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Functions of histone-modifying enzymes in development
Functions of histone-modifying enzymes in development Wenchu Lin and Sharon YR Dent Multiple histone-modifying enzymes have been identified in the past several years. Much has been learned regarding the biochemistry of these enzymes and their effects on gene expression in cultured cells. However, the functions of these factors during development are still largely unknown. Recent genetic studies indicate that specific histone modifications and modifying enzymes play essential roles in both global and tissue-specific chromatin organization. In particular, these studies indicate that enzymes that control levels and patterns of histone acetylation and methylation are required for normal embryo patterning, organogenesis, and survival. Addresses Department of Biochemistry and Molecular Biology, Program in Genes and Development, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA Corresponding author: Dent, Sharon YR ([email protected])
Current Opinion in Genetics & Development 2006, 16:137–142 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Susan Parkhurst and Toshio Tsukiyama Available online 28th February 2006 0959-437X/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2006.02.002
Introduction Dynamic programs of gene expression are required for the differentiation of pluripotent stem cells into specific tissue lineages. Studies over the past 20 years have defined several transcription factors that initiate and control these changing transcription programs through the interaction with cis-acting DNA sequences. These factors, however, are not sufficient to regulate transcription on their own. They recruit co-activators or co-repressors that, in turn, reorganize chromatin through the recruitment of enzymes that post-translationally modify histones or mobilize nucleosomes in an ATP-dependent manner (Figure 1). Alterations in DNA methylation and incorporation of histone variants also impact the overall state of chromatin-folding and the association of transcription factors with chromatin. Here, we review several recent studies that define changes in global patterns of gene expression during early development, as well as focussing on genetic experiments that indicate that specific histone-modifying enzymes are crucial for normal development of particular tissue lineages. www.sciencedirect.com
Global alterations to histone modifications in embryonic stem cells and early embryos Cell differentiation is largely a process of restricting specific gene expression patterns to particular types of cells. This restriction involves both activation and repression of tissue-specific genes. Histone modifications affect both of these regulatory processes through effects on chromatin at individual gene promoters and on large-scale chromatin domains. Histones are subject to several post-translational modifications, including acetylation, methylation, ubiquitination and phosphorylation [1]. Acetylation occurs on lysine residues, but methylation can occur on either lysine or arginine. In addition, lysines can be monomethylated (me1), dimethylated (me2) or trimethylated (me3), and arginines can be dimethylated in a symmetrical or asymmetrical fashion, providing further regulatory potential [2]. Recent studies indicate that global levels of histone acetylation and histone H3K4 methylation, which are generally associated with gene activation, transiently decline once embryonic stem (ES) cells start to differentiate [3]. By contrast, levels of histone H3K9 methylation, a mark associated with gene silencing, increase during ES cell differentiation. These results suggest that ‘active’ histone marks are replaced as the global transcriptional potential of ES cells becomes more restricted, and that more silent, heterochromatic regions are created upon initiation of differentiation. Treatment of ES cells with the histone deacetylase (HDAC) inhibitor trichostatin A blocks differentiation [3], confirming the functional importance of changes in histone acetylation patterns. Moreover, the re-establishment of pluripotency in somatic cells is associated with the re-establishment of ‘active’ histone marks, including hyperacetylation and high levels of H3K4 methylation [4]. Dynamic changes in levels and locations of histone modifications also occur in the developing embryo. For example, H3K9me3, H4K20me1 and H4K20me3 exhibit distinct distributions in cells derived from mid-gestation mouse embryos [5]. H3K9me3 and H4K20me1 are enriched in proliferating cells, whereas H4K20me3 is enriched in differentiating neurons [5]. As above, the distributions of these histone modifications probably reflect increased heterochromatin formation, which, in turn, reflects restriction of gene expression patterns and decreased cell division in differentiated cells. Both H3K9me3 and H4K20me3 are enriched in pericentric heterochromatin [4,6], as are factors such as HP1 that bind to the methylated moieties [6]. Current Opinion in Genetics & Development 2006, 16:137–142
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Figure 1
Chromatin dynamics. The assembly of chromatin into closed, transcriptionally silent states (top) is associated with specific histone modifications, including deacetylation and methylation of H3 at K9 and K27. These modifications are induced by specific HDACs and HMTs (red) such as PcG proteins, G9a and Suv39-h1/h2. ATP-dependent remodelers such as SWI–SNF and DNA methylation, induced by DNA methyltransferases, also contribute to gene repression. Open, transcriptionally active states of chromatin (bottom) are associated with increased acetylation mediated by HATs, and increased H3K4 methylation mediated by different HMTs (green) such as MLL1. SWI–SNF also acts to mobilize nucleosomes to facilitate gene expression. The open state enables association of transcription factors and the basal transcription machinery. These states can be highly dynamic.
Histone modification changes at specific genes Global alterations in histone modification sites and levels reflect not only large-scale shifts in heterochromatic domains but also localized alterations in euchromatic structures. Locus-specific alterations in histone modifications that are associated with tissue-specific transcription programs are probably initiated well before differentiation is achieved. For example, the transcriptional competence of the B-cell specific genes l5 and VpreB1 appears to be established in ES cells by H3 acetylation and H3K4 methylation of histones associated with a particular cisacting sequence. These modifications then spread along the l5–VpreB1 locus, creating an extended open domain that enables maximal activation of these genes in pre-B cells [7]. A temporal and directional spreading of ‘open’ chromatin marks is also associated with sequential activation of genes within the mouse HoxD4 locus during neurogenesis. In this case, H3K4 methylation occurs first, followed by H3 and then H4 acetylation [8]. Colinear Hox gene activation parallels establishment of the anterior– posterior axis of the embryo, so these sequential changes in histone modifications might be crucially important for proper anterior–posterior patterning. Sequential changes in histone modification patterns are also associated with gene repression. Downregulation of the Dntt gene during Current Opinion in Genetics & Development 2006, 16:137–142
thymocyte maturation, for example, is associated with ordered H3K9-deacetylation, loss of H3K4 methylation and finally increased H3K9 methylation [9]. Altogether, these findings highlight not only the intimate connections between the alterations in histone modification patterns and the regulation of gene expression but also the coordinated regulation of particular types and sites of histone modification.
Chromatin modifiers in embryogenesis The rate of embryonic growth increases dramatically from the time of implantation through to gastrulation, when the embryo differentiates into the three primary germlayers. Subsequently, the endoderm, mesoderm and ectoderm are further organized and differentiated into organs. At each of these stages, gene expression is subject to a high degree of temporal and spatial regulation. Genetic studies in mice demonstrate that various chromatin-modifying activities are important throughout these stages (Figure 2) [10–25], and that specific histone acetyltransferases (HATs), HDACs and histone methyltransferases (HMTs) have tissue-specific and dose-dependent functions in the developing embryo. Few mutations affecting histone-modifying activities cause problems at or before embryo implantation, www.sciencedirect.com
Functions of histone-modifying enzymes in development Lin and Dent 139
Figure 2
Chromatin-modifying enzymes are essential for normal mouse development. The different stages of mouse development are depicted relative to days of gestation. The time of embryonic lethality caused by homozygous deletion of individual chromatin-modifying activities or associated proteins is shown. Abnormal phenotypes are usually observed somewhat earlier, reflecting developmental time-points and processes that require the deleted factors.
highlighting the role of these enzymes in the control of specific genes rather than in global transcription. By contrast, loss of general transcription factors such as Srb7 [26] causes death soon after the onset of zygotic transcription in two-cell embryos. The diverse phenotypes caused by mutations in histone-modifying enzymes further indicate that particular developmental pathways require particular chromatin states. For example, loss of the GCN5 HAT results in loss of paraxial mesoderm, whereas loss of another HAT, p300, causes cardiac and skeletal defects [22,23]. Even enzymes that modify the same residue in a given histone might have unique functions during development. Double deletion of Suv39-h1 and Suv39-h2 HMT genes in mice leads to chromosomal segregation defects and lymphoma development [27]. However, deletion of G9a or ESET, two other H3K9 HMTs, causes death at an early stage of embryonic development [13,17]. In a similar manner to HATs, individual HDACs are required for specific functions during embryogenesis. Mice lacking class II HDACs survive to term but display a range of phenotypes. HDAC4 deficiency causes chondrocyte hypertrophy [28], whereas inactivation of either HDAC5 or HDAC9 results in increased sensitivity to cardiac stress signals, and cardiac hypertrophy [29]. Class II HDACs, therefore, might be central modulators of cell growth and organ size. Class I HDACs, by contrast, appear to limit tissue differentiation. Levels of HDAC1 and HDAC2, which are primarily located in prospective epithelium within the developing intestine, decrease as differentiation begins [30]. Overexpression of these HDACs in tissue explants inhibits the expression of differentiation markers, whereas treatment with valproic acid, a selective inhibitor of these enzymes, activates these genes and promotes differentiation. These studies www.sciencedirect.com
also indicate that certain class II (HDAC5 and HDAC9) and class I (HDAC1 and HDAC2) HDACs have redundant as well as unique functions in cell growth and development.
Polycomb group and Trithorax genes encode HMTs important in development Polycomb group (PcG) and Trithorax (Trx) genes were first identified in Drosophila as mutations that affect homeobox gene expression and body axis patterning [31]. PcG complexes are transcriptional repressors, whereas Trx complexes activate transcription. Both PcG and Trx proteins house HMT activities specific for K27 and K4 in H3, respectively [32–34]. H3K27 methylation contributes to PcG-mediated gene-silencing, but PcG complexes can also repress transcription by other means. The mammalian PcG protein PRC1 (Polycomb repressor complex 1) can block chromatin-remodeling mediated by SWI–SNF complexes [35]. Moreover, a PRC1 core complex can compact nucleosome arrays in vitro [36] in a manner that is independent of the histone tails. Nonetheless, PcG-mediated methylation is associated with target gene silencing and must be overcome by Trx complexes to enable gene activation. Interestingly, mis-expression of Hox genes in flies bearing Trx or Ash1 mutations can be corrected by subsequent loss of the PcG protein E(z) [37]. These data indicate that Trx proteins do not function directly as co-activators but as anti-repressors. Methylation of K4 in H3 by these proteins might inhibit methylation of K27 by PcG complexes, thereby preventing improper Hox gene silencing. The mammalian Trx homolog ALL-1 (acute lymphoblastic leukemia-1; also known as MLL1 [mixed lineage leukemia 1]) also methylates K4 in H3, and it promotes expression of Hox genes important for hematopoietic Current Opinion in Genetics & Development 2006, 16:137–142
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differentiation [38]. The MLL1 gene is often subject to translocations associated with acute leukemias. WDR5 (WD40 repeat 5), another component of the MLL1 complex, is the only mammalian protein identified to date that binds specifically to H3K4me1 and H3K4me2. WDR5 is also required for HOX gene activation, and it may help tether MLL1 to target promoters [39]. Interestingly, the only histone lysine de-methylase activity discovered to date — that of Lsd1 — de-methylates H3K4me1 and H3K4me2 [40]. The activity and specificity of this enzyme is altered upon association with cofactors [41] such as CoREST, which is important for preventing expression of neuronal genes in non-neural tissues. The Lsd1 complex has HDAC activities as well, highlighting the importance of removing both activating methyl and activating acetyl marks from histones associated with genes destined for repression. Whether alterations in Lsd1 functions lead to abnormal differentiation or cancer development is a question of some interest for future studies.
Alterations in histone modifications associated with X-inactivation The process of X-inactivation during female mammalian development is highly regulated and nicely illustrates the functional connections between alterations in histone modification patterns and alterations in gene expression levels. X-inactivation requires a non-coding RNA (Xist) expressed from the inactivation center on the inactive X chromosome. Xist RNA coats the inactive X, a process correlated with recruitment of PRC1 and PRC2 complexes [42,43]. During early cell cleavages, before the onset of cellular differentiation, the paternal X chromosome is selectively inactivated by a process that involves hypomethylation of H3K4 and hypoacetylation of H3K9, followed by increased methylation at both K9 and K27 by the PRC complexes [44]. However, this epigenetic state is reversed as the paternal X is reactivated in the inner cell mass, from which the embryo develops. Random X-inactivation then ensues, accompanied again by the reprogramming of H3 modifications into a silent state. Expression of the antisense Tsix RNA is important for the conversion of the imprinted, inactive X back to a transcription-competent state. Interestingly, Tsix does not appear to affect transcription of Xist directly, but it triggers an increase in H3K4 methylation [45].
Conclusion Several histone modifications are known to be important for the regulation of gene expression. Knockout approaches have revealed many chromatin-modifying proteins that are essential for normal development in mice (Figure 1). However, early embryonic lethality occurs upon depletion of many of these factors, precluding assessment of their functions in later developmental stages. The use of conditional alleles of these genes that would enable deletion at later developmental time points Current Opinion in Genetics & Development 2006, 16:137–142
or in specific tissues will greatly enhance our understanding of their functions during embryogenesis and in adult tissues. Moreover, future studies defining the developmental functions of enzymes that govern histone ubiquitination, sumoylation and phosphorylation will nicely complement findings described here regarding the functions of HATs, HDACs and HMTs. Importantly, all of these enzymes probably modify several non-histone substrates. Identification of such factors and their functions is crucial to the interpretation of the phenotypes caused by null alleles of these enzymes. Finally, the highly dynamic and coordinated networks of chromatin modifiers and transcription factors that orchestrate cell fate decisions and growth controls identified by these collective genetic studies will no doubt provide key insights for the design of stem cell-based therapies for human diseases.
Acknowledgements This work was supported by grants from the March of Dimes Foundation (1-FY03-82) and the National Institutes of Health (GM067718) to SYRD.
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38. Ernst P, Fisher JK, Avery W, Wade S, Foy D, Korsmeyer SJ: Definitive hematopoiesis requires the mixed-lineage leukemia gene. Dev Cell 2004, 6:437-443. Using a mouse system, the authors demonstrate that Mll-deficient cells cannot develop into lymphocytes or B-cells, and that Mll-deficient embryos lack hematopoietic stem cell activity. These studies show that the Mll protooncogene is required for definitive hematopoiesis. 39. Wysocka J, Swigut T, Milne TA, Dou YL, Zhang X, Burlingame AL, Roeder RG, Brivanlou AH, Allis CD: WDR5 associates with histone H3 methylated at K4 and is essential for H3K4 methylation and vertebrate development. Cell 2005, 121:859-872. 40. Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA, Shi Y: Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 2004, 119:941-953. Prior to this work, the existence of histone de-methylating activities was questionable. The authors demonstrate that a conserved amine oxidase performs this function. 41. Wysocka J, Milne TA, Allis CD: Taking LSD1 to a new high. Cell 2005, 122:654-658. 42. Mak W, Baxter J, Silva J, Newall AE, Otte AP, Brockdorff N: Mitotically stable association of polycomb group proteins Eed and Enx1 with the inactive X chromosome in trophoblast stem cells. Curr Biol 2002, 12:1016-1020. Current Opinion in Genetics & Development 2006, 16:137–142
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45. Navarro P, Pichard S, Ciaudo C, Avner P, Rougeulle C: Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation. Genes Dev 2005, 19:1474-1484. This study provides direct evidence that Xist RNA expression is regulated at the level of basal transcription factor recruitment. The authors also provide evidence that the antisense Tsix RNA somehow triggers H3K4 methylation over the entire Xist–Tsix locus.
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Current Opinion in Genetics & Development 2006, 16:137–142 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Susan Parkhurst and Toshio Tsukiyama Available online 28th February 2006 0959-437X/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2006.02.002
Introduction Dynamic programs of gene expression are required for the differentiation of pluripotent stem cells into specific tissue lineages. Studies over the past 20 years have defined several transcription factors that initiate and control these changing transcription programs through the interaction with cis-acting DNA sequences. These factors, however, are not sufficient to regulate transcription on their own. They recruit co-activators or co-repressors that, in turn, reorganize chromatin through the recruitment of enzymes that post-translationally modify histones or mobilize nucleosomes in an ATP-dependent manner (Figure 1). Alterations in DNA methylation and incorporation of histone variants also impact the overall state of chromatin-folding and the association of transcription factors with chromatin. Here, we review several recent studies that define changes in global patterns of gene expression during early development, as well as focussing on genetic experiments that indicate that specific histone-modifying enzymes are crucial for normal development of particular tissue lineages. www.sciencedirect.com
Global alterations to histone modifications in embryonic stem cells and early embryos Cell differentiation is largely a process of restricting specific gene expression patterns to particular types of cells. This restriction involves both activation and repression of tissue-specific genes. Histone modifications affect both of these regulatory processes through effects on chromatin at individual gene promoters and on large-scale chromatin domains. Histones are subject to several post-translational modifications, including acetylation, methylation, ubiquitination and phosphorylation [1]. Acetylation occurs on lysine residues, but methylation can occur on either lysine or arginine. In addition, lysines can be monomethylated (me1), dimethylated (me2) or trimethylated (me3), and arginines can be dimethylated in a symmetrical or asymmetrical fashion, providing further regulatory potential [2]. Recent studies indicate that global levels of histone acetylation and histone H3K4 methylation, which are generally associated with gene activation, transiently decline once embryonic stem (ES) cells start to differentiate [3]. By contrast, levels of histone H3K9 methylation, a mark associated with gene silencing, increase during ES cell differentiation. These results suggest that ‘active’ histone marks are replaced as the global transcriptional potential of ES cells becomes more restricted, and that more silent, heterochromatic regions are created upon initiation of differentiation. Treatment of ES cells with the histone deacetylase (HDAC) inhibitor trichostatin A blocks differentiation [3], confirming the functional importance of changes in histone acetylation patterns. Moreover, the re-establishment of pluripotency in somatic cells is associated with the re-establishment of ‘active’ histone marks, including hyperacetylation and high levels of H3K4 methylation [4]. Dynamic changes in levels and locations of histone modifications also occur in the developing embryo. For example, H3K9me3, H4K20me1 and H4K20me3 exhibit distinct distributions in cells derived from mid-gestation mouse embryos [5]. H3K9me3 and H4K20me1 are enriched in proliferating cells, whereas H4K20me3 is enriched in differentiating neurons [5]. As above, the distributions of these histone modifications probably reflect increased heterochromatin formation, which, in turn, reflects restriction of gene expression patterns and decreased cell division in differentiated cells. Both H3K9me3 and H4K20me3 are enriched in pericentric heterochromatin [4,6], as are factors such as HP1 that bind to the methylated moieties [6]. Current Opinion in Genetics & Development 2006, 16:137–142
138 Chromosomes and expression mechanisms
Figure 1
Chromatin dynamics. The assembly of chromatin into closed, transcriptionally silent states (top) is associated with specific histone modifications, including deacetylation and methylation of H3 at K9 and K27. These modifications are induced by specific HDACs and HMTs (red) such as PcG proteins, G9a and Suv39-h1/h2. ATP-dependent remodelers such as SWI–SNF and DNA methylation, induced by DNA methyltransferases, also contribute to gene repression. Open, transcriptionally active states of chromatin (bottom) are associated with increased acetylation mediated by HATs, and increased H3K4 methylation mediated by different HMTs (green) such as MLL1. SWI–SNF also acts to mobilize nucleosomes to facilitate gene expression. The open state enables association of transcription factors and the basal transcription machinery. These states can be highly dynamic.
Histone modification changes at specific genes Global alterations in histone modification sites and levels reflect not only large-scale shifts in heterochromatic domains but also localized alterations in euchromatic structures. Locus-specific alterations in histone modifications that are associated with tissue-specific transcription programs are probably initiated well before differentiation is achieved. For example, the transcriptional competence of the B-cell specific genes l5 and VpreB1 appears to be established in ES cells by H3 acetylation and H3K4 methylation of histones associated with a particular cisacting sequence. These modifications then spread along the l5–VpreB1 locus, creating an extended open domain that enables maximal activation of these genes in pre-B cells [7]. A temporal and directional spreading of ‘open’ chromatin marks is also associated with sequential activation of genes within the mouse HoxD4 locus during neurogenesis. In this case, H3K4 methylation occurs first, followed by H3 and then H4 acetylation [8]. Colinear Hox gene activation parallels establishment of the anterior– posterior axis of the embryo, so these sequential changes in histone modifications might be crucially important for proper anterior–posterior patterning. Sequential changes in histone modification patterns are also associated with gene repression. Downregulation of the Dntt gene during Current Opinion in Genetics & Development 2006, 16:137–142
thymocyte maturation, for example, is associated with ordered H3K9-deacetylation, loss of H3K4 methylation and finally increased H3K9 methylation [9]. Altogether, these findings highlight not only the intimate connections between the alterations in histone modification patterns and the regulation of gene expression but also the coordinated regulation of particular types and sites of histone modification.
Chromatin modifiers in embryogenesis The rate of embryonic growth increases dramatically from the time of implantation through to gastrulation, when the embryo differentiates into the three primary germlayers. Subsequently, the endoderm, mesoderm and ectoderm are further organized and differentiated into organs. At each of these stages, gene expression is subject to a high degree of temporal and spatial regulation. Genetic studies in mice demonstrate that various chromatin-modifying activities are important throughout these stages (Figure 2) [10–25], and that specific histone acetyltransferases (HATs), HDACs and histone methyltransferases (HMTs) have tissue-specific and dose-dependent functions in the developing embryo. Few mutations affecting histone-modifying activities cause problems at or before embryo implantation, www.sciencedirect.com
Functions of histone-modifying enzymes in development Lin and Dent 139
Figure 2
Chromatin-modifying enzymes are essential for normal mouse development. The different stages of mouse development are depicted relative to days of gestation. The time of embryonic lethality caused by homozygous deletion of individual chromatin-modifying activities or associated proteins is shown. Abnormal phenotypes are usually observed somewhat earlier, reflecting developmental time-points and processes that require the deleted factors.
highlighting the role of these enzymes in the control of specific genes rather than in global transcription. By contrast, loss of general transcription factors such as Srb7 [26] causes death soon after the onset of zygotic transcription in two-cell embryos. The diverse phenotypes caused by mutations in histone-modifying enzymes further indicate that particular developmental pathways require particular chromatin states. For example, loss of the GCN5 HAT results in loss of paraxial mesoderm, whereas loss of another HAT, p300, causes cardiac and skeletal defects [22,23]. Even enzymes that modify the same residue in a given histone might have unique functions during development. Double deletion of Suv39-h1 and Suv39-h2 HMT genes in mice leads to chromosomal segregation defects and lymphoma development [27]. However, deletion of G9a or ESET, two other H3K9 HMTs, causes death at an early stage of embryonic development [13,17]. In a similar manner to HATs, individual HDACs are required for specific functions during embryogenesis. Mice lacking class II HDACs survive to term but display a range of phenotypes. HDAC4 deficiency causes chondrocyte hypertrophy [28], whereas inactivation of either HDAC5 or HDAC9 results in increased sensitivity to cardiac stress signals, and cardiac hypertrophy [29]. Class II HDACs, therefore, might be central modulators of cell growth and organ size. Class I HDACs, by contrast, appear to limit tissue differentiation. Levels of HDAC1 and HDAC2, which are primarily located in prospective epithelium within the developing intestine, decrease as differentiation begins [30]. Overexpression of these HDACs in tissue explants inhibits the expression of differentiation markers, whereas treatment with valproic acid, a selective inhibitor of these enzymes, activates these genes and promotes differentiation. These studies www.sciencedirect.com
also indicate that certain class II (HDAC5 and HDAC9) and class I (HDAC1 and HDAC2) HDACs have redundant as well as unique functions in cell growth and development.
Polycomb group and Trithorax genes encode HMTs important in development Polycomb group (PcG) and Trithorax (Trx) genes were first identified in Drosophila as mutations that affect homeobox gene expression and body axis patterning [31]. PcG complexes are transcriptional repressors, whereas Trx complexes activate transcription. Both PcG and Trx proteins house HMT activities specific for K27 and K4 in H3, respectively [32–34]. H3K27 methylation contributes to PcG-mediated gene-silencing, but PcG complexes can also repress transcription by other means. The mammalian PcG protein PRC1 (Polycomb repressor complex 1) can block chromatin-remodeling mediated by SWI–SNF complexes [35]. Moreover, a PRC1 core complex can compact nucleosome arrays in vitro [36] in a manner that is independent of the histone tails. Nonetheless, PcG-mediated methylation is associated with target gene silencing and must be overcome by Trx complexes to enable gene activation. Interestingly, mis-expression of Hox genes in flies bearing Trx or Ash1 mutations can be corrected by subsequent loss of the PcG protein E(z) [37]. These data indicate that Trx proteins do not function directly as co-activators but as anti-repressors. Methylation of K4 in H3 by these proteins might inhibit methylation of K27 by PcG complexes, thereby preventing improper Hox gene silencing. The mammalian Trx homolog ALL-1 (acute lymphoblastic leukemia-1; also known as MLL1 [mixed lineage leukemia 1]) also methylates K4 in H3, and it promotes expression of Hox genes important for hematopoietic Current Opinion in Genetics & Development 2006, 16:137–142
140 Chromosomes and expression mechanisms
differentiation [38]. The MLL1 gene is often subject to translocations associated with acute leukemias. WDR5 (WD40 repeat 5), another component of the MLL1 complex, is the only mammalian protein identified to date that binds specifically to H3K4me1 and H3K4me2. WDR5 is also required for HOX gene activation, and it may help tether MLL1 to target promoters [39]. Interestingly, the only histone lysine de-methylase activity discovered to date — that of Lsd1 — de-methylates H3K4me1 and H3K4me2 [40]. The activity and specificity of this enzyme is altered upon association with cofactors [41] such as CoREST, which is important for preventing expression of neuronal genes in non-neural tissues. The Lsd1 complex has HDAC activities as well, highlighting the importance of removing both activating methyl and activating acetyl marks from histones associated with genes destined for repression. Whether alterations in Lsd1 functions lead to abnormal differentiation or cancer development is a question of some interest for future studies.
Alterations in histone modifications associated with X-inactivation The process of X-inactivation during female mammalian development is highly regulated and nicely illustrates the functional connections between alterations in histone modification patterns and alterations in gene expression levels. X-inactivation requires a non-coding RNA (Xist) expressed from the inactivation center on the inactive X chromosome. Xist RNA coats the inactive X, a process correlated with recruitment of PRC1 and PRC2 complexes [42,43]. During early cell cleavages, before the onset of cellular differentiation, the paternal X chromosome is selectively inactivated by a process that involves hypomethylation of H3K4 and hypoacetylation of H3K9, followed by increased methylation at both K9 and K27 by the PRC complexes [44]. However, this epigenetic state is reversed as the paternal X is reactivated in the inner cell mass, from which the embryo develops. Random X-inactivation then ensues, accompanied again by the reprogramming of H3 modifications into a silent state. Expression of the antisense Tsix RNA is important for the conversion of the imprinted, inactive X back to a transcription-competent state. Interestingly, Tsix does not appear to affect transcription of Xist directly, but it triggers an increase in H3K4 methylation [45].
Conclusion Several histone modifications are known to be important for the regulation of gene expression. Knockout approaches have revealed many chromatin-modifying proteins that are essential for normal development in mice (Figure 1). However, early embryonic lethality occurs upon depletion of many of these factors, precluding assessment of their functions in later developmental stages. The use of conditional alleles of these genes that would enable deletion at later developmental time points Current Opinion in Genetics & Development 2006, 16:137–142
or in specific tissues will greatly enhance our understanding of their functions during embryogenesis and in adult tissues. Moreover, future studies defining the developmental functions of enzymes that govern histone ubiquitination, sumoylation and phosphorylation will nicely complement findings described here regarding the functions of HATs, HDACs and HMTs. Importantly, all of these enzymes probably modify several non-histone substrates. Identification of such factors and their functions is crucial to the interpretation of the phenotypes caused by null alleles of these enzymes. Finally, the highly dynamic and coordinated networks of chromatin modifiers and transcription factors that orchestrate cell fate decisions and growth controls identified by these collective genetic studies will no doubt provide key insights for the design of stem cell-based therapies for human diseases.
Acknowledgements This work was supported by grants from the March of Dimes Foundation (1-FY03-82) and the National Institutes of Health (GM067718) to SYRD.
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