the synthesis of nuclear membrane phospholipids does not vary in relation to the S phase, chromatin phospholipid synthesis does increase in relation to DNA synthesis.'.' During the first few days after birth, hepatocytes undergo a biochemical maturation accompanied by an increase in the amount of DNA/diploid nucleus as a consequence of a de nouo synthesis of a DNA aliquot characterized by a different AT content.'-1° Synthesis of chromatin phospholipids also increases during cell duplication following
Addressee lor correspondence: Peter B. Gahan, King's College London, Carnpden Hill Road. Kensington, London W8 7AH, U.K. 0263-6484/9 1!020119-05 $05.00 Q') 1991 by John Wiley & Sons, Ltd.
hepatectomy and is correlated with DNA synthesk6 However, the rates of synthesis of the component phospholipids vary: phosphatidylserine and phosphatidylcholine increasing greatly at the beginning of S phase and others, such as phosphatidylethanolamine, doing so at the end of S phase (unpublished data), indicating the possibility of a different role for each phospholipid. It may be that the changes in phospholipids could be related to two possible, but not mutually exclusive, events. Either the duplication of some DNA strands might be regulated by specific linkages to different phospholipids, or the higher levels of newly synthesized phospholipids may be involved in transcriptional control of the newly synthesized DNA through an on/off switching controlled by phosphatidylserine In view and phosphatidylcholine, respectively.' of the indication of the possible involvement of the chromatin-associated phospholipid with RNA synthesis,' ' - 1 3 the hepatocytes from newborn rats could provide a suitable model on which to investigate further the role(s) of these phospholipids. As a first stage, we report the analysis of nuclear and chromatin phospholipids, extracted from hepatocytes isolated at 0 and 6 days. These results are compared with those from hepatocytes of 45-dayold rats.
'-'
120 MATERIALS AND METHODS
E. ALBl ET A L .
Biochemical Determinations
The lipids were extracted with chloroformmethanol16 and analysed chromatographically Sprague Dawley rats of both sexes, either at birth on thin layer silica gels in a bidimensional (100 animals per experiment) or 6 days (90 animals system using chloroform: methano1:ammonia per experiment) or 45 days (6 animals per experi- (65:25:4v/v) for the first run. The gels were ment) old, were killed and the pooled livers kept dried and exposed for 10min to concentrated in liquid nitrogen. A small portion of the liver HCl vapour and dried again. The second run was was used for the isolation of microsomes,6 the made with ch1oroform:methanol:acetone:acetic acid:distilled water (70: 15:30:15:7.5 v/v). The remainder for nuclei and chromatin preparations. lipids were detected with iodine vapour and scraped into test tubes for inorganic phosphorus d e t e r m i n a t i ~ n .The ~ ~ ~exposure to HCl vapour Nuclei Isolarion between the two runs allows a better separation of The nuclei were prepared according to the meth- phosphatidylinositol and phosphatidylserine. The concentrations of DNA,” RNA’* and protein” od of Bresnick et a l l 4 Briefly, the liver was homogenized in a cold were determined in both isolated nuclei and chrosolution of 2.2 M sucrose containing 3.3 mM CaCI, matin. The purity of the single preparations was and 1 mM phenylmethylsulfonyfluoride (PMFS) determined as previously described.I3 and centrifuged at 50 000 g for 60 min. The sediment is formed by an homogeneous population of RESULTS hepatocyte nuclei without contamination from other kinds of nuclei present in the liver. The Nuclei and Chromatin Composition (Table l a separated nuclei were washed twice in the physio- and b ) logical solution as previously de~cribed.~ No great differences in nuclear composition between 0-6 and 45 days were observed, only the RNA changing with age, increasing in 6-day-old Chromatin Extraction and decreasing in 45-day-old rats. The results reThe nuclei were gently suspended for 5 min in ported for 45-day-old animals are similar to those 1 mM PMFS, pH 7.4 containing 0.3 per cent Triton reported previ~usly.~ X- 100, 0.25 M sucrose, 10 mM Tris and centrifuged The isolated chromatin contained more DNA for 5 min at 5000 g. The swelling of the nuclei and and less protein than the nuclei. Nevertheless, the extraction of chromatin was done according to the amount of DNA differed with respect to age, being method of Shaw and Huang.‘’ The nuclei were higher in the newborn than in adult rats. The RNA washed four times with hypotonic solution increased significantly during the first six days of (0.075 M NaClO.024 M EDTA, 1 mM PMHS pH 8) life in relation to the cell maturation process.20 and with progressively less concentrated Tris sol- However, the most striking variation occurred in utions (from 5 0 m to ~ 0 . 4 ~ ) All . solutions con- the level of phospholipids which at 0-6 days was tained 1 mM PMFS and all procedures were double that observed in 45-day-old rats (Table 1). performed at 0-4°C. Note that the composition reported for 45 days Finally, the material was resuspended in 1 mM remains unchanged with age.21 This cannot be PMFS (100 ml for about 20 g of liver), brought to attributed to a faulty preparation of the chromatin pH 8 with ammonium hydroxide and stirred over- as then a similar increase in protein levels should night at 14°C. The solution was then centrifuged at have been observed. 90 000 g. The sediment containing chromatin was resuspended in 10 mM Tris pH 8. The preparation Phospholipid Composition (Table 2 ) was controlled: (i) by light microscopy after methyThe analysis of the composition of phospholipids lene blue staining in order to verify the swelling of the nuclei and the separation of chromatin; (ii) by from the microsomal fraction showed no significant the biochemical marker previously described (glu- changes with age for phosphatidylcholine, phosco~e-6-phosphatase).~ phatidylserine and phosphatidylethanolamine, Animals
121
CHROMATIN PHOSPHOLIPIDS IN DEVELOPMENT
Table la. Composition of isolated hepatocyte nuclei and chromatin at different ages.
DNA 0 days 6 days 45 dys RNA 0 days 6 days 45 days Proteins 0 days 6 days 45 days Phospholipids 0 days 6 days 45 days
Days DNA RNA Prot. Phosp.
Nuclei
Chromatin
%
%
18.70 f 1.76 (5) 17.77 & 2.09 (3) 23.74 f 0.65 (3)
+
38.01 f 3.20 (5) 34.02 f 3.49 (3) 30.30 f 2.56 (3)
+
3.43 1.05 ( 5 ) 5.25 k 1.03 (3) 3.39 f 0.30 (3)
5.58 1.40 (5) 8.81 f 0.54 (3) 4.50 0.34 (3)
76.10 2.36 (5) 73.53 3.72 (3) 71.33 f 2.30 (3)
55.77 f 3.70 (5) 57.31 f 2.69 (3) 64.80 rt 2.57 (3)
+
1-73f 0.34 ( 5 ) 1.33 f 0.26 (3) 1.93 0.32 (3)
+
+
0.47 +_ 0.015 (5) 0.59 & 0.10 (3) 0.22 & 0.06 (3)
Nuclei Chromatin N./Chr. 016 6/45 0145 016 6/45 0145 0 6 45 S S NS NS S S* S S NS S NS NS S S S NS S S* S NS S S S* S S NS NS S* S* S S* NS NS NS S S
Table 1b.
RNA 0 days 6 days 45 dys Proteins 0 days 6 days 45 days Phospholipids 0 days 6 days 45 days
Days RNA Prot. Phosp.
016 NS NS NS
Nuclei
Chromatin
04
%
0.1838 & 0.0513 (5) 0.2990 & 0,0392 (3) 0.1467 0.0134 (3)
0.1466 rt 0.0338 ( 5 ) 0.2630 0.0308 (3) 0.1507 k 0.0137 (3)
4.1028 k 0.4283 ( 5 ) 4-1870 f 0.5648(3) 2.9457 f 0.1 308 (3)
1.4796 f 0.0338 (5) 1.6810 & 0.2379 (3) 2.0713 0.2338 (3)
0.0924 & 0.0 144 ( 5 ) 0.0803 f 0.0133 (3) 0.0813 +0.0111 (3)
0.0120 k 0~0011( 5 ) 0.0176 OC037 (3) 0.0086 k 0,0022 (3)
Nuclei 6/45 S NS NS
0145 NS S NS
+
+
016 S NS S
Chromatin 6/45 0145 S NS S S S S
The values are expressed as mg of RNA or proteins or phospholipids per mg DNA. In parenthesis the number of experiments and at foot of table significance values, S: p < 0.05 and S: p < 0.001.
~
The v'iluec expressed as "6 of different components, reprcbent the average + S D In parenthesis the number ofexperlments and at foot of table significance values. S. p < 0 05 a n d S * p
