Epigenetic reprogramming is thought to play an important role in the development of cloned embryos reconstructed by somatic cell nuclear transfer (SCNT). In the present study, dynamic reprogramming of histone acetylation and methylation modifications was investigated in the first cell cycle of cloned embryos. The results demonstrated that part of somatic inherited lysine acetylation on core histones (H3K9, H3K14, H4K16) could be quickly deacetylated following SCNT, and reacetylation occurred following activation treatment. However, acetylation marks of the other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos with only mild deacetylation occurring in the process of SCNT and activation treatment. The somatic cloned embryos established histone acetylation modifications resembling those in normal embryos produced by intracytoplasmic sperm injection through these two different programs. Moreover, treatment of cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA), improved the histone acetylation in a manner similar to that in normal embryos, and the improved histone acetylation in cloned embryos treated with TSA might contribute to improved development of TSA-treated clones. In contrast to the asymmetric histone H3K9 tri- and dimethylation present in the parental genomes of fertilized embryos, the tri- and dimethylations of H3K9 were gradually demethylated in the cloned embryos, and this histone H3K9 demethylation may be crucial for gene activation of cloned embryos. Together, these results indicate that dynamic reprogramming of histone acetylation and methylation modifications in cloned embryos is developmentally regulated (Wang, 2007).
It has been generally accepted that the mammalian embryo starts its development with all cells identical, and only when inside and outside cells form do differences between cells first emerge. However, recent findings show that cells in the mouse embryo can differ in their developmental fate and potency as early as the four-cell stage. These differences depend on the orientation and order of the cleavage divisions that generated them. Because epigenetic marks are suggested to be involved in sustaining pluripotency, such developmental properties might be achieved through epigenetic mechanisms. This study shows that modification of histone H3, through the methylation of specific arginine residues, is correlated with cell fate and potency. Levels of H3 methylation at specific arginine residues are maximal in four-cell blastomeres that will contribute to the inner cell mass (ICM) and polar trophectoderm and undertake full development when combined together in chimaeras. Arginine methylation of H3 is minimal in cells whose progeny contributes more to the mural trophectoderm and that show compromised development when combined in chimaeras. This suggests that higher levels of H3 arginine methylation predispose blastomeres to contribute to the pluripotent cells of the ICM. This prediction was confirmed by overexpressing the H3-specific arginine methyltransferase CARM1 in individual blastomeres; this directs their progeny to the ICM and results in a dramatic upregulation of Nanog and Sox2. Thus, these results identify specific histone modifications as the earliest known epigenetic marker contributing to development of ICM and show that manipulation of epigenetic information influences cell fate determination (Torres-Padilla, 2007).
A genome-wide analysis of human cells suggests that most protein-coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. Nucleosomes with H3K4me3 and H3K9,14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein-coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full-length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at postinitiation steps. Genes encoding most developmental regulators fall into this group. These results also identify a class of genes that are excluded from experiencing transcription initiation, at which mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells (Guenther, 2007).
The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. This study demonstrates that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, these results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi (Plath, 2003a).
Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, since mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). This study demonstrates that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, since not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. These results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process (Plath, 2003b).
The Eed-Enx1 Polycomb group complex has been implicated in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure (Silva, 2003).
In eed-/- XX embryos Enx1 protein does not localize to Xi. This result is consistent with in vitro analysis demonstrating that the eed3354SB mutation disrupts a WD40 domain required for the interaction of Eed with Enx1. Thus, in the absence of functional Eed protein, the Enx1 HMTase cannot be directed toward specific targets. Eed/Enx1 complexes methylate H3-K9 and K27 in vitro, with a strong preference for K27. Failure to localize Enx1 clearly accounts for the absence of H3-K9/K27 methylation on Xi in eed-/- embryos (Silva, 2003).
It should be noted that, while previous studies have identified H3-K9 methylation as an early mark of silent chromatin on the inactive X chromosome, more recent data indicates that this could be attributable to cross-reactivity of di/tri-meH3-K9 antisera toward di/tri-meH3-K27. New antisera highly specific for di/tri-meH3-K9 detect pericentromeric heterochromatin, but not Xi, while the tri-meH3-K27 antibody used in this study detects Xi, but not pericentromeric heterochromatin. It is also possible that Xi is di/trimethylated both at K9 and K27 and that this configuration is not recognized by the novel di/trimethylH3-K9 antisera (Silva, 2003).
H3-K9/K27 methylation is shown to serve to stabilize the Xi chromatin structure. Thus, in eed-/- embryos, H3-K9 hypoacetylation and loss of H3-K4 methylation on Xi are not seen in a significant number of cells. Moreover, both the Xa and Xi alleles of two X-linked genes were seen to be expressed in a similar proportion of cells. These observations provide a basis for explaining reactivation of the X-linked GFP transgene in trophectoderm cells of eed-/- embryos. However, other studies have not observe reactivation of the GFP transgene in cells of the embryo proper, leading to the conclusion that Eed is required for the maintenance of imprinted, but not random, X inactivation. This difference can be accounted for by two factors. First, it is probable that reactivation of any given locus on Xi is sporadic and progressive. Since embryos exhibit mosaic expression of the XGFP transgene because of random X inactivation, a relatively small increase in the proportion of cells expressing the transgene, as observed for endogenous X-linked genes in this study, would be difficult to quantify. A second factor, is that additional levels of epigenetic silencing, for example, DNA methylation, play a more significant role in maintenance of X inactivation in cells of the embryo proper compared with the trophectoderm, potentially masking the effects of failure to establish H3-K9/K27 methylation (Silva, 2003 and references therein).
Initiation and propagation of X inactivation occurs coincident with cellular differentiation and involves a large nonprotein-coding RNA, the X inactive-specific transcript (Xist). Xist RNA spreads over the X chromosome and is thought to induce chromosome inactivation through recruitment of as yet unidentified silencing factors. The silencing function of Xist RNA has been shown to map to a short tandemly repeated element at the 5' end of the transcript (Silva, 2003 and references therein).
The banded localization of Eed-Enx1 complexes on Xi parallels that observed for Xist RNA. There could be an interaction, either direct or indirect, between Xist and the Eed-Enx1 complex. This view is further supported by observations reported in this study. (1) Recruitment of Eed-Enx1 complexes occurs extremely rapidly after the onset of stable Xist RNA accumulation in differentiating XX ES cells. (2) Eed-Enx1 recruitment occurs in response to expression of ectopic Xist RNA transgenes, both in undifferentiated XY ES cells and also in XYTg15 blastocysts. (3) Eed-Enx1 complexes are not recruited in response to expression of Xistinv mutant RNA, which fails to elicit X inactivation (Silva, 2003).
While the data support the idea that Eed-Enx1 complexes are recruited by Xist RNA, they do not prove that this interaction is direct. In fact, localization of Eed-Enx1 to Xi is first seen to occur at the morula stage, when imprinted X inactivation is initiated, whereas Xist RNA expression is detected earlier, in cleavage stage embryos. High levels of Eed-Enx1 complexes are available during the cleavage stages, suggesting that a factor(s) required for the interaction of the complex with Xist is absent (Silva, 2003).
Also relevant to the question of whether or not the Eed-Enx1 complex interacts directly with Xist RNA is the observation that eed-/- embryos are able to initiate X inactivation. This is, presumably, at least in part, attributable to recruitment of an HDAC complex that can deacetylate H3-K9. It is possible that components of the PcG complex other than Eed and Enx1 are still assembled in eed-/- embryos and that these confer H3-K9 HDAC activity. In the Drosophila ESC (Eed) protein, an equivalent mutation disrupts EZ (Enx1) recruitment but does not appear to ablate complex formation. An alternative scenario is that the complex responsible for H3-K9 deacetylation is a direct target of Xist RNA and that the Eed-Enx1 HMTase is then recruited by this complex. In support of this idea, heritable silencing at Drosophila homeotic genes is initiated by recruitment of the dMi2 protein, a component of an HDAC and chromatin remodeling complex, by Hunchback. Moreover, Eed protein can interact with type 1 HDACs, both in mammalian cells and in Drosophila. Thus, HDAC complexes may recruit Eed-Enx1 to target sites, including Xi, rather than vice versa (Silva, 2003).
It is interesting to note that the time during which high levels of Eed-Enx1 complex are available corresponds closely to the window of opportunity during which cells are responsive to Xist RNA. On the basis of this consideration, a model is proposed that speculates that Xist RNA recruits HDAC and Eed-Enx1 complexes, which lead to establishment of a primary level of chromatin silencing. Only during early differentiation stages would levels of these complexes be sufficient to establish chromosome-wide primary silencing. This would explain why expression of Xist in more differentiated cell types cannot induce X inactivation. It is further suggested that maintaining localization of the HDAC/Eed-Enx1 complexes is Xist RNA dependent. This would account for reversibility and Xist dependence of silencing in undifferentiated ES cells or during early differentiation stages. Extinguishing Xist expression would result in delocalization of HDAC/Eed-Enx1 complexes, loss of H3-K9/K27 methylation, increased H3-K9 acetylation, and, hence, reactivation of Xi (Silva, 2003).
To account for the fact that X inactivation does subsequently become stabilized and Xist independent, it is suggested that the chromatin modifications induced by the HDAC and Eed-Enx1 complex provide a template for recruitment of other silencing components. These could be responsible for further histone N-terminal modifications, for example, global H4 deacetylation, and also for DNA methylation at CpG islands and recruitment of macroH2A1.2. It should be noted that proteins of the PRC1 PcG group complex are not localized to Xi at any stage and are therefore unlikely to be involved in maintaining X inactivation in late development (Silva, 2003).
A key finding from these experiments is that recruitment of Eed-Enx1 to Xi is temporally regulated, rather than lineage specific, and that this, in turn, appears to relate to temporal regulation of overall levels of Eed and, to a lesser extent, Enx1 proteins. A similar expression profile has been reported for ESC (Eed) protein in Drosophila embryogenesis. It is striking that these factors are expressed at highest levels in totipotent or multipotent precursors and during early stages of differentiation. One interpretation of this observation is that Eed-Enx1 complexes are components of the machinery required to confer genome plasticity. Thus, like X inactivation during the window of opportunity, silent chromatin at other Eed-Enx1 targets may be reversible if the primary signal (for example Hunchback at homeotic loci in Drosophila) is removed. This would provide cells of the early embryo with the capacity to activate regions of the genome in response to specific differentiation signals, contrasting with the situation in differentiated cells, where heritable silencing is highly stable and is normally irreversible (Silva, 2003).
Heterochromatin is defined classically by condensation throughout the cell cycle, replication in late S phase and gene inactivity. Facultative heterochromatin is of particular interest, because its formation is developmentally regulated as a result of cellular differentiation. The most extensive example of facultative heterochromatin is the mammalian inactive X chromosome (Xi). A variety of histone variants and covalent histone modifications have been implicated in defining the organization of the Xi heterochromatic state, and the features of Xi heterochromatin have been widely interpreted as reflecting a redundant system of gene silencing. The human Xi is packaged into at least two nonoverlapping heterochromatin types, each characterized by specific Xi features: one defined by the presence of Xi-specific transcript RNA, the histone variant macroH2A, and histone H3 trimethylated at lysine 27 and the other defined by H3 trimethylated at lysine 9, heterochromatin protein 1, and histone H4 trimethylated at lysine 20. Furthermore, regions of the Xi packaged in different heterochromatin types are characterized by different patterns of replication in late S phase. The arrangement of facultative heterochromatin into spatially and temporally distinct domains has implications for both the establishment and maintenance of the Xi and adds a previously unsuspected degree of epigenetic complexity (Chadwick, 2004).
The nucleosomal response refers to the rapid phosphorylation of histone H3 on serine 10 and HMG-14 on serine 6 that occurs concomitantly with immediate-early (IE) gene induction in response to a wide variety of stimuli. Using antibodies against the phosphorylated residues, it has been shown that H3 and HMG-14 phosphorylation is mediated via different MAP kinase (MAPK) cascades, depending on the stimulus. The nucleosomal response elicited by TPA is ERK-dependent, whereas that elicited by anisomycin is p38 MAPK-dependent. In intact cells, the nucleosomal response can be selectively inhibited using the protein kinase inhibitor H89. MAPK activation and phosphorylation of transcription factors are largely unaffected by H89, whereas induction of IE genes is inhibited and its characteristics markedly altered. MSK1 (Drosophila homolog: JIL-1) is considered the most likely kinase to mediate this response because (1) it is activated by both ERK and p38 MAPKs; (2) it is an extremely efficient kinase for HMG-14 and H3, utilizing the physiologically relevant sites; and (3) its activity towards H3/HMG-14 is uniquely sensitive to H89 inhibition. Thus, the nucleosomal response is an invariable consequence of ERK and p38 but not JNK/SAPK activation, and MSK1 potentially provides a link to complete the circuit between cell surface and nucleosome (Thomson, 1999).
NF-kappaB is a principal transcriptional regulator of diverse cytokine- mediated processes and is tightly controlled by the IkappaB kinase complex (IKK-alpha/beta/gamma). IKK-beta and IKK-gamma are critical for cytokine-induced NF-kappaB function, whereas IKK-alpha is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-alpha in NF-kappa B-dependent gene expression in response to cytokine treatment1. Nuclear accumulation of IKK-alpha after cytokine exposure is demonstrated, suggests a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-alpha is recruited to the promoter regions of NF-kappaB-regulated genes on stimulation with tumor-necrosis factor-alpha. Notably, NF-kappaB regulated gene expression is suppressed by the loss of IKK-alpha and this correlates with a complete loss of gene-specific phosphorylation of histone H3 on serine 10, a modification associated with positive gene expression. Furthermore, IKK-alpha is shown to directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. It is proposed that IKK-alpha is an essential regulator of NF-kappaB-dependent gene expression through control of promoter-associated histone phosphorylation after cytokine exposure. These findings provide additional insight into the role of the IKK complex in NF-kappaB regulated gene expression (Anest, 2003).
Post-translational modifications of conserved N-terminal tail residues in histones regulate many aspects of chromosome activity. Thr 3 of histone H3 is highly conserved, but the significance of its phosphorylation is unclear, and the identity of the corresponding kinase unknown. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation of H3 Thr 3 in prophase and its dephosphorylation during anaphase. Haspin, a member of a distinctive group of protein kinases present in diverse eukaryotes, phosphorylates H3 at Thr 3 in vitro. Importantly, depletion of haspin by RNA interference reveals that this kinase is required for H3 Thr 3 phosphorylation in mitotic cells. In addition to its chromosomal association, haspin is found at the centrosomes and spindle during mitosis. Haspin RNA interference causes misalignment of metaphase chromosomes, and overexpression delays progression through early mitosis. This work reveals a new kinase involved in composing the histone code and adds haspin to the select group of kinases that integrate regulation of chromosome and spindle function during mitosis and meiosis (Dai, 2005 ).
Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase regulates INO1 gene activation in part via Gcn5/SAGA complex-mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. This study examines the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA-bound activators, and in its role in modifying histones and recruiting TATA-binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10's role in recruiting SAGA may substitute for recruitment by DNA-bound activator. These results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter-specific requirements (Lo, 2005).
Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h. During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. This study shows that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin (Hirota, 2005).
The phosphorylation of the serine 10 at histone H3 has been shown to be important for transcriptional activation. This study reports the molecular mechanism through which H3S10ph triggers transcript elongation of the FOSL1 gene. Serum stimulation induces the PIM1 kinase to phosphorylate the preacetylated histone H3 at the FOSL1 enhancer. The adaptor protein 14-3-3 binds the phosphorylated nucleosome and recruits the histone acetyltransferase MOF, which triggers the acetylation of histone H4 at lysine 16 (H4K16ac). This histone crosstalk generates the nucleosomal recognition code composed of H3K9acS10ph/H4K16ac determining a nucleosome platform for the bromodomain protein BRD4 binding. The recruitment of the positive transcription elongation factor b (P-TEFb) via BRD4 induces the release of the promoter-proximal paused RNA polymerase II and the increase of its processivity. Thus, the single phosphorylation H3S10ph at the FOSL1 enhancer triggers a cascade of events which activate transcriptional elongation (Zippo, 2009).
Activation of Janus kinase 2 (JAK2) by chromosomal translocations or point mutations is a frequent event in haematological malignancies. JAK2 is a non-receptor tyrosine kinase that regulates several cellular processes by inducing cytoplasmic signalling cascades. This study shows that human JAK2 is present in the nucleus of haematopoietic cells and directly phosphorylates Tyr 41 (Y41) on histone H3. Heterochromatin protein 1alpha (HP1alpha), but not HP1beta, specifically binds to this region of H3 through its chromo-shadow domain. Phosphorylation of H3Y41 by JAK2 prevents this binding. Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1alpha at the same site. These results identify a previously unrecognized nuclear role for JAK2 in the phosphorylation of H3Y41 and reveal a direct mechanistic link between two genes, jak2 and lmo2, involved in normal haematopoiesis and leukaemia (Dawson, 2009).
One of the predictions of the histone code hypothesis is the existence of functional interactions between chromatin remodeling complexes, such as SWI/SNF, and histone acetylase complexes, such as GCN5. Recent studies have elucidated the temporal sequence in which these coactivators of transcription are recruited to promoters in vivo and how their enzymatic properties contribute to gene activation. The best-characterized example in mammals is provided by the human IFN-ß gene. The gene is switched on by three transcription factors (NF-kappaB, IRFs, and ATF-2/c-Jun), and an architectural protein [HMG I(Y)], all of which bind cooperatively to the nucleosome-free enhancer DNA to form an enhanceosome. The enhanceosome targets the modification and repositioning of a nucleosome that blocks the formation of a transcriptional preinitiation complex on the IFN-ß promoter. This is accomplished by the ordered recruitment of HATs, SWI/SNF, and basal transcription factors. Initially, the GCN5 HAT-containing complex is recruited, and it acetylates the nucleosome. This is followed by the recruitment of the CBP-PolII holoenzyme complex. Next, the SWI/SNF remodeling machine arrives at the promoter via bivalent interactions with CBP and the acetylated histone N tails. SWI/SNF alters the structure of the nucleosome via an unknown mechanism, thus allowing recruitment and DNA binding of TFIID to the TATA box. The DNA bending induced upon TFIID binding to the promoter causes sliding of the SWI/SNF-modified nucleosome to a new position 36 bp downstream, thus allowing the initiation of transcription. This ordered recruitment and nucleosome sliding is consistent with the view that histone acetylation sets the stage for ATP-dependent remodeling by establishing a recognition surface for the bromodomains present in SWI/SNF-like remodeling machines. Furthermore, since histone acetylation precedes the recruitment of additional complexes bearing bromodomains, such as TFIID, it is possible that this modification also regulates recruitment of TFIID to promoters (Agalioti, 2002).
Experiments were carried out to test the histone code hypothesis. Only a small subset of lysines in histones H4 and H3 are acetylated in vivo during viral infection, and this modification is carried out by the GCN5 transcriptional coactivator complex. Reconstitution of recombinant nucleosomes bearing mutations in these lysine residues reveals a biochemical cascade for gene activation via a point-by-point interpretation of the histone code through the ordered recruitment of bromodomain transcription complexes. More specifically, acetylation of H4 lysine 8 is required for recruitment of the SWI/SNF complex, whereas acetylation of lysines 9 and 14 in histone H3 is critical for the recruitment of the general transcription factor TFIID. Thus, the information contained in the DNA address of the enhancer is extended (transferred) to the histone N termini by generating novel adhesive surfaces that participate in the recruitment of transcription complexes (Agalioti, 2002).
The precision by which the histone code is decoded is remarkable. Most likely, the code is translated via specific interactions of bromodomains with the acetylated histone N termini. The bromodomain in BRG1 associates with the H4 tail acetylated at K8, whereas the double bromodomain in TAFII250 binds to the doubly acetylated (at K9 and K14) H3 tail. The competition assays using either acetylated histone N termini peptides or bromodomain polypeptides as competitors revealed an unprecedented level of specificity for the interactions between bromodomains and acetyl-lysine histone N termini. Again, this remarkable degree of specificity may be dictated by the conformational changes forced upon these complexes by their interactions with other proteins in the complex. Thus, the point-by-point interpretation of the histone acetylation code may rely on the precise allosteric changes induced in many proteins upon their association with transcription factor complexes. For example, the initial recruitment of SWI/SNF via its association with CBP is stabilized on the promoter through the association of the BRG1 bromodomain with the H4 K8 acetylated tail. Although, BRG1's bromodomain could interact at least in principle with other acetylated lysine residues on H3, these interactions may not be of sufficient strength to ensure stable binding of the SWI/SNF complex to the promoter. Similarly, recruitment of TFIID to the SWI/SNF-modified promoter is stabilized via two types of interactions. The first with various enhanceosome components and the second with the interaction between the two bromodomains of TAFII250 and the two acetyl groups on residues K9 and K14 of histone H3. Several observations support this notion: (1) both TBP and TAFII250 are simultaneously recruited to the promoter with almost identical kinetics in vivo; (2) recruitment of TFIID in vivo occurs only when both H3 K9 and K14 are acetylated; (3) mutations in either K9 or K14 abrogate TFIID recruitment. The data show that the TAFII250 double bromodomain, when recruited to the natural IFN-ß promoter, interacts specifically with the H3 K9 and K14 acetylated residues and not with the acetylated H4 tails. However, when tested in isolation and out of the promoter/chromatin natural context, the double TAFII250 bromodomain interacts with similar affinities to both H4 and H3 acetylated tails, a result consistent with in vitro observations. It is proposed that the inordinate set of interactions occurring with purified bromodomains and acetylated histone tails is 'fixed' when present in natural promoter/chromatin contexts. In the latter case, it is possible that the competing interactions cannot take place either because the alternative target is occupied (e.g., the H4 tail is already bound by SWI/SNF) or the specific three-dimensional conformation of the transcription complex does not permit these interactions to occur (Agalioti, 2002).
A model is presented that depicts the ordered biochemical cascade decoding the DNA and histone acetylation code during activation of human IFN-ß gene transcription following Sendai virus infection. It is thought that the promoter DNA code contains all the information for the assembly of the enhanceosome in response to virus infection. The enhanceosome that assembles at the promoter element recruits the GCN5 histone acetyltransferase. Subsequently GCN5 acetylates initially H4K8 and H3K9. An unknown kinase recruited by the enhanceosome phosphorylates H3 Ser 10, a prerequisite for H3K14 acetylation by GCN5. The histone code is subsequently translated by recruiting of additional components required for transcription. The bromodomain containing transcription complexes SWI/SNF and TFIID are recruited to the promoter via bivalent interactions between the enhanceosome and specifically acetylated histone N termini, and this subsequently stimulates transcription of the IFN-ß gene (Agalioti, 2002).
Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, it has been shown that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacts with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone is essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persists on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions (Kanno, 2004).
Given that H3 is a preferred substrate for many HATs in in vitro assays, the absence of FRET between H3 and Brd2 was striking. This could not be explained by a lack of H3 acetylation, since HeLa cells contained significant amounts of acetylated H3. This led to a test of another bromodomain protein, TAFII250, for interaction with H3. TAFII250 is a component of the basal transcription factor TFIID, and its double bromodomain module binds to an acetylated H4 peptide in vitro and acetylated H3 in a reconstituted chromatin target (Agalioti, 2002). Transfected CFP-TAFII250 associated with other components of TFIID in HeLa cells, indicating that it was incorporated into a stable TFIID complex. It was found that TAFII250 produced a significant FRET signal with H3 as well as H4 and to a lesser extent H2B but not with H2A. Because TAFII250 has a HAT activity and contains a histone recognition site outside the bromodomains (Mizzen, 1996), deletions were tested that removed the HAT region, but retained the bromodomains. The deletions produced FRET with H3 and H4 as well as did full length TAFII250. It is of note that these deletions also lacked the TBP binding surface, and TAFII250-BD is further devoid of the putative HMG-like region, indicating that DNA binding of TAFII250 is dispensable for its histone association. Thus, H3 is recognized by the bromodomains of TAFII250 but not of Brd2. Moreover, in contrast to FRET with Brd2, both mutants H4-K(5,12)G and H4-K(8,16)G partially reduced FRET with TAFII250, indicating that K5/K12 and K8/K16 of H4 both critically contribute to the interaction with TAFII250. Peptide precipitation analysis showed that TAFII250 bound H4 peptides mono-acetylated at K8, K12, or K16 as well as H3 peptides acetylated at K14. It is concluded that specific lysine residues of both H3 and H4 are recognized by the TAFII250 bromodomains in vivo and that TAFII250 has broader recognition specificity than Brd2 (Kanno, 2004).
The N-terminal tails of the core histones play important roles in transcriptional regulation, but their mechanism(s) of action are poorly understood. Pure chromatin templates assembled with varied combinations of recombinant wild-type and mutant core histones have been employed to ascertain the role of individual histone tails, both in overall acetylation patterns and in transcription. In vitro assays show an indispensable role for H3 and H4 tails, especially major lysine substrates, in p300-dependent transcriptional activation, as well as activator-targeted acetylation of promoter-proximal histone tails by p300. These results indicate, first, that constraints to transcription are imposed by nucleosomal histone components other than histone N-terminal tails and, second, that the histone N-terminal tails have selective roles, which can be modulated by targeted acetylation, in transcriptional activation by p300 (An, 2002).
The first significant conclusion from these results is that the tails do not simply and uniquely impose constraints to the binding and function of either gene-specific transcriptional activators or components of the general transcriptional machinery. Instead, it seems clear that the globular domains themselves maintain a repressed state and that specific N-terminal tails and corresponding natural acetylatable lysine residues are actively required for the reversal of these effects. Another significant conclusion from the present study is that the H3 and H4 tails are selectively required for the observed derepression and net activation by Gal4-VP16 and p300 and, that these tails are not redundant for transcription. These results are consistent with differential effects of H3 versus H4 tail mutations on the transcriptional regulation of specific genes and differential functions for H3 and H4 tails versus H2A and H2B tails both in transcription and in higher-order chromatin structure (An, 2002 and references therein).
These results also establish a direct link between activator-dependent acetylation of histones by p300 and activator-dependent transcription. Beyond the fact that activator-dependent transcription requires activator- and p300-dependent histone tail acetylation, the selective requirement for H3 and H4 tails and corresponding acetylation sites for transcription correlates with the observations (1) that H3 is the preferred p300 substrate in chromatin, (2) that optimal H3 and H4 acetylation occurs independently of H2A and H2B tails, whereas maximal H2A and H2B acetylation is dependent upon H3 and H4 tails, and (3) that there is a strong activator-mediated targeting of acetylation to promoter-proximal H3 and H4 (An, 2002).
Dynamic changes in the modification pattern of histones, such as acetylation, phosphorylation, methylation, and ubiquitination, are thought to provide a code for the correct regulation of gene expression mostly by affecting chromatin structure and interactions of non-histone regulatory factors with chromatin. Recent studies have suggested the existence of an interplay between histone modifications during transcription. The CBP/p300 acetylase and cofactor-associated arginine [R] methyltransferase 1 (CARM1) can positively regulate the expression of estrogen-responsive genes, but the existence of a crosstalk between lysine acetylation and arginine methylation on chromatin has not yet been established in vivo. By following the in vivo pattern of modifications on histone H3, following estrogen stimulation of the pS2 promoter, it has been shown that arginine methylation follows prior acetylation of H3. Within 15 min after estrogen stimulation, CBP is bound to chromatin, and acetylation of K18 takes place. Following these events, K23 is acetylated, CARM1 associates with chromatin, and methylation at R17 takes place. Exogenous expression of CBP is sufficient to drive the association of CARM1 with chromatin and methylation of R17 in vivo, whereas an acetylase-deficient CBP mutant is unable to induce these events. A mechanism for the observed cooperation between acetylation and arginine methylation comes from the finding that acetylation at K18 and K23, but not K14, tethers recombinant CARM1 to the H3 tail and allows it to act as a more efficient arginine methyltransferase. These results reveal an ordered and interdependent deposition of acetylation and arginine methylation during estrogen-regulated transcription and provides support for a combinatorial role of histone modifications in gene expression (Daujat, 2002).
The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. This study demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. Autoacetylation of CBP is critical for the bromodomain-H3 K56Ac interaction, and it is proposed that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1-bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein-Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP (Das, 2014).
Rb associates with histone deacetylase. This association serves to repress transcription by promoting formation of nucleosomes that inhibit transcription. However, mSin3A (see Drosophila Sin3A), a corepressor that binds to the transcription factor Mad and appears to tether MAD to histone deacetylase, in contrast to other repressors, is not detected in the complex between Rb and histone deacetylase. Interaction between domain A and B in the Rb pocket forms a site for association with histone deacetylase. Recruitment of histone deacetylase by either Rb or Mad results in a decrease in acetylated histone H3 associated with the promoter in vivo, consistent with the idea that this recruitment indeed results in deacetylation of histones bound to the promoter. This Rb-mediated recruitment of histone deacetylase can only repress a subset of promoters and transcription factors. Repression of the adenovirus major late promoter by Rb and Mad is dependent on histone deacetylase activity, while repression of the tyrosine kinase promoter and the SV40 enhancer by Rb is independent of histone deacetylase activity. The activity of other promoters and transcription factors appears resistant to recruitment of histone deacetylase, but these promoters and transcription factors are still blocked by Rb through direct inhibition of these transcription factors. Thus, Rb can block transcription through two separate mechanisms (direct action and recruitment of histone deacetylase), and both mechanisms are required to account for the pattern of promoters repressed by Rb. Surprisingly, even though Rb and p107 appear to share significant structural similarity within the pocket repressor motif, p107 does not interact with histone deacetylase and does not depend on histone deacetylase activity to repress transcription. The results demonstrate fundamental differences in the mechanism of transcriptional repression by Rb and p107 and suggest that p107 may only have a subset of the repressor activities of Rb (Luo, 1998).
The induction of immediate-early (IE) genes, including proto-oncogenes c-fos and c-jun, correlates well with a nucleosomal response, with the phosphorylation of histone H3 and is HMG-14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Direct evidence is provided that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly-modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c-fos- and c-jun-associated nucleosomes become doubly-modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site-specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)-induced hyperacetylation than non-phosphorylated H3. Because MAP kinase-mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance (Clayton, 2000).
In cultured mammalian cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1, creating a binding site for the chromo domain of HP1. SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter. In fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins (Nielsen, 2001).
The Rb protein functions as a repressor, at least partly, through the recruitment of histone deacetylase activity. Whether histone methylation might also be involved in Rb-mediated repression is considered in this study, since the SUV39H1 methylase has repressive potential. To establish whether Rb can associate with histone-methylase activity, a glutathione S-transferase (GST)-Rb fusion was incubated with nuclear extract, and any bound methylase activity was assayed on bulk histones as a substrate. GST-Rb can purify histone-methylase activity, whereas GST fusions to transcriptional activators such as P/CAF, E2F1, p53 and ATF2 do not. The Rb-associated methylase activity is specific for histone H3 and does not recognize the GAR substrate for arginine methylases (Nielsen, 2001).
An antibody directed against Rb can precipitate histone-methylase activity that is specific for histone H3. This methylase binds the pocket domain of Rb because tumor-derived mutations in the pocket (F706C), or truncations of the pocket (928 and 737), abolish binding to the methylase. The Rb-associated methylase has specificity for Lys 9 of histone H3 (Nielsen, 2001).
The SUV39H1 protein possesses lysine methylase activity, which resides within its conserved SET domain. Since this enzyme has specificity for Lys 9 of histone H3 an investigation was carried out to see whether SUV39H1 could be the methylase associated with Rb. A GST-Rb fusion can bind to transfected, hemagglutinin (HA)-tagged SUV39H1. Endogenous Rb also associates with endogenous SUV39H1, as shown by a co-immunoprecipitation analysis (Nielsen, 2001).
Whether SUV39H1 can act as co-repressor with Rb was investigated. SUV39H1 represses the activity of a promoter bearing GAL4 sites in a concentration-dependent manner in vivo, but only when Gal4-Rb is present at the promoter. The co-repressor functions of SUV39H1 can also be seen on the cyclin E promoter, a natural target for Rb-mediated repression. This promoter can be stimulated by E2F and is not affected by SUV39H1 alone. Under limiting conditions, where Rb represses E2F activity slightly, the SUV39H1 enzyme can further repress E2F activity in cooperation with Rb. When the methylase domain of SUV39H1 is removed, the resulting SUV39H1 SET domain is unable to mediate repression. These results suggest that SUV39H1 uses its methylase activity to repress the cyclin E promoter when it is targeted there by Rb (Nielsen, 2001).
SUV39H1 is known to form a complex with the HP1 protein. Recently, HP1 function has been placed downstream of SUV39H1 histone methylation, since HP1 recognizes specifically, and binds to, histone H3 methylated at Lys 9. This mechanistic link has prompted an investigation of the role of HP1 in Rb/SUV39H1-mediated repression. Rb and HP1 can interact in a two-hybrid screen in yeast, and it has been shown that there is an LXCXE motif (X is any amino acid) in HP1. It was therefore asked if HP1 binds to Rb in mammalian cells. A GST-HP1 fusion can bind Rb that is present in nuclear extracts; Rb and HP1 can associate in vivo, as determined by co-immunoprecipitation analysis. An LXCXE motif peptide can compete for the binding of histone H3 methylase activity to Rb, but does not affect the binding of H3 methylase activity to HP1, which is consistent with the finding that the methylase activity is associated with the Rb pocket (Nielsen, 2001).
Whether HP1 can recognize methylated lysines while associated with Rb was tested. To address this, a histone H3 peptide methylated at Lys 9 was used as an affinity resin. Recombinant Rb does not bind to this methylated peptide, but it can do so efficiently in the presence of recombinant HP1. This result confirms that HP1 can bind directly to Rb and that it can recognize Rb and methylated lysine simultaneously. A similar experiment was attempted using nuclear extracts as the source of protein. The H3 peptide methylated at Lys 9 binds to HP1, SUV39H1 and Rb, as detected by Western blotting (Nielsen, 2001).
These results suggest that an Rb-regulated promoter such as cyclin E should be associated with HP1. To test this chromatin immunoprecipitation analysis of the cyclin E promoter was performed. A nucleosome encompassing the cyclin E initiation site (cyclin Epr) that is known to be deacetylated is associated with HP1 in fibroblast cells of mouse embryos. Since the cyclin Epr nucleosome binds HP1, whether this nucleosome contains histone H3 that is methylated at Lys 9 was examined. To test this an antibody was produced that recognizes histone H3 when methylated at Lys 9. In Rb+/+ cells the cyclin Epr nucleosome contains methylated histone H3 and is associated with HP1. However, in Rb-/- cells histone H3 methylation and HP1 binding is significantly reduced. Thus, in the presence of Rb, methylase activity and HP1 are targeted to the cyclin E promoter (Nielsen, 2001).
Collectively, these results implicate each of the components of the SUV39H1-HP1 complex in the repression functions of the Rb protein. In this model Rb brings to the promoter the SUV39H1 enzyme (and possibly other members of this family) to methylate Lys 9 of histone H3 and provides a binding site for HP1. Methylation by SUV39H1 cannot take place on an already acetylated lysine. Thus the deacetylase activity associated with Rb may be a necessary preceding step to SUV39H1-mediated methylation. The precise function of HP1 in repression is unclear. HP1 may protect the methyl group on Lys 9 from attack from potential demethylases; it may bring in other repressive functions, or it may enhance the stability of the Rb-associated repressor complex (Nielsen, 2001).
HP1 is found associated with a number of transcriptional repressors, suggesting that it may have a role in repressing many other promoters. Thus, the results presented here extend the role of SUV39H1 and HP1 beyond heterochromatic gene silencing to a more general, genome-wide function in repressing gene transcription (Nielsen, 2001).
Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. TRRAP is a component of TIP60 (see Tip60) and PCAF/GCN5 (see Drosophila Pcaf) histone acetyl transferase (HAT) complexes. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc (Bouchard, 2001).
The aim of this study was to resolve the role of MBII (an effector domain of Myc that binds TRRAP) and TRRAP in gene activation by Myc, using an endogenous target gene of Myc, cyclin D2, as model. Upon binding to the cyclin D2 promoter, Myc recruits TRRAP and induces the preferential acetylation of histone H4 at a single nucleosome. Conversely, loss of endogenous Myc binding correlates with histone deacetylation and loss of TRRAP binding during the TPA-induced differentiation of a human promyelocytic cell line, HL60. The integrity of MBII is required for TRRAP recruitment, histone acetylation, and transcriptional activation at the cyclin D2 locus. Therefore, previous suggestions that MBII has no role in transcriptional activation based on transient reporter assays need to be reevaluated. Deletion of the entire N terminus of Myc up to MBII (s-Myc) renders Myc unable to induce cell cycle progression and expression of either cyclin A or cyclin D2 in 3T3 fibroblasts, consistent with recent results that the N terminus of Myc is required for regulation of proliferation and induction of gene expression in a cell-type-dependent manner. Most likely, this is because stable association with TRRAP requires sequences in the N terminus of Myc in addition to MBII (Bouchard, 2001 and references therein).
Mad proteins are thought to antagonize the function of Myc by recruiting a repressor complex that contains histone deacetylase activity. Observations suggest that this model applies to the cyclin D2 promoter: (1) repression of the cyclin D2 promoter by Mad1 requires the integrity of an N-terminal domain, which mediates recruitment of histone deacetylase complexes through interaction with Sin3A; (2) during HL60 differentiation, Mad1 and HDAC1 are corecruited to the cyclin D2 promoter, correlating with histone deacetylation of both histones H3 and H4 at the cyclin D2 promoter. Taken together, these data strongly support a model in which endogenous Myc/Max and Mad/Max complexes contribute to the regulation of transcription of the cyclin D2 gene through their antagonistic effects on histone acetylation. In addition, these findings show the functional relevance of the switch between Myc/Max and Mad/Max complexes during differentiation of hematopoietic cells. Recent work on the gene encoding the catalytic subunit of telomerase, htert, suggests that this model also may apply to this promoter (Bouchard, 2001 and references therein).
Up-regulation of the CAD (carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase) gene by Myc does not involve changes in histone acetylation. Instead, high levels of histone acetylation at the promoter were found in both quiescent and proliferating cells, showing that Myc can control at least one step in addition to histone acetylation to promote active transcription. Additional proteins have been identified that bind to different domains of Myc and that are candidates for such an activity: for example, the C terminus of Myc binds to Ini1, a component of the Swi/Snf family of chromatin-remodeling complexes. Clearly, a detailed analysis of the role of Myc in activation of individual promoters will be required before the role of each interaction in Myc biology can be resolved fully (Bouchard, 2001 and references therein).
Histone deacetylases (HDACs) modulate chromatin structure and transcription, but little is known about their function in mammalian development. Previously, HDACs have been shown to be required for embryonic development of invertebrates. In addition, loss of specific components of the Sin3 and the NuRD complexes such as RbAp46/48 (lin-53, rba-1), Sin3 (dSin3A), Mi-2 (dMi-2, chd-3, chd-4) and MTA1/MTA2 (egl-27, egr1) affect embryonic viability and development of Drosophila melanogaster and Caenorhabditis elegans. Mammalian HDAC1 (Drosophila homolog: Rpd3) has been implicated in the repression of genes required for cell proliferation and differentiation. Targeted disruption of both HDAC1 alleles results in embryonic lethality before E10.5 due to severe proliferation defects and retardation in development. HDAC1-deficient embryonic stem cells show reduced proliferation rates, which correlate with decreased cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1. Similarly, expression of p21 and p27 is up-regulated in HDAC1-null embryos. In addition, loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 and concomitant changes in other histone modifications. The expression of HDAC2 and HDAC3 is induced in HDAC1-deficient cells, but cannot compensate for loss of the enzyme, suggesting a unique function for HDAC1. This study provides the first evidence that a histone deacetylase is essential for unrestricted cell proliferation by repressing the expression of selective cell cycle inhibitors (Lagger, 2002).
HDAC1-deficient embryos and HDAC1-null cells show proliferation defects. Together with previous data showing high expression levels of HDAC1 in proliferating cells, these results are suggestive of a proliferation-linked function for the enzyme. Paradoxically, the recruitment of class I HDACs by Rb seems to be important for the repression of proliferation-associated genes, and HDAC1 should therefore act rather as a growth inhibitor. However, the data shown in this report demonstrate that one of the key functions of HDAC1 is to prevent the expression of antiproliferative genes in cycling cells. These findings indicate that deacetylases other than HDAC1 as well as deacetylase-independent mechanisms ensure the proper regulation of Rb target genes in HDAC1-null cells. Evidence has been presented that HDAC1 controls the expression of a specific subset of CDK inhibitors. The induction of p21 and p27 in HDAC1-null cells correlates with the hyperacetylation of the corresponding promoters. The specificity of this response is underlined by the fact that only the proximal but not the distal portion of the p21 promoter was found to be associated with hyperacetylated histone H3. The proximal p21 promoter contains Sp1-binding sites that are required for the induction of the p21 gene by deacetylase inhibitors. Activation of tumor suppressors has been shown to be a crucial function of HDAC inhibitors as anti-cancer drugs in human cells. These results strongly support the idea that HDAC1 might be a relevant target in tumor treatment (Lagger, 2002).
Purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. The conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. These findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm (Kondo, 2004).
Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. This study shows that the state of acetylated histones fluctuate in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) represses transcription with HDACs and mSin3B, which is relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induces endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which both interact with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulates rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. TSA administration into the lateral ventricle induces mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation (Naruse, 2004).
Mammalian circadian rhythms are based on transcriptional and post-translational feedback loops. Essentially, the activity of the transcription factors BMAL1 (also known as MOP3) and CLOCK is rhythmically counterbalanced by Period (PER) and Cryptochrome (CRY) proteins to govern time of day-dependent gene expression. Circadian regulation of the mouse albumin D element-binding protein (Dbp) gene involves rhythmic binding of BMAL1 and CLOCK and marked daily chromatin transitions. Thus, the Dbp transcription cycle is paralleled by binding of BMAL1 and CLOCK to multiple extra- and intra-genic E boxes, acetylation of Lys9 of histone H3, trimethylation of Lys4 of histone H3 and a reduction of histone density. In contrast, the antiphasic daily repression cycle is accompanied by dimethylation of Lys9 of histone H3, the binding of heterochromatin protein 1alpha and an increase in histone density. The rhythmic conversion of transcriptionally permissive chromatin to facultative heterochromatin relies on the presence of functional BMAL1-CLOCK binding sites (Ripperger, 2006).
Methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, evidence is provided that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA (Cowell, 2002).
Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. The methylation patterns of histone H3 at lysine 9 (H3/K9) have been investigated, as well as the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persists until the two-cell stage. However, de novo methylation of H3/K9 occurs and the asymmetry is lost during the four-cell stage. The unmethylated male pronucleus undergoes de novo methylation when it is transferred into enucleated GV- or MII-stage oocytes: this suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurs when either protein synthesis or gene expression is inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurs when the male pronucleus is transferred to an enucleated GV oocyte. These results suggest that H3/K9 methylation is an epigenetic marker of parental genome origin during early preimplantation development (Liu, 2003).
In mouse embryos, transcription is initiated during the one-cell stage, at which stage transcriptional activity is much higher in the male pronucleus than in the female pronucleus. Studies have suggested that this difference is caused by differences in the repressive states of chromatin; the chromatin in the male pronucleus is not repressed, whereas that in the female pronucleus is partially repressed. However, the mechanism regulating this differential repression is not clear. It has been suggested that H3/K9 methylation is involved in the repression of gene expression in both euchromatic and heterochromatic regions. It is possible that the asymmetric methylation of H3/K9 is involved in the different repressive states of the two parental genomes (Liu, 2003).
During the one-cell stage, DNA replication occurs asynchronously between the two pronuclei, in that the female pronucleus requires a longer time to complete replication. This suggests that heterochromatin is asymmetrically constituted in the two pronuclei, as H3K9 methylation is involved in the formation of heterochromatin, which is late-replicating. A higher level of H3K9 methylation would promote the formation of heterochromatin in the female pronucleus, resulting in late DNA replication. Supporting this hypothesis are the facts that methylated H3K9 recruits heterochromatin protein 1 (HP1), an essential protein constituting heterochromatin, and that this protein is accumulated only in the female pronucleus before S phase of the one-cell stage. The biological significance of this asymmetric heterochromatin formation is unclear. It does not directly involve asymmetric X chromosome inactivation, since HP1 does not accumulate with the inactive X chromosome (Liu, 2003 and references therein).
The histone H3 Lys 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using chromatin immmunoprecipitation (ChIP) and sequencing (ChIP-seq) analyses, Eset target genes with Eset-dependent H3K9 trimethylation were identified. Genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single-cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and, subsequently, placental tissues. Coimmunoprecipitation and ChIP assays further demonstrate that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. These results suggest that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor (Yuan, 2009).
Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes, which is limited by the compaction of DNA in chromatin. Althought insight has been gained into how core histones and their modifications influence this process, the role of linker histones remains unclear. This study show that, within the first minute of progesterone action, a complex cooperation between different enzymes acting on chromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First, activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2 [activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 by the MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylase KDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, and kinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histone H2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) and expression arrays show that H1 displacement is required for hormone induction of most hormone target genes, some of which are involved in cell proliferation (Vicent, 2011).
These results contribute to a better comprehension of the molecular mechanisms of gene induction by describing the very initial steps of hormonal promoter activation. The data reveal an unexpected complexity in the interactions between enzymatic activities implicated in preparing the chromatin for full access of transcription factors. Apart from previously described enzymatic activities, at least four complexes act 1 min after hormone addition. An ATP-dependent chromatin remodeling complex (NURF), a protein kinase complex (Cdk2/CyclinA), a histone lysine demethylase (JARID1B/KDM5B), and a histone lysine methylase (MLL2 or MLL3)-containing complex cooperate in the displacement of histone H1 from the promoter, an important early step in gene induction by progestins (Vicent, 2011).
It has been shown, in T47D-MTVL cells treated with hormone for 5 min, PR interacts with an exposed HRE on the surface of a nucleosome positioned over the MMTV promoter and recruits Brg1/Brm-containing BAF complexes. This study demonstrates that NURF interacts with PR, and that recruitment of the NURF complex in the first minute following hormone addition is a requisite for subsequent binding of BAF and activation of mammary tumor virus (MMTV) and other progesterone target genes. NURF is anchored at the promoter of progesterone target genes by an interaction with H3K4me3, likely generated by the MLL2/3 histone lysine methylases of the ASCOM complex. This is reminiscent of the role of hormone-induced acetylation of H3K14 in anchoring the BAF complex. At both phases in activation of the promoter, a histone tail modification stabilizes the binding of an ATP-dependent chromatin remodeling complex to the target promoters (Vicent, 2011).
Another similarity between the two subsequent cycles of promoter chromatin remodeling relates to the role of protein kinases. It was found previously that hormone-induced activation of the Src/Ras/Erk cascade leads to phosphorylation of PR at S294 and activation of Msk1, which is targeted to the promoter by PR and phosphorylates H3 at S10, contributing to the displacement of a repressive complex containing HP1γ. This study shows that, prior to this event, 1 min after hormone, PR interacts with a complex of Cdk2 and CyclinA that phosphorylates PR at S400, is recruited to the promoter, and phosphorylates histone H1, leading to its displacement. Thus, there are two similar and consecutive cycles essential for transcriptional activation of hormone-dependent genes, both involving the collaboration between protein kinases, histone-modifying enzymes, and ATP-dependent chromatin remodelers. Each of the remodeling complexes is anchored at the promoter by different epigenetic marks: H3K4me3 established by MLL2/3 anchors NURF, and H3K14ac established by PCAF anchors BAF. The final output of the first cycle is to decompact the chromatin fiber by displacing histone H1, and the outcome of the second cycle is to open the nucleosome by displacing H2A/H2B dimers (Vicent, 2011).
The chromatin remodeling complex NURF has been shown to be necessary for both transcription activation and repression in vivo. Most reports on the role of NURF in gene regulation come from studies in Drosophila, where NURF is involved in the activation of several genes, including the homeotic selector gene engrailed, ultrabithorax, ecdysone-responsive genes, and the roX noncoding RNA. These studies were complemented with mechanistic studies using recombinant Drosophila NURF complex. In contrast, little is known regarding the mechanism of action of NURF in human cells, except for reports on a role in neuronal physiology. It was found that, in T47D-MTVL human breast cancer cells, NURF is essential for efficient hormone-dependent activation of several PR target genes, and is recruited to the target promoters via an interaction with PR. The BAF complex is also recruited to the MMTV promoter within minutes after progestin treatment, but the kinetics of loading of both chromatin remodelers are different. NURF is recruited after 1 min of hormone treatment, while BAF is loaded only after 5 min and its recruitment depends on NURF action. These findings highlight the notion of transcription initiation as a process involving consecutive cycles of enzymatic chromatin remodeling, where each enzyme complex is necessary at a given time point and catalyzes a particular remodeling step. These results support the existence in T47D-MTVL cells of several pools of PR, associated with the different chromatin remodelers. How the coordinated action of each PR population on target promoters is orchestrated is not well understood, but phosphorylation of the receptor by different kinases and post-translational modifications of nucleosomal histones could provide possible mechanisms (Vicent, 2011).
Although H3K4me3 marks transcription start sites (TSSs) of virtually all active genes the role of this modification during MMTV activation has been questioned. This study shows that, in T47D-MTVL cells, the MLL2/3-containing complex ASCOM is recruited to a target promoter after 1 min of hormone and increases H3K4me3. Experiments with siRNA knockdown, ChIP, and peptide pull-down assays showed that H3K4me3 is critical for NURF anchoring at the promoter. The very early and transient appearance of the H3K4me3 mark could explain the apparent controversy with previously published studies. It was found that the H3K4me3 signal observed at the MMTV promoter is due to the concerted recruitment of the ASCOM complex and the localized displacement of the H3K4me3/2/1 demethylase KDM5B. Knockdown of KDM5B increased the basal and hormone-dependent activity of PR target genes and caused an increase in H3K4me3 levels at the promoters in the absence of hormone (Vicent, 2011).
The molecular mechanism underlying hormone-induced displacement of KDM5B is unclear. It has been reported that KDM5B forms a complex with histone deacetylases (HDACs). Ir was shown previously that an HP1γ-containing complex is bound to the MMTV promoter prior to induction, and is displaced by phosphorylation of H3S10 catalyzed by hormone-activated Msk1. However, in coimmunoprecipitation experiments, no interaction between KDM5B and HP1γ was detected. Recently, it has been reported that PARP1 can parylate and inactivate KDM5B catalytic activity. Since nuclear receptors are known to activate PARP1, it is possible that this pathway participates in the inactivation and displacement of KDM5B following progestin treatment (Vicent, 2011).
The PHD finger present in the BPTF subunit of NURF acts as a highly specialized methyl lysine-binding domain critical for NURF loading. H3S10ph and H3K14ac, two other post-translational modifications present in the MMTV promoter chromatin after hormone addition, increase the binding of the PHD domain to H3K4me3. Binding of BPTF to acetylated lysines could be expected, as the protein contains a bromodomain in its C terminus, but the interaction with H3 phosphopeptides was not predicted, as BPTF does not encompass a consensus 14-3-3-like domain. Regarding the role of the H3K9me3 signal in NURF recruitment, peptide pull-down experiments showed no interaction of NURF components with the H3K9me3 mark. Moreover, either knockdown or inhibition of the methyltransferase G9a increased the basal level of transcription in several target genes without affecting the fold induction after hormone. The same effect was observed when cells were depleted of HP1γ, indicating that the H3K9me3 signal anchors HP1γ at the target chromatin (Vicent, 2011).
The NURF complex is recruited after 1 min of hormone, decreased after 2 min, and is almost undetectable after 5 min. How NURF is released from target chromatin is still unknown. Binding of NURF correlates closely with H3K4me3, and therefore a decrease in the trimethylation of H3K4 would explain NURF displacement. It has been proposed that methylation of histone H3R2 by PRMT6 and methylation of H3K4 by MLLs are mutually exclusive. Moreover, H3R2 methylation has been reported to block the binding of effectors that harbor methyl-specific binding domains, including PHD domains, chromodomains, and Tudor domains. Thus, the presence of the H3R2me2 mark could cooperate in erasing the H3K4me3 signal from the promoters and in competing for NURF binding, thus triggering NURF displacement (Vicent, 2011).
MMTV minichromosomes reconstituted with Drosophila embryo extracts were used previously to address the role of histone H1. Histone H1 increases nucleosome spacing and compacts the chromatin, hinders access of general transcription factors to the MMTV promoter, and thus inhibits basal transcription. In the presence of bound PR, H1 is phosphorylated by Cdk2 and subsequently is removed from the promoter upon transcription initiation. The kinase Cdk2 is known to phosphorylate histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions. Histone H1 phosphorylation by Cdk2 has been associated with hormone-dependent transcriptional activation. This study found that NURF facilitates the access of Cdk2/CyclinA to target promoter chromatin, and this could explain its role in H1 displacement from the MMTV promoter and from 15 other PR-binding sites that also contain NURF and recruit Cdk2 after hormone treatment. Along with the general effect of Cdk2 inhibition on gene regulation by progestins, these results support a very general role of Cdk2/CyclinA in histone H1 eviction during the initial steps of hormonal chromatin remodeling (Vicent, 2011).
There is evidence for a direct interaction between PR and Cdk2, CyclinA, or cyclinE that could explain how Cdk2/CyclinA is recruited to the target promoters. In the T47D-MTVL breast cancer cell line, a hormone-independent association of PR with Cdk2 was found and recruitment of CyclinA to this complex upon hormone addition. Therefore, PR could recruit Cdk2/CyclinA to the target promoter upon hormone addition. It is not known whether NURF and Cdk2/CyclinA form a single ternary complex with PR, or rather are in two different PR-associated complexes. Although by coimmunoprecipitation interaction of PR with both complexes was detected after 1 min of hormone addition, a more in-depth analysis performed at 1-min intervals at 30°C revealed that NURF is recruited before Cdk2/CyclinA. These results suggest that NURF recruitment is required for Cdk2/CyclinA loading at target promoters, and support the existence of two independent complexes (Vicent, 2011).
Although H1 displacement takes place locally, it could have a long-range effect on chromatin decompaction, as demonstrated with in vitro assembled condensed chromatin. Displacement of histone H1 could be a prerequisite for all subsequent steps in remodeling, as SWI/SNF remodeling has been reported to be inhibited by the presence of histone H1. A connection between ISWI-containing remodeling machineries and histone H1 dynamics has been reported previously in Drosophila. In this system, ISWI promotes the association of the linker histone H1 with chromatin. Along these lines, it is still possible that NURF is also involved in later steps during hormone induction by helping histone H1 deposition back at the promoter (Vicent, 2011).
How H1 binding is regulated and leads to a more open chromatin structure remains unclear. Some models proposed that Cdk2-dependent H1 phosphorylation leads to the decondensation of chromatin during interphase by disrupting the association of HP1γ with the chromatin fiber. A hormone-dependent displacement of HP1γ from the MMTV promoter was observed without changes in H3K9me3 levels. Whether H1 and Hp1γ are interacting as part of a common repressive complex requires further studies but constitutes an attracting hypothesis. In contrast, PARP-1 possesses the ability to disrupt chromatin structure by PARylating histones (e.g., H1 and H2B) and a variety of nuclear proteins involved in gene regulation. Both PARP-1 and H1 compete for binding to nucleosomes and exhibit a reciprocal pattern of binding at actively transcribed promoters: H1 is depleted and PARP-1 is enriched. Other post-translational modifications of H1 have been proposed to influence its binding and function. Histone H1 is acetylated at Lys 26 in vivo and can be deacetylated by the NAD+-dependent HDAC SirT1, promoting the formation of repressive heterochromatin. This effect was accompanied by an enrichment of H1 at the promoter, and the spreading of heterochromatin marks like H3K9me3 and H4K20me1 throughout the coding region (Vicent, 2011).
Regarding the NURF-mediated changes in chromatin structure, analysis of nucleosome profiles obtained by MNase digestion before hormone treatment showed a preferential location of nucleosomes overlapping with NURF and PR sites that is less pronounced after hormone activation, indicating that chromatin remodeling is involved (Vicent, 2011).
Analysis of the hormone-regulated genes that are affected by depletion of NURF reveals many genes involved in cell cycle and cell proliferation, which could mediate the proliferative response of breast cancer cells to progestins. This may explain the inhibition of cell proliferation in response to progestins that was observed in T47D cells depleted of NURF. A similar inhibition of the proliferative response has been observed in cells depleted of Cdk2. These results indicate that histone H1 displacement may be a prerequisite for the effects of progestins on cell proliferation, and therefore the enzymes involved in this process would be novel targets for the pharmacological control of breast cancer cell proliferation (Vicent, 2011).
A model of the current view of the initial steps in progesterone activation of the MMTV promoter is presented. Although the different steps of remodeling are depicted as a linear time sequence, it cannot be excluded that some of these process occur in parallel and in different time sequences in different target promoters. The model reflects the average time sequence in the cell population. Briefly, after hormone induction, activated PR carrying Erk and Msk1 binds first to the exposed HRE1 on the surface of the MMTV promoter nucleosome in a process that does not require chromatin remodeling. Along with the activated PR kinases, the NURF and ASCOM complexes are recruited to the promoter chromatin in one or several complexes. The combined action of ASCOM recruitment and KDM5B displacement enhances H3K4me3 and stabilizes NURF at the promoter. Other modifications, such as H3S10phos and H3K14ac produced by Msk1 and PCAF, could also contribute to NURF anchoring. Once at the promoter, NURF remodels the nucleosome and facilitates the access of PR and the associated Cdk2/CyclinA kinase, which phosphorylates histone H1 and promotes its displacement, contributing to unfolding of the chromatin fiber. Although it was observed that H3S10 phosphorylation by Msk1 plays a role in HP1γ displacement, it is possible that phosphorylation of histone H1 also contributes to this process. H1-depleted nucleosomes constitute a suitable substrate for recruitment of PR-BAF complexes and further remodeling events catalyzed by BAF and PCAF. H3K14 acetylation by PCAF promotes BAF anchoring. BAF mediates ATP-dependent displacement of histones H2A/H2B, and thus facilitates binding of NF1. Bound NF1 stabilizes the open conformation of the H3/H4 tetramer particle that exposes the previously hidden HREs, allowing synergistic binding of further PR-BAF-kinase complexes and PCAF (Vicent, 2011).
Finally, given that NURF is also recruited to the promoter 30 min after hormone addition, when no H1 is present, it cannot be excluded that NURF catalyzes later steps in chromatin remodeling involving histones or nonhistone chromatin proteins. Indeed, the current results indicate that NURF can act on MMTV minichromosomes lacking histone H1. In this respect, it remains to be established whether NURF and BAF fulfill partly redundant functions, cooperate on the same promoter, or, rather, are mutually exclusive (Vicent, 2011).
An emerging concept in development is that transcriptional poising presets patterns of gene expression in a manner that reflects a cell's developmental potential. However, it is not known how certain loci are specified in the embryo to establish poised chromatin architecture as the developmental program unfolds. This study found that, in the context of transcriptional quiescence prior to the midblastula transition in Xenopus, dorsal specification by the Wnt/beta-catenin pathway is temporally uncoupled from the onset of dorsal target gene expression, and that beta-catenin establishes poised chromatin architecture at target promoters. beta-catenin recruits the arginine methyltransferase Prmt2 to target promoters, thereby establishing asymmetrically dimethylated H3 arginine 8 (R8). Recruitment of Prmt2 to beta-catenin target genes is necessary and sufficient to establish the dorsal developmental program, indicating that Prmt2-mediated histone H3(R8) methylation plays a critical role downstream of beta-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression (Blythe, 2010).
The targets of the maternal Wnt/beta-catenin pathway demonstrate different latencies between dorsal specification and the onset of gene expression. The preMBT genes xnr5 and xnr6 require additional input from the maternal transcription factor VegT. Thus, VegT could function as a 'release factor' for these genes, recruiting elongation-promoting factors to the xnr5 and xnr6 loci downstream of β-catenin. In contrast, factors that regulate the global activation of the zygotic genome at the MBT could be responsible for the release of the later responding siamois and xnr3 genes. Indeed, transcriptional poising is an attractive mechanism to account for the synchronous activation of large-scale zygotic gene expression at the MBT. While such global activating factors have yet to be identified in Xenopus, in Drosophila, both Smaug and Zelda have been shown to be essential factors for zygotic genome activation. Smaug activity is essential for the establishment of elongating (CTD pSer2) RNA Pol II at the MBT, and could thereby promote 'release' of such preMBT poised loci, albeit indirectly. The mechanism of action for Zelda is unknown, but it binds DNA sequences present in the majority of immediate-early zygotic transcripts. Further investigation is needed to determine the extent of transcriptional poising at immediate-early zygotic loci and the mechanism of action for such global zygotic gene activators at the MBT (Blythe, 2010).
While preMBT Xenopus embryos are transcriptionally competent, several overlapping mechanisms dominantly suppress zygotic gene expression. Interfering with these repressive activities can reveal a suppressed protranscriptional activity. Depleting embryos of the DNA methyltransferase Dnmt1 causes precocious expression of many genes, suggesting that these genes are poised for activation prior to the MBT but are repressed by Dnmt1-mediated DNA methylation. Also, embryos generated from transplantation of transcriptionally active nuclei will display preMBT expression of genes that were active in the original donor cells. This transcriptional memory is linked to chromatin modifications that correlate with active transcription, particularly the incorporation of the histone variant H3.3. These observations demonstrate the competency of preMBT embryos to establish and maintain active-but-repressed chromatin. It is further speculated that transcriptional poising is a major mechanism underlying the activation of the zygotic genome at the MBT (Blythe, 2010).
Throughout development, cell fate decisions are converted into epigenetic information that determines cellular identity. Covalent histone modifications are heritable epigenetic marks and are hypothesized to play a central role in this process. This report assesses the concordance of histone H3 lysine 4 dimethylation (H3K4me2) and trimethylation (H3K4me3) on a genome-wide scale in erythroid development by analyzing pluripotent, multipotent, and unipotent cell types. Although H3K4me2 and H3K4me3 are concordant at most genes, multipotential hematopoietic cells have a subset of genes that are differentially methylated (H3K4me2+/me3-). These genes are transcriptionally silent, highly enriched in lineage-specific hematopoietic genes, and uniquely susceptible to differentiation-induced H3K4 demethylation. Self-renewing embryonic stem cells, which restrict H3K4 methylation to genes that contain CpG islands (CGIs), lack H3K4me2+/me3- genes. These data reveal distinct epigenetic regulation of CGI and non-CGI genes during development and indicate an interactive relationship between DNA sequence and differential H3K4 methylation in lineage-specific differentiation (Orford, 2008).
Epigenetic regulation of chromatin structure is essential for the expression of genes determining cellular specification and function. The Polycomb repressive complex 2 (PRC2) di- and trimethylates histone H3 on lysine 27 (H3K27me2/me3) to establish repression of specific genes in embryonic stem cells and during differentiation. How the Polycomb group (PcG) target genes are regulated by environmental cues and signaling pathways is quite unexplored. This study, carried out in mammalian cells, shows that the mitogen- and stress-activated kinases (MSK), through a mechanism that involves promoter recruitment, histone H3K27me3S28 phosphorylation, and displacement of PcG proteins, lead to gene activation. Evidence is presented that the H3K27me3S28 phosphorylation is functioning in response to stress signaling, mitogenic signaling, and retinoic acid (RA)-induced neuronal differentiation. It is proposed that MSK-mediated H3K27me3S28 phosphorylation serves as a mechanism to activate a subset of PcG target genes determined by the biological stimuli and thereby modulate the gene expression program determining cell fate (Gehani, 2010)
In both mammalian and Drosophila spermatids, the completely histone-based chromatin structure is reorganized to a largely protamine-based structure. During this histone-to-protamine switch, transition proteins are expressed, for example TNP1 and TNP2 in mammals and Tpl94D in Drosophila. Recent studies have demonstrated that in Drosophila spermatids, H3K79 methylation accompanies histone H4 hyperacetylation during chromatin reorganization. Preceding the histone-to-protamine transition, the H3K79 methyltransferase Grappa is expressed, and the predominant isoform bears a C-terminal extension. This study has shown that isoforms of the Grappa-equivalent protein in humans, rats and mice, that is DOT1L, have a C-terminal extension. In mice, the transcript of this isoform was enriched in the post-meiotic stages of spermatogenesis. In human and mice spermatids, di- and tri-methylated H3K79 temporally overlapped with hyperacetylated H4 and thus accompanied chromatin reorganization. In rat spermatids, trimethylated H3K79 directly precedes transition protein loading on chromatin. This study analysed the impact of bacterial infections on spermatid chromatin using a uropathogenic Escherichia coli-elicited epididymo-orchitis rat model; it was shown that these infections caused aberrant spermatid chromatin. Bacterial infections led to premature emergence of trimethylated H3K79 and hyperacetylated H4. Trimethylated H3K79 and hyperacetylated H4 simultaneously occurred with transition protein TNP1, which was never observed in spermatids of mock-infected rats. Upon bacterial infection, only histone-based spermatid chromatin showed abnormalities, whereas protamine-compacted chromatin seemed to be unaffected. These results indicated that H3K79 methylation is a histone modification conserved in Drosophila, mouse, rat and human spermatids and may be a prerequisite for proper chromatin reorganization (Dottermusch-Heidel, 2014).
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