Repeat to gene expression ratios in leukemic blast cells can stratify risk prediction in acute myeloid leukemia.

BACKGROUND: Repeat elements constitute a large proportion of the human genome and recent evidence indicates that repeat element expression has functional roles in both physiological and pathological states. Specifically for cancer, transcription of endogenous retrotransposons is often suppressed to attenuate an anti-tumor immune response, whereas aberrant expression of heterochromatin-derived satellite RNA has been identified as a tumor driver. These insights demonstrate separate functions for the dysregulation of distinct repeat subclasses in either the attenuation or progression of human solid tumors. For hematopoietic malignancies, such as Acute Myeloid Leukemia (AML), only very few studies on the expression/dysregulation of repeat elements were done. METHODS: To study the expression of repeat elements in AML, we performed total-RNA sequencing of healthy CD34 + cells and of leukemic blast cells from primary AML patient material. We also developed an integrative bioinformatic approach that can quantify the expression of repeat transcripts from all repeat subclasses (SINE/ALU, LINE, ERV and satellites) in relation to the expression of gene and other non-repeat transcripts (i.e. R/G ratio). This novel approach can be used as an instructive signature for repeat element expression and has been extended to the analysis of poly(A)-RNA sequencing datasets from Blueprint and TCGA consortia that together comprise 120 AML patient samples. RESULTS: We identified that repeat element expression is generally down-regulated during hematopoietic differentiation and that relative changes in repeat to gene expression can stratify risk prediction of AML patients and correlate with overall survival probabilities. A high R/G ratio identifies AML patient subgroups with a favorable prognosis, whereas a low R/G ratio is prevalent in AML patient subgroups with a poor prognosis. CONCLUSIONS: We developed an integrative bioinformatic approach that defines a general model for the analysis of repeat element dysregulation in physiological and pathological development. We find that changes in repeat to gene expression (i.e. R/G ratios) correlate with hematopoietic differentiation and can sub-stratify AML patients into low-risk and high-risk subgroups. Thus, the definition of a R/G ratio can serve as a valuable biomarker for AML and could also provide insights into differential patient response to epigenetic drug treatment.

Re-SET-ting heterochromatin by histone methyltransferases.

Histone methylation was first described more than 35 years ago, but its role has remained enigmatic. Proposed functions range from transcriptional regulation to the higher-order packaging of chromatin in preparation for mitotic condensation. Histone methylation can occur on Arg or Lys residues, with an exquisite site selectivity for Lys methylation at specific positions in the N-termini of histones H3 and H4. Thus, Lys methylation joins acetylation and phosphorylation as a third component of a 'histone code' that modifies the underlying chromatin structure of the genetic information. Notably, in contrast to acetylation and phosphorylation, Lys methylation appears to be a relatively stable histone modification, thereby providing an ideal epigenetic mark for more long-term maintenance of chromatin states. The recent discovery of the first histone Lys methyltransferase has allowed the identification of a molecular mechanism in which the specific methylation of histone H3 at Lys9 generates a binding site for heterochromatin-associated proteins. These findings have broad implications for the overall functional organization of chromosome structure at constitutive heterochromatin (e.g. centromeres) and for chromatin-dependent inheritance of gene expression patterns. This review discusses how understanding this methylation system should address some of the long-standing mysteries of heterochromatin.

Translating the histone code.

Chromatin, the physiological template of all eukaryotic genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA. Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a "histone code" that considerably extends the information potential of the genetic code. We propose that this epigenetic marking system represents a fundamental regulatory mechanism that has an impact on most, if not all, chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development.

Dependence of enhancer-mediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions.

Transcription of the immunoglobulin mu heavy chain locus is regulated by an intronic enhancer that is flanked on both sides by nuclear matrix attachment regions (MARs). These MARs have now been shown to be essential for transcription of a rearranged mu gene in transgenic B lymphocytes, but they were not required in stably transfected tissue culture cells. Normal rates of transcriptional initiation at a variable region promoter and the formation of an extended deoxyribonuclease I (DNase I)--sensitive chromatin domain were dependent on MARs, although DNase I hypersensitivity at the enhancer was detected in the absence of MARs. Thus, transcriptional activation of the mu gene during normal lymphoid development requires a synergistic collaboration between the enhancer and flanking MARs.

Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression.

SUV39H1, a human homologue of the Drosophila position effect variegation modifier Su(var)3-9 and of the Schizosaccharomyces pombe silencing factor clr4, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis. Using a detailed structure-function analysis of SUV39H1 mutant proteins in transfected cells, we now show that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organization. First, forced expression of full-length SUV39H1 (412 amino acids) redistributes endogenous M31 (HP1beta) and induces abundant associations with inter- and metaphase chromatin. These properties depend on the C-terminal SET domain, although the major portion of the SUV39H1 protein (amino acids 89 to 412) does not display affinity for nuclear chromatin. By contrast, the M31 interaction surface, which was mapped to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. Second, cells overexpressing full-length SUV39H1 display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G(2)-specific distribution of phosphorylated histone H3 at serine 10 (phosH3) is dispersed in these cells. This phosH3 shift is not observed with C-terminally truncated mutant SUV39H1 proteins or with deregulated M31. Together, our data reveal a dominant role(s) for the SET domain of SUV39H1 in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal SU(VAR) protein and histone H3.

The polycomb-group gene Ezh2 is required for early mouse development.

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.

Isolation and characterization of Suv39h2, a second histone H3 methyltransferase gene that displays testis-specific expression.

Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1, Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, the Suv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although both Suv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body) which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line.

The role of histone modifications in epigenetic transitions during normal and perturbed development.

Epigenetic mechanisms control eukaryotic development beyond DNA-stored information. DNA methylation, histone modifications and variants, nucleosome remodeling and noncoding RNAs all contribute to the dynamic make-up of chromatin under distinct developmental options. In particular, the great diversity of covalent histone tail modifications has been proposed to be ideally suited for imparting epigenetic information. While most of the histone tail modifications represent transient marks at transcriptionally permissive chromatin, some modifications appear more robust at silent chromatin regions, where they index repressive epigenetic states with functions also outside transcriptional regulation. Under-representation of repressive histone marks could be indicative of epigenetic plasticity in stem, young and tumor cells, while committed and senescent (old) cells often display increased levels of these more stable modifications. Here, we discuss profiles of normal and aberrant histone lysine methylation patterns, as they occur during the transition of an embryonic to a differentiated cell or in controlled self-renewal vs pro-neoplastic or metastatic conditions. Elucidating these histone modification patterns promises to have important implications for novel advances in stem cell research, nuclear reprogramming and cancer, and may offer novel targets for the combat of tumor cells, potentially leading to new diagnostic and therapeutic avenues in human biology and disease.

Isolation and structural analysis of a biologically active chicken c-fos cDNA: identification of evolutionarily conserved domains in fos protein.

To identify functionally important domains in the fos gene product we have studied the evolutionary divergence between chicken and mammalian fos proteins. A cDNA containing the entire chicken c-fos coding region was isolated and its nucleotide sequence determined. The deduced 367-amino acid sequence was compared to that of the mouse and human proteins. This comparison revealed a highly conserved domain (98% homology between mouse and chicken) in the center of the protein (85 amino acids) that coincides with a region known to be indispensible for transforming activity. This highly charged domain presumably contains contact sites for DNA and other proteins as well as a nuclear location signal sequence. Two other regions, that are dispensable for transformation, are also highly conserved and may thus be important for the physiological function of c-fos. These are the N-terminal 88 amino acids (85% homology) and the C-terminal 62 amino acids (92% homology). The C-terminus not only contains a potential DNA-binding Zn-finger structure but is also the least divergent region in the protein at the nucleotide level (92% conservation between chicken and mouse), supporting the hypothesis that mRNA secondary structures in this region may contribute to post-transcriptional regulatory mechanisms. In contrast, the domains between the terminal sequences and the center region of fos protein show considerable divergence (39% and 45% homology, respectively), indicating a minor role, if any, for these sequences. The significance of these conclusions is emphasized by the observation that the chicken c-fos protein, expressed from the cDNA inserted into a retrovirally-derived expression vector, efficiently induces morphological transformation in rat fibroblasts. The chicken c-fos gene product could be identified by immunoprecipitation and in vitro transcription/translation of the isolated cDNA as a protein of Mr approximately 60 K.

Over-expression of the SUV39H1 histone methyltransferase induces altered proliferation and differentiation in transgenic mice.

The development of multi-cellular organisms is regulated by the ordered definition of gene expression programmes that govern cell proliferation and differentiation. Although differential gene activity is mainly controlled by transcription factors, it is also dependent upon the underlying chromatin structure, which can stabilize transcriptional "on" or "off" states. We have recently isolated human (SUV39H1) and mouse (Suv39h1) histone methyltransferases (HMTases) and shown that they are important regulators for the organization of repressive chromatin domains. To investigate whether a SUV39H1-induced modulation of heterochromatin would affect mammalian development, we generated transgenic mice that over-express the SUV39H1 HMTase early during embryogenesis. SUV39H1 transgenic mice are growth retarded, display a weak penetrance of skeletal transformations and are largely characterized by impaired erythroid differentiation, consistent with highest transgene expression in foetal liver. Ex vivo transgenic foetal liver cultures initially contain reduced numbers of cells in G1 but progress to immortalized erythroblasts that are compromised in executing an erythroid differentiation programme. The outgrowing SUV39H1-immortalized erythroblasts can maintain a diploid karyotype despite deregulation of several tumour suppressor proteins and dispersed distribution of the heterochromatin component HP1. Together, these data provide evidence for a role of the SUV39H1 HMTase during the mammalian development and indicate a possible function for higher-order chromatin in contributing to the balance between proliferation and differentiation potentials of progenitor cells.

Complex pattern of immunoglobulin mu gene expression in normal and transgenic mice: nonoverlapping regulatory sequences govern distinct tissue specificities.

Analysis of normal mice and transgenic mice carrying a rearranged immunoglobulin mu gene revealed a more complex tissue-specific pattern of mu gene expression than anticipated from previous observations. Expression of the endogenous mu locus and the mu transgene was detected both in lymphoid tissues and in skeletal muscle. Analysis of the expression pattern of mu transgenes containing intragenic deletions or point mutations in binding sites for Oct transcription factors (OCTA sites) indicated that distinct regulatory sequences control lymphoid- and muscle-specific mu gene expression. Consistent with previous transfection experiments, mu gene expression in lymphoid tissues is dependent on the intragenic enhancer and the OCTA site in the promoter. However, neither of these regulatory sequences is required for mu gene expression in skeletal muscle that is governed by a muscle-specific control region located 3' of the enhancer. An "off-state" of the mu transgene was observed only in liver and embryonal fibroblasts, whereas enhancer-dependent mu transgene expression was detected at low levels in other nonlymphoid tissues. From these data we suggest a model for the regulation of tissue-specific mu gene expression in which a "ubiquitous" competence for basal transcription is up-regulated in lymphoid and muscle tissues by distinct cell type-specific regulatory sequences and down-regulated in liver and fibroblastic cells by putative negative sequence elements.

Structure-function analysis of fos protein: a single amino acid change activates the immortalizing potential of v-fos.

We have undertaken a detailed structure-function analysis of v-fos protein, taking the ability to induce transformation and immortalization as criteria of biological activity. Our results demonstrate that the evolutionarily conserved center region of fos, comprising ca. 28% of the protein, is indispensable for its function. A single amino acid change (Glu 138----Val 138) in this region activates the immortalizing potential of v-fos without affecting its transforming capacity. The presence of additional either N- or C-terminal amino acids, however, is required for efficient expression of a stable protein, and significantly increases its biological activity. In contrast, sequences in the C-terminal half (between positions 228 and 267) strongly downmodulate the transforming activity of v-fos protein without decreasing its immortalizing potential.

Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres.

Centromeres of eukaryotes are frequently associated with constitutive heterochromatin and their activity appears to be coregulated by epigenetic modification of higher order chromatin. Recently, we isolated murine (Suv39h1) and human (SUV39H1) homologues of the dominant Drosophila suppressor of position effect variegation Su(var)3-9, which is also related to the S. pombe silencing factor Clr4. We have shown that mammalian Su(var)3-9 homologues encode novel centromeric proteins on metaphase-arrested chromosomes. Here, we describe a detailed analysis of the chromatin distribution of human SUV39H1 during the cell cycle. Although there is significant heterochromatic overlap between SUV39H1 and M31 (HP1(beta)) during interphase, mitotic SUV39H1 displays a more restricted spatial and temporal association pattern with metaphase chromosomes than M31 (HP1(beta)), or the related HP1(&agr;) gene product. SUV39H1 specifically accumulates at the centromere during prometaphase but dissociates from centromeric positions at the meta- to anaphase transition. In addition, SUV39H1 selectively associates with the active centromere of a dicentric chromosome and also with a neocentromere. Interestingly, SUV39H1 is shown to be a phosphoprotein with modifications at serine and, to a lesser degree, also at threonine residues. Whereas SUV39H1 steady-state protein levels appear constant during the cell cycle, two additional phosphorylated isoforms are detected in mitotic extracts. This intriguing localisation and modification pattern would be consistent with a regulatory role(s) for SUV39H1 in participating in higher order chromatin organisation at mammalian centromeres.

SET domain proteins modulate chromatin domains in eu- and heterochromatin.

The SET domain is a 130-amino acid, evolutionarily conserved sequence motif present in chromosomal proteins that function in modulating gene activities from yeast to mammals. Initially identified as members of the Polycomb- and trithorax-group (Pc-G and trx-G) gene families, which are required to maintain expression boundaries of homeotic selector (HOM-C) genes, SET domain proteins are also involved in position-effect-variegation (PEV), telomeric and centromeric gene silencing, and possibly in determining chromosome architecture. These observations implicate SET domain proteins as multifunctional chromatin regulators with activities in both eu- and heterochromatin--a role consistent with their modular structure, which combines the SET domain with additional sequence motifs of either a cysteine-rich region/zinc-finger type or the chromo domain. Multiple functions for chromatin regulators are not restricted to the SET protein family, since many trx-G (but only very few Pc-G) genes are also modifiers of PEV. Together, these data establish a model in which the modulation of chromatin domains is mechanistically linked with the regulation of key developmental loci (e.g. HOM-C).

Extended life span and tumorigenicity of nonestablished mouse connective tissue cells transformed by the fos oncogene of FBR-MuSV.

We have analyzed the transforming potential of two fos oncogene products in nonestablished cultures of mouse connective tissue cells: p55fos of FBJ-MuSV and p75gag-fos of FBR-MuSV. Although both proteins induced morphological transformation and colony formation at low cell density in a G418 resistance selection assay, p75gag-fos exhibited more pronounced transforming potential than p55fos. In addition, p75gag-fos-transformed cells overcame crisis with a high probability and were tumorigenic in syngenic mice. These properties of the FBR-MuSV appear to be linked to structural alterations in the p75gag-fos oncogene product. Polyoma virus large T protein complemented the transforming potential of fos, in that it not only increased the probability of establishment of fos-transformed cells but also enhanced fos-induced morphological transformation. Our results suggest that different oncogenes affect morphological transformation, low cell density growth, establishment, and tumorigenicity to various degrees.

Multiple regions of v-Fos protein involved in the activation of AP1-dependent transcription: is trans-activation crucial for transformation?

We show that trans-activation by v-Fos requires several functionally separable regions, including the leucine repeat, the basic DNA-binding region, a directly adjacent acidic cluster, and additional flanking sequences. Structural alterations in the flanking regions are in part responsible for the greater trans-activating potential of the fos gene product of the Finkel-Biskis-Reilly mouse osteosarcoma virus, FBR-MuSV. A point mutation in the acidic cluster, which is known to activate the immortalizing potential of Fos, leads to a significant increase in trans-activation. However, comparison of the trans-activating and transforming properties of mutant Fos proteins suggests that functions other than trans-activation are involved in the induction of transformation.

The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation.

Factor access in chromatin has been proposed to be facilitated by transcriptional enhancers. With the aim of uncoupling factor access from transcriptional stimulation by protein-protein contacts, we analyzed the potential of enhancer fragments to confer accessibility upon a linked promoter for prokaryotic T7 RNA polymerase. Access to the T7 promoter in pre-B cells from transgenic mice was examined by transcribing chromatin of isolated nuclei with T7 RNA polymerase. A 95-bp immunoglobulin mu enhancer core element was necessary and sufficient to confer accessibility upon the T7 promoter independent of its chromosomal position. This enhancer-dependent factor access could be uncoupled from an active transcriptional state of the transgene and was not accompanied by the formation of pronounced DNase I hypersensitive sites. Additional mu enhancer sequences comprising previously identified matrix attachment regions and a cryptic promoter were required to induce DNase I hypersensitivity. Together, these data provide evidence that the 95-bp mu enhancer core can establish localized factor access in nuclear chromatin independent of detectable transcription by endogenous polymerases and suggest that multiple steps are involved in the alteration of chromatin structure.

Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.

Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. In addition to histone modifications that facilitate gene activity, it is of similar importance to restrict inappropriate gene expression if cellular and developmental programmes are to proceed unperturbed. Here we show that mammalian methyltransferases that selectively methylate histone H3 on lysine 9 (Suv39h HMTases) generate a binding site for HP1 proteins--a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated histone H3 peptide by HP1 requires a functional chromo domain; thus, the HP1 chromo domain is a specific interaction motif for the methyl epitope on lysine9 of histone H3. In vivo, heterochromatin association of HP1 proteins is lost in Suv39h double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active SWUV39H1 HMTase. Our data define a molecular mechanism through which the SUV39H-HP1 methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin.

Role of the OCTA site in regulation of IgH chain gene transcription during B cell activation.

We have evaluated the importance of the OCTA site in both the promoter and enhancer regions for the induction of enhancement of IgH chain gene transcription after B cell activation. These studies show that although occupancy of the OCTA site in the promoter is critical for basal transcription of the mu gene, it is not necessary for the increase in transcription induced by in vivo activation. On the other hand, the OCTA site in the enhancer is necessary for neither basal transcription nor in vivo activation of transcription; however, occupancy of this site is required for further up-regulation in transcription of the mu gene in pre-activated cells. These results indicate that different mechanisms may be involved in the activation of resting versus in vivo stimulated B lymphocytes. The findings are discussed in relation to the phenotype described for Oct-2-deficient mice.

The leucine repeat motif in Fos protein mediates complex formation with Jun/AP-1 and is required for transformation.

Cellular and viral Fos proteins form a tight complex with other nuclear proteins, including the transcription factor and proto-oncogene AP-1/Jun. We have mapped the c-Jun binding site in Fos to a region containing regularly spaced leucine residues recently suggested to interdigitate with a similar structure in Jun. Substitution of single or multiple leucine residues or the alteration of leucine phasing by insertion of additional amino acids reduces or abolishes the binding to Jun, while the substitution of other amino acids has no noticeable effect. These results strongly suggest that the formation of a "leucine zipper" mediates the interaction between Fos and Jun. We also show that the differential binding of the various Fos mutants correlates with their potential to trans-activate AP-1-dependent transcription and to induce morphological transformation.