The Role of Histone Acetylation in Regulating Early Gene Expression Patterns during Early Embryonic Stem Cell Differentiation

We have examined the role of histone acetylation in the very earliest steps of differentiation of mouse embryonic stem cells in response to withdrawal of leukemia inhibitory factor (LIF) as a differentiation signal. The cells undergo dramatic changes in morphology and an ordered program of gene expression changes representing differentiation to all three germ layers over the first 3-5 days of LIF withdrawal. We observed a global increase in acetylation on histone H4 and to a lesser extent on histone H3 over this time period. Treatment of the cells with trichostatin A (TSA), a histone deacetylase inhibitor, induced changes in morphology, gene expression, and histone acetylation that mimicked differentiation induced by withdrawal of LIF. We examined localized histone acetylation in the regulatory regions of genes that were transcriptionally either active in undifferentiated cells, induced during differentiation, or inactive under all treatments. There was striking concordance in the histone acetylation patterns of specific genes induced by both TSA and LIF withdrawal. Increased histone acetylation in local regions correlated best with induction of gene expression. Finally, TSA treatment did not support the maintenance or progression of differentiation. Upon removal of TSA, the cells reverted to the undifferentiated phenotype. We concluded that increased histone acetylation at specific genes played a role in their expression, but additional events are required for maintenance of differentiated gene expression and loss of the pluripotent state. We have examined the role of histone acetylation in the very earliest steps of differentiation of mouse embryonic stem cells in response to withdrawal of leukemia inhibitory factor (LIF) as a differentiation signal. The cells undergo dramatic changes in morphology and an ordered program of gene expression changes representing differentiation to all three germ layers over the first 3-5 days of LIF withdrawal. We observed a global increase in acetylation on histone H4 and to a lesser extent on histone H3 over this time period. Treatment of the cells with trichostatin A (TSA), a histone deacetylase inhibitor, induced changes in morphology, gene expression, and histone acetylation that mimicked differentiation induced by withdrawal of LIF. We examined localized histone acetylation in the regulatory regions of genes that were transcriptionally either active in undifferentiated cells, induced during differentiation, or inactive under all treatments. There was striking concordance in the histone acetylation patterns of specific genes induced by both TSA and LIF withdrawal. Increased histone acetylation in local regions correlated best with induction of gene expression. Finally, TSA treatment did not support the maintenance or progression of differentiation. Upon removal of TSA, the cells reverted to the undifferentiated phenotype. We concluded that increased histone acetylation at specific genes played a role in their expression, but additional events are required for maintenance of differentiated gene expression and loss of the pluripotent state. Mouse embryonic stem (mES) 2The abbreviations used are: mES cells, mouse embryonic stem cells; LIF, leukemia inhibitory factor; TSA, trichostatin A; Fgf, fibroblast growth factor; PR, progesterone receptor; Brachy, brachyury; Afp, α-fetoprotein; Ins, insulin; Prl, prolactin; ChIP, chromatin immunoprecipitation; Hoxb1, homeobox b1; PR, progesterone receptor; PBS, phosphate-buffered saline; RT, room temperature; AcH3, acetylated histone H3. cells are characterized by pluripotency and their capacity for self-renewal (1Solter D. Nat. Rev. Genet. 2006; 7: 319-327Crossref PubMed Scopus (248) Google Scholar). Upon exposure to a differentiation signal these cells are capable of multi-lineage differentiation into cells of the three germ layers: ectoderm, mesoderm, and endoderm (2Gadue P. Huber T.L. Nostro M.C. Kattman S. Keller G.M. Exp. Hematol. 2005; 33: 955-964Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The term “commitment” is frequently used with regard to achieving a new differentiated phenotype, assayed by changes in morphology and patterns of gene expression (3Keller G.M. Curr. Opin. Cell Biol. 1995; 7: 862-869Crossref PubMed Scopus (781) Google Scholar, 4Loebel D.A. Watson C.M. De Young R.A. Tam P.P. Dev. Biol. 2003; 264: 1-14Crossref PubMed Scopus (213) Google Scholar, 5Keller G. Genes Dev. 2005; 19: 1129-1155Crossref PubMed Scopus (951) Google Scholar). Commitment to the differentiated phenotype is probably a different, although not completely independent cellular process than loss of the undifferentiated state. The loss of the undifferentiated state occurs after exposure to a differentiation signal that results in an irreversible loss of the mES cell phenotype, characterized by loss of tight colony growth, altered self-renewal, and a pattern of marker gene expression that no longer resembles undifferentiated mES cells. We are interested in identifying the molecular events that regulate loss of the undifferentiated state of mES cells and commitment to lineage specific differentiation. Mouse ES cells are maintained in the undifferentiated state by adding leukemia inhibitory factor (LIF) to the culture media. A common method of inducing differentiation is withdrawal of LIF from the culture media, resulting in multi-lineage differentiation, indicated by the induced expression of lineage marker genes (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). During early mES cell differentiation, the rate of transcription of large numbers of genes is substantially altered in a time-dependent fashion. Epigenetic changes are proposed to play a role in regulating both local and global gene expression through post-translational histone modifications (7Huebert D.J. Bernstein B.E. Curr. Opin. Genet. Dev. 2005; 15: 476-481Crossref PubMed Scopus (28) Google Scholar). Some histone modifications result in result in Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Some of these modifications to over resulting in large modifications of the histone have proposed to as that or of the state of specific genes or regions of the Full Text Full Text PDF PubMed Scopus Google Scholar, D. G. Cell Biol. 2005; PubMed Scopus Google Scholar). with both and histone modifications are in undifferentiated mES cells B.E. X. D.J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). We that commitment and changes in transcription with very early differentiation of mES cells are at in by local global modifications of histone We examined the role of acetylation of H3 and H4 in regulating the early events during differentiation of mES cells. increased acetylation of H3 and H4 is with increased gene acetylation increase transcription factor to the by with the histone and of acetylation chromatin Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Cell Biol. 2005; PubMed Scopus Google Scholar). We observed a global increase in acetylation of H3 and H4 during the first days of mES cell differentiation induced by LIF withdrawal. of a of marker genes that increased histone acetylation was not at all local the regulatory regions of specific genes and correlated with the increase in transcription from We that of H3 and H4 by treatment with the histone deacetylase trichostatin A induced a and molecular expression program that mimicked early differentiation induced by LIF withdrawal. the cells were not to a differentiation of TSA in a to the undifferentiated cell phenotype. We that global acetylation of H3 and H4 the expression of marker but is not for early commitment to differentiation and loss of the pluripotent state. Cell mES cells were used for all and were from with from Keller PubMed Scopus Google Scholar, G. Biol. PubMed Scopus Google Scholar). were as (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). of were from cells as (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). of and of was as (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). used and are in the were with phosphate-buffered with into and at at was and the cells were at The cells were in of to for at and in a with of a The was at for at in a and the was The was in at of for at and to at was for at and at the for at in a of was and and were to for at by at for at in a The histone was with of at with and The resulting was with at and The was to in of at room and at were the from were by on and as were as a cell by of the growth The cells were a at for at room were with in for at were with in and with in for at at was in for at in a The were with for at RT, and at was for at The cells were with at and were and Cell was of was to in and used at in were by to and were acetylated histone H3 acetylated histone H4 histone H3 and of histone H3 for histone H3 was used as a were and was by either or was as with as in the X. Murdoch F.E. Fritsch M.K. PubMed Scopus Google Scholar). was on from the and the chromatin used for of by for regulatory regions are in The were on a with and the with for or no were from the numbers and than of the signal with specific The of the over the for the mES was to and all were to this as a acetylation to of the of acetylation of H3 and H4 at in the cells, the were at the regulatory and this was to and all were to independent were and the were as the Increased during of mES by LIF were from undifferentiated mES cells maintained in the of LIF and from cells induced to by LIF withdrawal for in of acetylated H3 and H4 increased over the days of differentiation. The of histone H4 an increase in global acetylation with localized on after LIF on and through The of acetylation of histone H3 but and a increase by The of on of histone H3 did not during this period. in LIF withdrawal a specific pattern of gene expression and of induction and of marker genes over this time days of LIF this pattern of gene expression was from the undifferentiated mES cells at a for multi-lineage differentiation. The gene expression pattern at days of LIF withdrawal that of undifferentiated mES cells, with for and of marker genes was to their expression in the pluripotent stem cell state. The of all three of these genes to LIF withdrawal but at The of marker to as were not in the undifferentiated mES cell but a ordered increase in their expression during LIF withdrawal. for the increased of LIF for and days and The of were not during the early of differentiation examined in these and did not increase after days of LIF withdrawal (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). and are genes that were not after LIF withdrawal in after longer induction were on from cell and not was in undifferentiated cells, and did not over this period. was used as an in with and the changes in gene expression that during early differentiation with localized or global histone examined regulatory regions of the marker gene during early differentiation We to regulatory and of their in the gene increased histone acetylation in regions is to for regulatory regions of the marker gene in were after of the chromatin with either histone H3 or histone H4 and changes in of histone acetylation to the undifferentiated cells for the three gene and A and the acetylation of H3 and in regulatory regions of the are in undifferentiated mES cells but over days of differentiation and The regulatory of a in both H3 and H4 acetylation of LIF withdrawal. and a in histone H4 acetylation loss of histone although was in to the global increase in histone the of all localized for acetylated H3 and H4 at the regulatory regions of the gene as in The of acetylated at and and days of LIF withdrawal is as a to that for undifferentiated mES cells the of the from three independent in acetylated histone during early differentiation. in during early for acetylated H3 and H4 at the regulatory regions of the gene as in are as in A and in acetylated histone H3 during early differentiation. and in during early for acetylated H3 and H4 at the regulatory regions of the gene as in are as in A and in acetylated H3 during early differentiation. and in during early of acetylation of H3 and H4 in undifferentiated mES cells substantially gene regulatory acetylation in the regulatory regions in was assayed by on undifferentiated mES cells. The of histone acetylation at the was to and the of acetylation of histone H3 or histone H4 are as a to the of acetylation of the The the from three independent the histone acetylation in the regulatory regions of genes that were not in undifferentiated cells but were induced during LIF withdrawal in histone acetylation over the differentiation time were observed for all of these is a have is on in mES cells after LIF withdrawal (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). of PR, and are by a gene with Res. PubMed Scopus Google Scholar). The is of the the all the an additional The used in did not the in A and the a increase in histone H4 acetylation and a increase in histone H3 acetylation as early as after LIF withdrawal. acetylation of histone H4 at the a increase at days and in acetylation of histone H3. The increased histone acetylation of the increase in observed at (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). regulatory regions for the in the and the in the of the were examined D. Dev. PubMed Scopus Google Scholar). of histone H4 in the increased by by of differentiation, with in histone H3 acetylation A and H4 acetylation of the and to and of histone H3 a increase by The increase in histone H3 acetylation the increased expression of at days and in and acetylation of both H3 and H4 in the regulatory increased days after LIF with increased gene expression. of histone H3 in the regulatory increased days after LIF withdrawal with increased of Increased acetylation of histone H4 in this was not after days increased acetylation of either or both H3 and H4 was observed in at regulatory of gene or with A and the acetylation of H3 and in three regulatory regions of after days of LIF withdrawal (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). The gene is genes and as as regulatory is for of the S. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, P.P. 2006; PubMed Scopus Google Scholar). The gene is early in Keller G. PubMed Google Scholar). The local of this gene a increase in histone H3 acetylation after days of LIF but in the of H4 acetylation over the days A and The a with increased acetylation of both H3 and H4 after LIF withdrawal by a of acetylated histone H4 to the in undifferentiated cells and a in acetylated histone H3 to that in undifferentiated cells. was maintained the days of LIF withdrawal The regulatory an increase in the acetylation of histone H3 over the days of LIF withdrawal and at H4 A and We have that both and are in this cell (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google and these local in histone acetylation to these genes in a chromatin during this early differentiation period. Finally, of histone acetylation were not altered in the regulatory regions of genes and that were not at in this mES cell and of H3 and H4 in mES the of the used to a of the acetylation of the histone at the regulatory regions of the marker gene in the undifferentiated mES cells as in all the of acetylation of both H3 and H4 was in the regulatory this to and in regulatory regions are to the The three genes of the of the of acetylation of H3 and H4 The of histone acetylation in the regulatory regions of the of the marker gene were There was no the of histone acetylation in the undifferentiated state and the of that is in response to LIF withdrawal for the was indicated by the of histone acetylation for the induced expression in both regulatory and acetylation of H3 and H4 than the of genes were by LIF withdrawal. The acetylation of H3 and H4 for the of genes was than for the the results from with and differentiation induced the in local histone acetylation in gene regulatory regions that both a of histone acetylation and were transcriptionally by differentiation. TSA Treatment an LIF on the that global histone acetylation increased during differentiation induced by LIF withdrawal induction of global histone acetylation was for early differentiation of mES cells. TSA is a of and histone of these histone results in increased global acetylation of in cell mES cells S. Biol. Full Text Full Text PDF PubMed Scopus Google and in TSA treatment induced dramatic changes to cell morphology in mES cells maintained in LIF as by and The morphology of cells that of LIF cells the tight were cellular were and cell were TSA treatment of cells in the of LIF in a differentiated morphology The changes induced by TSA were removal from as in the of and were for phenotype, and are in of TSA, in an undifferentiated of a differentiated morphology after TSA treatment in the of LIF. than of the an undifferentiated morphology after withdrawal of TSA for days in TSA treatment of mES cells in the of LIF induced an ordered in gene expression that mimicked LIF although was a gene from the was in undifferentiated mES cells and after of TSA TSA treatment a ordered induction of the genes in the PR, Hoxb1, and by The genes and were not induced by TSA a of was in at The pattern of gene expression observed after of TSA in the of LIF that induced by LIF withdrawal for days with results support a in the induced by TSA is for the of a gene expression in the of TSA were at the of gene expression. The expression pattern for genes altered by TSA in was over a time after removal of were with TSA for time after TSA was and cells were for the indicated in media. in the gene expression pattern reverted to that of the undifferentiated cells as early as after TSA withdrawal and was maintained to TSA Treatment Commitment to in treatment of mES cells mimicked differentiation induced by LIF withdrawal. The results from a of cells. of cells did not to a differentiation as by an irreversible in gene expression and There are for these this to a of cells undifferentiated during TSA and cells after TSA cells the differentiated phenotype or TSA have induced a in all the cells that was was the to of the that indicated changes to all on the cell and days the time of in the or of LIF LIF were the not the molecular changes induced by TSA were at the of cells, for marker of undifferentiated and marker of differentiated expression in of were in undifferentiated TSA treatment in the of LIF in a dramatic in in cell LIF withdrawal for days in a in cells after TSA was in of cells, and this was maintained at days of TSA withdrawal of of cells from the of is in no was in undifferentiated cell TSA treatment in the of LIF in a of in of cells of LIF for days induced in than of cells days after TSA withdrawal than of cells and the of was substantially of three independent is in the differentiated phenotype induced by TSA treatment was at the cell We the that a of undifferentiated mES cells in the culture were by TSA, and these cells the culture as undifferentiated cells withdrawal of the of this cell cell during a time of TSA treatment and withdrawal. in during the of TSA treatment was no on the cell growth rate time of A in cell growth was observed for the after TSA withdrawal time and the time to the rate of in cell growth rate are an of cell and cell that was a of by TSA the in time for not for the of cells with in There were at cells in the TSA than of the cells from and not from a phenotype. Increased of H3 and H4 after TSA Treatment at the of used to a of the acetylation of the histone at the regulatory regions of the marker gene in the undifferentiated mES cells and after TSA the increase in histone acetylation for gene regulatory induced by TSA treatment to undifferentiated cells. are as acetylation and with the of histone acetylation from The three genes of the no increased acetylation of H3 and H4 in response to gene regions of histone acetylation in and were in the undifferentiated cells The of TSA to increase the histone acetylation of regulatory regions of the genes was to their acetylation state in The regions with the of as and and the induced by was the of the of TSA induced a increase in histone acetylation in the regulatory regions of the genes that were at in culture and The of acetylation did not for the genes that were completely transcriptionally in this and the of TSA on localized histone acetylation were very to the of LIF withdrawal. We concluded that increased histone acetylation was an active in the expression of early differentiation genes but was not for of very maintenance of the differentiated phenotype, or irreversible loss of the undifferentiated state. of the chromatin changes in histone modifications and are of early embryonic as as of ES cell loss of pluripotency and commitment to differentiation Nat. Genet. 2003; 33: PubMed Scopus Google Scholar, Biol. 2003; PubMed Scopus Google Scholar, Curr. Opin. Genet. Dev. 2006; PubMed Scopus Google Scholar, Nat. Rev. Cell Biol. 2006; 7: PubMed Scopus Google Scholar, Res. 2006; PubMed Scopus Google Scholar). on chromatin changes during differentiation, and in germ cells an increase in is a on the very early time after an mES cell is to a differentiation signal. of colony morphology and dramatic changes in the expression of a of marker genes the phenotype with this very early differentiation of mES cells in response to LIF withdrawal. We have on this early time of the changes in gene expression and loss of pluripotency that are and by changes in the We changes are of the of gene expression but are to genes for changes in expression and for of the program of that the cell into a differentiation to to specific cell for The were to the role of histone with in the very earliest steps of the differentiation induced by LIF withdrawal. During this early time the expression of of all three germ cell is but the cells are not to specific (3Keller G.M. Curr. Opin. Cell Biol. 1995; 7: 862-869Crossref PubMed Scopus (781) Google Scholar, C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). We observed a global increase in the acetylation of both histone H3 and H4 by either LIF withdrawal or TSA induction of histone acetylation by either treatment to the gene expression changes of the very early differentiation is for genes that in differentiation as and histone acetylation is not for irreversible from the stem cell state and progression to a lineage specific differentiation program as by the TSA withdrawal results and of in during mES Cell global in acetylation of the histone observed in is an of the response to LIF withdrawal regions of and The that have changes in global histone acetylation in mES cells during differentiation have on differentiated cells or on and A on changes mES cells and cells a increase in the and of of on histone H3 and a in acetylation on both H3 and H4 on of their differentiation D. Dev. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The time examined was of LIF withdrawal at a increase in H4 acetylation was observed to A of have that a of differentiation in mES cells to of of the and over Dev. Genet. PubMed Scopus Google Scholar). on of a in histone acetylation is observed at Dev. Biol. PubMed Scopus Google Scholar). examined global histone acetylation during differentiation of mES cells and an and of histone acetylation PubMed Scopus Google Scholar). The differentiation of used in that is from of the and results that is a very early increase in global histone events to active chromatin histone histone and time acetylation is a process resulting from the and deacetylase A.L. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the and of these to specific by differentiation. We for this very early increase in global histone acetylation during mES cell differentiation. The first is changes in transcription at specific The is as a for genes that and to during events to The is as of a to the cell of the pluripotent state. We a striking in the pattern of local histone acetylation changes cells are with either LIF withdrawal or The global in histone acetylation are not regulatory regions of marker genes and and but best with the of that marker of the genes in the an increase in either or both histone H3 and H4 acetylation in at regulatory with genes that are transcriptionally active or inactive in and or no increase in histone acetylation and for the results are with the first role for the global increase in histone that changes in the of specific We observed an increase in acetylated histone H3 in the regulatory of and the of by days of LIF withdrawal or TSA treatment the of expression of these genes during this time expression of and is days of LIF withdrawal in (6Sauter C.N. McDermid R.L. Weinberg A.L. Greco T.L. Xu X. Murdoch F.E. Fritsch M.K. Exp. Cell Res. 2005; 311: 251-264Crossref PubMed Scopus (27) Google Scholar). the expression pattern of these genes from and for no expression is with for days not genes are an of the role of early increased histone the genes are for expression, but increased histone acetylation is not to is a histone Increased for mES to to of mES cells with TSA changes that the withdrawal of LIF for 3-5 that have in the of LIF for days are to differentiation. D. and cells differentiated by days of LIF withdrawal to culture in with LIF not the tight colony morphology or gene expression pattern in the pluripotent mES TSA treatment results in a and the cells to the pluripotent mES cell phenotype removal of We from these that the increase in histone acetylation observed LIF withdrawal and induced by histone deacetylase with TSA is not for maintenance or progression of differentiation. additional events are required that with LIF withdrawal but that are not induced by We that a signal is the cells TSA treatment signal a in the of a specific transcription factor or or specific changes from the histone acetylation changes histone of histone into and at either a local or global Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, P. D. Curr. Opin. Genet. Dev. 2005; 15: PubMed Scopus Google Scholar, D. Nat. Rev. Cell Biol. 2005; PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar). the of a chromatin with in the regulatory regions of a large of genes in mES cells B.E. X. D.J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The are on of histone H3 and on of histone H3. is with active is a that the of both genes but for in mES cells. cells, the to with a gene that is correlated with of and with Increased acetylation induced by TSA support the expression of a of genes for the removal of required to the expression of of or on additional genes required to the cell to a differentiation that of gene transcription during mES cell differentiation best by a of histone modifications removal of as B.E. X. D.J. 2006; Full Text Full Text PDF PubMed Scopus Google and the maintenance or of as B.E. X. D.J. 2006; Full Text Full Text PDF PubMed Scopus Google and acetylation to of the role for in mES cell differentiation. The of ES cells is the of and cells for treatment of of the program of required to a specific cell to this We and for with