Early-replicating heterochromatin
1 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA; 2 Department of Biotechnology, VBS Purvanchal University, Jaunpur 222001, India\, http://www.100md.com
ABSTRACT\, http://www.100md.com
Euchromatin, which has an open structure and is frequently transcribed, tends to replicate in early S phase. Heterochromatin, which is more condensed and rarely transcribed, usually replicates in late S phase. Here, we report significant deviation from this correlation in the fission yeast, Schizosaccharomyces pombe. We found that heterochromatic centromeres and silent mating-type cassettes replicate in early S phase. Only heterochromatic telomeres replicate in late S phase. Research in other laboratories has shown that occasionally other organisms also replicate some of their heterochromatin in early S phase. Thus, late replication is not an obligatory feature of heterochromatin.\, http://www.100md.com
Introduction\, http://www.100md.com
"Heterochromatin" was originally defined as chromatin that remains condensed during interphase, whereas "euchromatin" decondenses during interphase (Heitz 1928, 1929). Later, the discovery that genes from euchromatin can become epigenetically inactivated if they are translocated into heterochromatin (for review, see Grewal and Elgin 2002) provided an additional, functional definition for heterochromatin. Recent studies have begun to provide a molecular definition as well (for review, see Grewal and Elgin 2002). Many forms of heterochromatin are characterized by hypermethylation of Lys 9 on histone H3 (H3-K9). In these cases, a protein with a chromodomain and a chromo-shadow domain (similar to Drosophila Hp1 and fission yeast Swi6) binds to methylated H3-K9 with its chromodomain, and then recruits additional proteins to the heterochromatic region with its chromo-shadow domain.
Until now, replication in late S phase has been considered to be another distinguishing feature of heterochromatin. The paradigm of late heterochromatin replication was first articulated by Lima-de-Faria and Jaworska (1968) on the basis of studies in a wide range of eukaryotic organisms. With minor exceptions, this paradigm has withstood the test of time (for review, see Gilbert 2002). Now, however, we report significant deviation from this paradigm in fission yeast.8l00)u1, 百拇医药
Results and Discussion8l00)u1, 百拇医药
Previous results: telomeres8l00)u1, 百拇医药
Chromatin near fission yeast telomeres is heterochromatic, because bringing normally active genes into the proximity of telomeres epigenetically inactivates the genes, and this inactivation is partially dependent on standard heterochromatin proteins including Swi6 (Nimmo et al. 1994; Allshire et al. 1995; Ekwall et al. 1995, 1996). Epigenetic inactivation of euchromatic genes introduced into telomere-proximal positions is called Telomere Position Effect (TPE) and is conserved from budding yeast (Gottschling et al. 1990; Tham and Zakian 2002) to humans (Baur et al. 2001).
Using several independent synchronization procedures, we have demonstrated previously that the terminal HindIII restriction fragments of fission yeast chromosomes I and II replicate in very late S phase (Kim and Huberman 2001), consistent with the paradigm of late heterochromatin replication. However, we were surprised when the same studies revealed that the outer portions of fission yeast centromeres replicate in early S phase.!{%)f1[, 百拇医药
Previous results: outer centromeres!{%)f1[, 百拇医药
Fission yeast centromeres consist of variable numbers of outer repeat (otr) sequences arranged, in inverted orientation, around an inner portion consisting of chromosome-specific innermost repeats (imr) inverted around a central sequence (cnt) that is fully or partially unique for each chromosome (Chikashige et al. 1989; Takahashi et al. 1992; Smith et al. 1995).!{%)f1[, 百拇医药
The otr region consists primarily of dg and dh repeats. These repeats are heterochromatic, because an indicator gene transplaced into otr becomes silenced (Allshire et al. 1995), and otr is associated with the heterochromatin protein, Swi6 (Partridge et al. 2000). Our previous results led to the surprising conclusion that the HindIII fragments from the dg repeats (also called K repeats) within the otr portions of the centromeres contain active chromosomal replication origins and replicate in very early S phase (Kim and Huberman 2001).
Inner centromeresz0, 百拇医药
In fission yeast, the chromatin within the inner portions of centromeres is heterochromatic in the sense that euchromatic genes introduced into inner centromeres become epigenetically silenced (Allshire et al. 1994, 1995). However, in fission yeast as in all eukaryotic organisms, histone H3 within the inner centromeres is replaced by a histone H3 paralog, frequently called CENP-A (for review, see Smith 2002). Because the N-terminal portion of CENP-A differs substantially from that of histone H3, CENP-A is not subject to K9 methylation, and it does not bind Hp1/Swi6-like proteins. Instead, inner centromeres are associated with the Mis6 protein (Partridge et al. 2000; Takahashi et al. 2000; Kniola et al. 2001)z0, 百拇医药
The surprising early replication of the outer portions of the centromeres raised the following question: Do the inner portions, which have a different heterochromatin structure, also eplicate in early S phase? To answer this question, we used the cdc10 block and release procedure (Kim and Huberman 2001) to synchronize cells, and we used two-dimensional agarose gel electrophoresis (Brewer and Fangman 1987) to measure the abundance of replication intermediates (RIs). In (bottom panel), the course of S phase is indicated by the position of the flow cytometry peak, which shifts from a DNA content of 1N to 2N primarily between 50 and 90 min after temperature downshift. Examples of complete flow cytometry profiles for this experiment are shown in Figure 3 of Kim and Huberman (2001). We found that RIs (primarily Y arcs) from the cnt sequences were most abundant in early S phasebetween 50 and 70 min after shift to 25°C. The weaker signals from RIs at earlier and later time points may be due to imperfect synchrony or may be indicative of some heterogeneity of replication times.
fig.ommitted(|5u, 百拇医药
Figure 1. The central portions of the centromeres are replicated in early S phase. (A) The cdc10 temperature block and release procedure (Kim and Huberman 2001) was used to synchronize passage through S phase. This experiment is identical to the one shown in Figure 4 in Kim and Huberman (2001). The Southern membrane used in that experiment was stripped and rehybridized with probes specific for the cnt sequences. (B) The hydroxyurea (HU) block and release procedure (Kim and Huberman 2001) was used to synchronize passage through S phase. This experiment is identical to the one shown in Figure 7 in Kim and Huberman (2001). The Southern membrane used in that experiment was stripped and rehybridized with probes specific for the cnt sequences. Note that the cells in the zero-minute time point had been exposed to HU for 5 h at 25°C.(|5u, 百拇医药
We confirmed the early replication of cnt sequences by using the independent hydroxyurea (HU) block and release procedure. RIs corresponding to cnt sequences accumulate in the presence of HU ( zero-minute time point). After HU is removed, cnt RIs disappear as replication forks run off. This behavior is typical of sequences that replicate in early S phase (Kim and Huberman 2001). Examples of flow cytometry profiles for this experiment are in Figure 6 of Kim and Huberman (2001).
Comparison of the cdc10 block and release results for cnt sequences in with our previously published cdc10 results for outer centromere (dg) sequences (Fig. 4B in Kim and Huberman 2001) suggests that the dg sequences replicate in very early S phase, whereas the cnt sequences replicate about 10 min later, but still in early S phase. The later replication of the cnt sequences is consistent with their having few, if any, active replication origins (note the near absence of bubble arc signals in ). Our results suggest that the cnt sequences are primarily replicated by replication forks coming from active origins in the dg or dh sequences, all within early S phase.|m%1p+%, 百拇医药
Silent mating-type cassettes|m%1p+%, 百拇医药
The remaining well-characterized heterochromatic region in fission yeast is the region containing the silent mating-type cassettes. There are two mating types in fission yeast, plus and minus. Wild-type fission yeast cells have three sets of genes (cassettes) encoding mating-type information, Plus or Minus. The three cassettesmat1, mat2, and mat3are located within 30 kb of each other on chromosome II. This wild-type configuration, h90, is diagrammed in (top panel). The region between mat1 and mat2 is called the "L region," and that between mat2 and mat3 is called the "K region." The genes (Plus or Minus) in mat1 are expressed (mat1-P or mat1-M). Those in mat2 (usually Plus; mat2-P) and mat3 (usually Minus; mat3-M) are silenced by heterochromatinization that is partially dependent on Swi6. The silenced state is indicated in the diagram by a gray (rather than black) plus sign or minus sign. The silenced, heterochromatic region is located between two inverted repeats that form boundaries between heterochromatin and euchromatin (IR-L and IR-R; Noma et al. 2001; Singh and Klar 2002; Thon et al. 2002). Halfway between these inverted repeats is a stretch with strong sequence similarity to a portion of the otr of CEN2. This "CEN homology" domain contributes significantly to formation of regional heterochromatin (Hall et al. 2002).(Soo-Mi Kim Dharani D. Dubey,2 and Joel A. Huberman)
ABSTRACT\, http://www.100md.com
Euchromatin, which has an open structure and is frequently transcribed, tends to replicate in early S phase. Heterochromatin, which is more condensed and rarely transcribed, usually replicates in late S phase. Here, we report significant deviation from this correlation in the fission yeast, Schizosaccharomyces pombe. We found that heterochromatic centromeres and silent mating-type cassettes replicate in early S phase. Only heterochromatic telomeres replicate in late S phase. Research in other laboratories has shown that occasionally other organisms also replicate some of their heterochromatin in early S phase. Thus, late replication is not an obligatory feature of heterochromatin.\, http://www.100md.com
Introduction\, http://www.100md.com
"Heterochromatin" was originally defined as chromatin that remains condensed during interphase, whereas "euchromatin" decondenses during interphase (Heitz 1928, 1929). Later, the discovery that genes from euchromatin can become epigenetically inactivated if they are translocated into heterochromatin (for review, see Grewal and Elgin 2002) provided an additional, functional definition for heterochromatin. Recent studies have begun to provide a molecular definition as well (for review, see Grewal and Elgin 2002). Many forms of heterochromatin are characterized by hypermethylation of Lys 9 on histone H3 (H3-K9). In these cases, a protein with a chromodomain and a chromo-shadow domain (similar to Drosophila Hp1 and fission yeast Swi6) binds to methylated H3-K9 with its chromodomain, and then recruits additional proteins to the heterochromatic region with its chromo-shadow domain.
Until now, replication in late S phase has been considered to be another distinguishing feature of heterochromatin. The paradigm of late heterochromatin replication was first articulated by Lima-de-Faria and Jaworska (1968) on the basis of studies in a wide range of eukaryotic organisms. With minor exceptions, this paradigm has withstood the test of time (for review, see Gilbert 2002). Now, however, we report significant deviation from this paradigm in fission yeast.8l00)u1, 百拇医药
Results and Discussion8l00)u1, 百拇医药
Previous results: telomeres8l00)u1, 百拇医药
Chromatin near fission yeast telomeres is heterochromatic, because bringing normally active genes into the proximity of telomeres epigenetically inactivates the genes, and this inactivation is partially dependent on standard heterochromatin proteins including Swi6 (Nimmo et al. 1994; Allshire et al. 1995; Ekwall et al. 1995, 1996). Epigenetic inactivation of euchromatic genes introduced into telomere-proximal positions is called Telomere Position Effect (TPE) and is conserved from budding yeast (Gottschling et al. 1990; Tham and Zakian 2002) to humans (Baur et al. 2001).
Using several independent synchronization procedures, we have demonstrated previously that the terminal HindIII restriction fragments of fission yeast chromosomes I and II replicate in very late S phase (Kim and Huberman 2001), consistent with the paradigm of late heterochromatin replication. However, we were surprised when the same studies revealed that the outer portions of fission yeast centromeres replicate in early S phase.!{%)f1[, 百拇医药
Previous results: outer centromeres!{%)f1[, 百拇医药
Fission yeast centromeres consist of variable numbers of outer repeat (otr) sequences arranged, in inverted orientation, around an inner portion consisting of chromosome-specific innermost repeats (imr) inverted around a central sequence (cnt) that is fully or partially unique for each chromosome (Chikashige et al. 1989; Takahashi et al. 1992; Smith et al. 1995).!{%)f1[, 百拇医药
The otr region consists primarily of dg and dh repeats. These repeats are heterochromatic, because an indicator gene transplaced into otr becomes silenced (Allshire et al. 1995), and otr is associated with the heterochromatin protein, Swi6 (Partridge et al. 2000). Our previous results led to the surprising conclusion that the HindIII fragments from the dg repeats (also called K repeats) within the otr portions of the centromeres contain active chromosomal replication origins and replicate in very early S phase (Kim and Huberman 2001).
Inner centromeresz0, 百拇医药
In fission yeast, the chromatin within the inner portions of centromeres is heterochromatic in the sense that euchromatic genes introduced into inner centromeres become epigenetically silenced (Allshire et al. 1994, 1995). However, in fission yeast as in all eukaryotic organisms, histone H3 within the inner centromeres is replaced by a histone H3 paralog, frequently called CENP-A (for review, see Smith 2002). Because the N-terminal portion of CENP-A differs substantially from that of histone H3, CENP-A is not subject to K9 methylation, and it does not bind Hp1/Swi6-like proteins. Instead, inner centromeres are associated with the Mis6 protein (Partridge et al. 2000; Takahashi et al. 2000; Kniola et al. 2001)z0, 百拇医药
The surprising early replication of the outer portions of the centromeres raised the following question: Do the inner portions, which have a different heterochromatin structure, also eplicate in early S phase? To answer this question, we used the cdc10 block and release procedure (Kim and Huberman 2001) to synchronize cells, and we used two-dimensional agarose gel electrophoresis (Brewer and Fangman 1987) to measure the abundance of replication intermediates (RIs). In (bottom panel), the course of S phase is indicated by the position of the flow cytometry peak, which shifts from a DNA content of 1N to 2N primarily between 50 and 90 min after temperature downshift. Examples of complete flow cytometry profiles for this experiment are shown in Figure 3 of Kim and Huberman (2001). We found that RIs (primarily Y arcs) from the cnt sequences were most abundant in early S phasebetween 50 and 70 min after shift to 25°C. The weaker signals from RIs at earlier and later time points may be due to imperfect synchrony or may be indicative of some heterogeneity of replication times.
fig.ommitted(|5u, 百拇医药
Figure 1. The central portions of the centromeres are replicated in early S phase. (A) The cdc10 temperature block and release procedure (Kim and Huberman 2001) was used to synchronize passage through S phase. This experiment is identical to the one shown in Figure 4 in Kim and Huberman (2001). The Southern membrane used in that experiment was stripped and rehybridized with probes specific for the cnt sequences. (B) The hydroxyurea (HU) block and release procedure (Kim and Huberman 2001) was used to synchronize passage through S phase. This experiment is identical to the one shown in Figure 7 in Kim and Huberman (2001). The Southern membrane used in that experiment was stripped and rehybridized with probes specific for the cnt sequences. Note that the cells in the zero-minute time point had been exposed to HU for 5 h at 25°C.(|5u, 百拇医药
We confirmed the early replication of cnt sequences by using the independent hydroxyurea (HU) block and release procedure. RIs corresponding to cnt sequences accumulate in the presence of HU ( zero-minute time point). After HU is removed, cnt RIs disappear as replication forks run off. This behavior is typical of sequences that replicate in early S phase (Kim and Huberman 2001). Examples of flow cytometry profiles for this experiment are in Figure 6 of Kim and Huberman (2001).
Comparison of the cdc10 block and release results for cnt sequences in with our previously published cdc10 results for outer centromere (dg) sequences (Fig. 4B in Kim and Huberman 2001) suggests that the dg sequences replicate in very early S phase, whereas the cnt sequences replicate about 10 min later, but still in early S phase. The later replication of the cnt sequences is consistent with their having few, if any, active replication origins (note the near absence of bubble arc signals in ). Our results suggest that the cnt sequences are primarily replicated by replication forks coming from active origins in the dg or dh sequences, all within early S phase.|m%1p+%, 百拇医药
Silent mating-type cassettes|m%1p+%, 百拇医药
The remaining well-characterized heterochromatic region in fission yeast is the region containing the silent mating-type cassettes. There are two mating types in fission yeast, plus and minus. Wild-type fission yeast cells have three sets of genes (cassettes) encoding mating-type information, Plus or Minus. The three cassettesmat1, mat2, and mat3are located within 30 kb of each other on chromosome II. This wild-type configuration, h90, is diagrammed in (top panel). The region between mat1 and mat2 is called the "L region," and that between mat2 and mat3 is called the "K region." The genes (Plus or Minus) in mat1 are expressed (mat1-P or mat1-M). Those in mat2 (usually Plus; mat2-P) and mat3 (usually Minus; mat3-M) are silenced by heterochromatinization that is partially dependent on Swi6. The silenced state is indicated in the diagram by a gray (rather than black) plus sign or minus sign. The silenced, heterochromatic region is located between two inverted repeats that form boundaries between heterochromatin and euchromatin (IR-L and IR-R; Noma et al. 2001; Singh and Klar 2002; Thon et al. 2002). Halfway between these inverted repeats is a stretch with strong sequence similarity to a portion of the otr of CEN2. This "CEN homology" domain contributes significantly to formation of regional heterochromatin (Hall et al. 2002).(Soo-Mi Kim Dharani D. Dubey,2 and Joel A. Huberman)