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Exploring cellular memory molecules marking competent and active transcriptions.

Xin L, Zhou GL, Song W, Wu XS, Wei GH, Hao DL, Lv X, Liu DP, Liang CC - BMC Mol. Biol. (2007)

Bottom Line: In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome.Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription.Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: From National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China. xlgene@263.net <xlgene@263.net>

ABSTRACT

Background: Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation.

Results: We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription.

Conclusion: Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

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The distribution of active histone modifications on mouse α-like and β-like globin gene clusters in asynchronized (A) and mitotic (M) MEL cells. A, B. The changes of H3 and H4 acetylation level on the distant regulatory elements of globin loci in A and M cell populations. C, D. The changes of H3-K4 and H3-K79 dimethylataion on the distant regulatory elements of globin loci in A and M cell populations. The relative level of histone modifications at HS sites of globin clusters in A and M cell populations were analyzed after normalizing the DNA amount differences in A and M cell populations through DNA input (In-A and In-M).
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Figure 6: The distribution of active histone modifications on mouse α-like and β-like globin gene clusters in asynchronized (A) and mitotic (M) MEL cells. A, B. The changes of H3 and H4 acetylation level on the distant regulatory elements of globin loci in A and M cell populations. C, D. The changes of H3-K4 and H3-K79 dimethylataion on the distant regulatory elements of globin loci in A and M cell populations. The relative level of histone modifications at HS sites of globin clusters in A and M cell populations were analyzed after normalizing the DNA amount differences in A and M cell populations through DNA input (In-A and In-M).

Mentions: Besides protein factors, histone modification is recently identified as an efficient epigenetic factor involved in gene expression regulation through altering chromatin structure. Do the eukaryotic cells preserve some epigenetic marks on mitotic chromosomes to maintain its gene expression states? To verify the above-proposed possibility, we first observed the nuclear localization of four kinds of active histone modification including H3 acetylation, H4 acetylation, H3-K4 dimethylation and H3-K79 dimethylation in mitotic cells by in situ immunofluorescence. The results showed that all of the four modifications are retained on the mitotic chromosomes to some extent (Fig 4). We further detected the global changes of those modifications in both asynchronized and mitotic arrested MEL cell populations by western blotting. After correcting the variation in gel-loading and normalizing for the background, we found that there is a partial loss (about 20–30%) of H3 acetylation, H4 acetylation and H3-K4 dimethylation in mitotic cell extraction compared to those in asynchronous cell extraction but no obvious loss of H3-K79 dimethylation (Fig 5). This indicates that only part of histone acetylation and H3-K4 dimethylation signals can be inherited to mitotic chromatin through DNA replication-coupled chromatin assembly process. What is the detailed distribution of these preserved modifications on the mitotic chromosome? We therefore analysed the histone modification status across the distant hypersensitive sites and the adjacent promoter regions of mouse α- and β-globin genes (Fig 3A) during mitosis by the comparative ChIP analysis of the above mentioned asynchronous and mitotic arrested MEL cell populations. The results showed that HS26, HS21 and HS8 of mouse α-globin locus and HS3, HS2 and HS1 of mouse β-globin locus, as well as the promoters of α-globin and βmaj are acetylated at H3 and all of them except HS26 and HS3 are acetylated at H4 in asynchronous cells (Fig 6A, 6B). Higher H3-K4 dimethylation at HSs and α-globin promoter was noticed when compared to that at β-globin promoter(Fig 6C). HS8 and α-globin promoter are hypermethylated at H3-K79, but HS26 and HS21 show trace of signal. HS2 and HS1 of LCR are hypermethylated at H3-K79, while HS3 and βmaj promoter are just slightly methylated at H3-K79 (Fig 6D). In mitotic cells, the levels of H3 and H4 acetylation, H3-K4 dimethylation dropped at many analyzed regions, while H3-K79 dimethylation at all the analysed regions remain stable compared to those in asynchronous cells. Moreover H3-K79 dimethylation level on mitotic chromosomes is comparable to that in asynchronous cells, which consist with their chromatin states (Fig 6). The results suggested that, despite of some losses, the established active histone code at distant regulatory regions and globin gene promoters before mitotic chromatin inactivation can be stably inherited to mitotic chromosomes to mark the transcriptional competence of mouse globin genes in MEL cells.


Exploring cellular memory molecules marking competent and active transcriptions.

Xin L, Zhou GL, Song W, Wu XS, Wei GH, Hao DL, Lv X, Liu DP, Liang CC - BMC Mol. Biol. (2007)

The distribution of active histone modifications on mouse α-like and β-like globin gene clusters in asynchronized (A) and mitotic (M) MEL cells. A, B. The changes of H3 and H4 acetylation level on the distant regulatory elements of globin loci in A and M cell populations. C, D. The changes of H3-K4 and H3-K79 dimethylataion on the distant regulatory elements of globin loci in A and M cell populations. The relative level of histone modifications at HS sites of globin clusters in A and M cell populations were analyzed after normalizing the DNA amount differences in A and M cell populations through DNA input (In-A and In-M).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: The distribution of active histone modifications on mouse α-like and β-like globin gene clusters in asynchronized (A) and mitotic (M) MEL cells. A, B. The changes of H3 and H4 acetylation level on the distant regulatory elements of globin loci in A and M cell populations. C, D. The changes of H3-K4 and H3-K79 dimethylataion on the distant regulatory elements of globin loci in A and M cell populations. The relative level of histone modifications at HS sites of globin clusters in A and M cell populations were analyzed after normalizing the DNA amount differences in A and M cell populations through DNA input (In-A and In-M).
Mentions: Besides protein factors, histone modification is recently identified as an efficient epigenetic factor involved in gene expression regulation through altering chromatin structure. Do the eukaryotic cells preserve some epigenetic marks on mitotic chromosomes to maintain its gene expression states? To verify the above-proposed possibility, we first observed the nuclear localization of four kinds of active histone modification including H3 acetylation, H4 acetylation, H3-K4 dimethylation and H3-K79 dimethylation in mitotic cells by in situ immunofluorescence. The results showed that all of the four modifications are retained on the mitotic chromosomes to some extent (Fig 4). We further detected the global changes of those modifications in both asynchronized and mitotic arrested MEL cell populations by western blotting. After correcting the variation in gel-loading and normalizing for the background, we found that there is a partial loss (about 20–30%) of H3 acetylation, H4 acetylation and H3-K4 dimethylation in mitotic cell extraction compared to those in asynchronous cell extraction but no obvious loss of H3-K79 dimethylation (Fig 5). This indicates that only part of histone acetylation and H3-K4 dimethylation signals can be inherited to mitotic chromatin through DNA replication-coupled chromatin assembly process. What is the detailed distribution of these preserved modifications on the mitotic chromosome? We therefore analysed the histone modification status across the distant hypersensitive sites and the adjacent promoter regions of mouse α- and β-globin genes (Fig 3A) during mitosis by the comparative ChIP analysis of the above mentioned asynchronous and mitotic arrested MEL cell populations. The results showed that HS26, HS21 and HS8 of mouse α-globin locus and HS3, HS2 and HS1 of mouse β-globin locus, as well as the promoters of α-globin and βmaj are acetylated at H3 and all of them except HS26 and HS3 are acetylated at H4 in asynchronous cells (Fig 6A, 6B). Higher H3-K4 dimethylation at HSs and α-globin promoter was noticed when compared to that at β-globin promoter(Fig 6C). HS8 and α-globin promoter are hypermethylated at H3-K79, but HS26 and HS21 show trace of signal. HS2 and HS1 of LCR are hypermethylated at H3-K79, while HS3 and βmaj promoter are just slightly methylated at H3-K79 (Fig 6D). In mitotic cells, the levels of H3 and H4 acetylation, H3-K4 dimethylation dropped at many analyzed regions, while H3-K79 dimethylation at all the analysed regions remain stable compared to those in asynchronous cells. Moreover H3-K79 dimethylation level on mitotic chromosomes is comparable to that in asynchronous cells, which consist with their chromatin states (Fig 6). The results suggested that, despite of some losses, the established active histone code at distant regulatory regions and globin gene promoters before mitotic chromatin inactivation can be stably inherited to mitotic chromosomes to mark the transcriptional competence of mouse globin genes in MEL cells.

Bottom Line: In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome.Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription.Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: From National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China. xlgene@263.net <xlgene@263.net>

ABSTRACT

Background: Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation.

Results: We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription.

Conclusion: Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

Show MeSH
Related in: MedlinePlus