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PubMed2000

Characterisation of transcriptionally active and inactive chromatin domains in neurons.

The tandemly organised ribosomal DNA (rDNA) repeats are transcribed by a dedicated RNA polymerase in a specialised nuclear compartment, the nucleolus. There appears to be an intimate link between the maintenance of nucleolar structure and the presence of heterochromatic chromatin domains. This is particularly evident in many large neurons, where a single nucleolus is present, which is separated from the remainder of the nucleus by a characteristic shell of heterochromatin. Using a combined fluorescence in situ hybridisation and immunocytochemistry approach, we have analysed the molecular composition of this highly organised neuronal chromatin, to investigate its functional significance. We find that clusters of inactive, methylated rDNA repeats are present inside large neuronal nucleoli, which are often attached to the shell of heterochromatic DNA. Surprisingly, the methylated DNA-binding protein MeCP2, which is abundantly present in the centromeric and perinucleolar heterochromatin, does not associate significantly with the methylated rDNA repeats, whereas histone H1 does overlap partially with these clusters. Histone H1 also defines other, centromere-associated chromatin subdomains, together with the mammalian Polycomb group factor Eed. These data indicate that neuronal, perinucleolar heterochromatin consists of several classes of inactive DNA, that are linked to a fraction of the inactive rDNA repeats. These distinct chromatin domains may serve to regulate RNA transcription and processing efficiently and to protect rDNA repeats against unwanted silencing and/or homologous recombination events.

Animals↗

PubMed2001

CHMP1 is a novel nuclear matrix protein affecting chromatin structure and cell-cycle progression.

The Polycomb-group (PcG) is a diverse set of proteins required for maintenance of gene silencing during development. In a screen for conserved partners of the PcG protein Polycomblike (Pcl), we have identified a new protein, human CHMP1 (CHromatin Modifying Protein; CHarged Multivesicular body Protein), which is encoded by an alternative open reading frame in the PRSM1 gene and is conserved in both complex and simple eukaryotes. CHMP1 contains a predicted bipartite nuclear localization signal and distributes as distinct forms to the cytoplasm and the nuclear matrix in all cell lines tested. We have constructed a stable HEK293 cell line that inducibly overexpresses CHMP1 under ecdysone control. Overexpressed CHMP1 localizes to a punctate subnuclear pattern, encapsulating regions of nuclease-resistant, condensed chromatin. These novel structures are also frequently surrounded by increased histone H3 phosphorylation and acetylation. CHMP1 can recruit a PcG protein, BMI1, to these regions of condensed chromatin and can cooperate with co-expressed vertebrate Pcl in a Xenopus embryo PcG assay; this is consistent with a role in PcG function. In combination, these observations suggest that CHMP1 plays a role in stable gene silencing within the nucleus.

Amino Acid Sequence↗

PubMed2002

Loss of heterochromatin protein 1 (HP1) chromodomain function in mammalian cells by intracellular antibodies causes cell death.

The chromodomain (CD) is a highly conserved motif present in a variety of animal and plant proteins, and its probable role is to assemble a variety of macromolecular complexes in chromatin. The importance of the CD to the survival of mammalian cells has been tested. Accordingly, we have ablated CD function using two single-chain intracellular Fv (scFv) fragments directed against non-overlapping epitopes within the HP1 CD motif. The scFv fragments can recognize both CD motifs of HP1 and Polycomb (Pc) in vitro and, when expressed intracellularly, interact with and dislodge the HP1 protein(s) from their heterochromatin localization in vivo. Mouse and human fibroblasts expressing anti-chromodomain scFv fragments show a cell-lethal phenotype and an apoptotic morphology becomes apparent soon after transfection. The mechanism of cell death appears to be p53 independent, and the cells are only partly rescued by incubation with the wide spectrum caspase inhibitor Z-VAD fmk. We conclude that expression of anti-chromodomain intracellular antibodies is sufficient to trigger a p53-independent apoptotic pathway that is only partly dependent on the known Z-VAD-inhibitable caspases, suggesting that CD function is essential for cell survival.

3T3 Cells↗

PubMed2006

Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation.

The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.

Antimetabolites, Antineoplastic↗

PubMed2006

Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice.

There is increasing evidence in both plants and animals that epigenetic marks are not always cleared between generations. Incomplete erasure at genes associated with a measurable phenotype results in unusual patterns of inheritance from one generation to the next, termed transgenerational epigenetic inheritance. The Agouti viable yellow (A(vy)) allele is the best-studied example of this phenomenon in mice. The A(vy) allele is the result of a retrotransposon insertion upstream of the Agouti gene. Expression at this locus is controlled by the long terminal repeat (LTR) of the retrotransposon, and expression results in a yellow coat and correlates with hypomethylation of the LTR. Isogenic mice display variable expressivity, resulting in mice with a range of coat colours, from yellow through to agouti. Agouti mice have a methylated LTR. The locus displays epigenetic inheritance following maternal but not paternal transmission; yellow mothers produce more yellow offspring than agouti mothers. We have analysed the DNA methylation in mature gametes, zygotes, and blastocysts and found that the paternally and maternally inherited alleles are treated differently. The paternally inherited allele is demethylated rapidly, and the maternal allele is demethylated more slowly, in a manner similar to that of nonimprinted single-copy genes. Interestingly, following maternal transmission of the allele, there is no DNA methylation in the blastocyst, suggesting that DNA methylation is not the inherited mark. We have independent support for this conclusion from studies that do not involve direct analysis of DNA methylation. Haplo-insufficiency for Mel18, a polycomb group protein, introduces epigenetic inheritance at a paternally derived A(vy) allele, and the pedigrees reveal that this occurs after zygotic genome activation and, therefore, despite the rapid demethylation of the locus.

Alleles↗

PubMed2003

Genomic imprinting and endosperm development in flowering plants.

Genomic imprinting, the parent-of-origin-specific expression of genes, plays an important role in the seed development of flowering plants. As different sets of genes are imprinted and hence silenced in maternal and paternal gametophyte genomes, the contributions of the parental genomes to the offspring are not equal. Imbalance between paternally and maternally imprinted genes, for instance as a result of interploidy crosses, or in seeds in which imprinting has been manipulated, results in aberrant seed development. It is predominantly the endosperm, and not or to a far lesser extent the embryo, that is affected by such imbalance. Deviation from the normal 2m:1p ratio in the endosperm genome has a severe effect on endosperm development, and often leads to seed abortion. Molecular expression data for imprinted genes suggest that genomic imprinting takes place only in the endosperm of the developing seed. Although far from complete, a picture of how imprinting operates in flowering plants has begun to emerge. Imprinted genes on either the maternal or paternal side are marked and silenced in a process involving DNA methylation and chromatin condensation. In addition, on the maternal side, imprinted genes are most probably under control of the polycomb FIS genes.

Animals↗

PubMed2005

Roles of a trithorax group gene, MLL, in hematopoiesis.

The mixed-lineage leukemia (MLL) gene is a trithorax group (trxG) gene that was originally identified at chromosomal translocations in patients developing acute leukemia. Although Polycomb group (PcG) genes, which counteract trxG genes, were found to play essential roles in hematopoiesis, little has been understood about the roles of trxG genes in hematopoiesis except for MLL. MLL has been found fused with 1 of more than 30 different partner genes to yield a diverse collection of MLL fusion oncoproteins that lead to the aberrant expression of HOX genes. Recent studies have revealed that MLL assembles, as do some trxG proteins, into a chromatin-modifying transcriptional regulatory supercomplex to regulate epigenetic pathways, including the methylation of histone H3 lysine 4, which is conferred by the Su (var)3-9, enhancer of zeste, and tritho-rax (SET) domain. Other studies also indicated that MLL plays a nonredundant and essential role in definitive hematopoiesis and induces the proliferation and differentiation of hematopoietic progenitors by maintaining appropriate up-regulation of HOX genes. Further progress in the field will provide novel insights into trxG- and PcG-mediated hematopoiesis and help us understand the epigenetic process by which developing stem cells coordinate proliferation and differentiation.

Cell Differentiation↗

PubMed2004

The quantitative genetic basis of male mating behavior in Drosophila melanogaster.

Male mating behavior is an important component of fitness in Drosophila and displays segregating variation in natural populations. However, we know very little about the genes affecting naturally occurring variation in mating behavior, their effects, or their interactions. Here, we have mapped quantitative trait loci (QTL) affecting courtship occurrence, courtship latency, copulation occurrence, and copulation latency that segregate between a D. melanogaster strain selected for reduced male mating propensity (2b) and a standard wild-type strain (Oregon-R). Mating behavior was assessed in a population of 98 recombinant inbred lines derived from these two strains and QTL affecting mating behavior were mapped using composite interval mapping. We found four QTL affecting male mating behavior at cytological locations 1A;3E, 57C;57F, 72A;85F, and 96F;99A. We used deficiency complementation mapping to map the autosomal QTL with much higher resolution to five QTL at 56F5;56F8, 56F9;57A3, 70E1;71F4, 78C5;79A1, and 96F1;97B1. Quantitative complementation tests performed for 45 positional candidate genes within these intervals revealed 7 genes that failed to complement the QTL: eagle, 18 wheeler, Enhancer of split, Polycomb, spermatocyte arrest, l(2)05510, and l(2)k02206. None of these genes have been previously implicated in mating behavior, demonstrating that quantitative analysis of subtle variants can reveal novel pleiotropic effects of key developmental loci on behavior.

Animals↗

PubMed2000

Construction of macromolecular assemblages in eukaryotic processes and their role in human disease: linking RINGs together.

Members of the Really Interesting New Gene (RING) family of proteins are found throughout the cells of eukaryotes and function in processes as diverse as development, oncogenesis, viral replication and apoptosis. There are over 200 members of the RING family where membership is based on the presence of a consensus sequence of zinc binding residues. Outside of these residues there is little sequence homology; however, there are conserved structural features. Current evidence strongly suggests that RINGs are protein interaction domains. We examine the features of RING binding motifs in terms of individual cases and the potential for finding a universal consensus sequence for RING binding domains (FRODOs). This review examines known and potential functions of RINGs, and attempts to develop a framework within which their seemingly multivalent cellular roles can be consistently understood in their structural and biochemical context. Interestingly, some RINGs can self-associate as well as bind other RINGs. The ability to self-associate is typically translated into the annoying propensity of these domains to aggregate during biochemical characterization. The RINGs of PML, BRCA1, RAG1, KAP1/TIF1beta, Polycomb proteins, TRAFs and the viral protein Z have been well characterized in terms of both biochemical studies and functional data and so will serve as focal points for discussion. We suggest physiological functions for the oligomeric properties of these domains, such as their role in formation of macromolecular assemblages which function in an intricate interplay of coupled metal binding, folding and aggregation, and participate in diverse functions: epigenetic regulation of gene expression, RNA transport, cell cycle control, ubiquitination, signal transduction and organelle assembly.

Amino Acid Motifs↗

PubMed2005

Epigenetic modification as an enabling mechanism for leukemic transformation.

Cancer is now thought of as a fundamentally genetic disease, in that changes in the genome result in aberrant gene expression of oncogenes and tumor suppressor genes to promote oncogenesis. However, with our increasing knowledge of gene regulation, it is becoming obvious that changes in nucleotide sequence are not the sole mechanism for eliciting changes in transcription. An additional layer of regulation of gene expression, called epigenetics, is now being realized as increasingly important in oncogenesis. Epigenetics is defined as non-sequence based changes in chromatin that elicit changes in gene expression that are propagated through mitosis and/or meiosis. The alleles of the genes containing these epigenetic marks are termed epialleles. Epigenetics has been linked to cancer since 1983 by the work of Andy Feinberg and Bert Vogelstein, but has largely remained in the shadows. These changes in chromatin are now at the forefront of research in the field of oncogenesis, both as mechanisms of oncogenesis and as prognostic indicators of cancer risk. Leukemia, due to the defects in cellular differentiation associated with the disease, has important connections to epigenetic gene regulation. Cellular differentiation has been studied as a model system for epigenetic gene control in Drosophila. Homeobox genes in the antennapedia and bithorax gene clusters have long been known to be regulated by trithorax group and Polycomb group of genes, which regulate transcription through chromatin remodeling mechanisms. The ectopic expression of the mammalian homologs of the homeobox genes has been linked to leukemic transformation since 1988, and has continued to show extensive connections. These connections that leukemia has with cellular differentiation make this group of diseases amenable to exploring the mechanisms of epigenetic gene regulation as they pertain to oncogenesis. This review will examine leukemia, with an emphasis on myelogenous leukemia, as a defect in cellular differentiation and examine possibilities of epigenetic gene regulation of oncogenes and tumor suppressor genes.

Animals↗

PubMed2007

Geminin in embryonic development: coordinating transcription and the cell cycle during differentiation.

Geminin was initially characterized as a bifunctional protein with roles in regulating the fidelity of DNA replication and in controlling cell fate during embryonic nervous system formation. More recently, Geminin's roles have expanded, encompassing regulation of cell proliferation and differentiation during retinogenesis, control of Hox transcription factor function during vertebrate axial patterning, and regulation of the timing of neuronal differentiation. Geminin interacts with homeodomain-containing transcription factors and with protein complexes that regulate chromatin structure, including Polycomb complexes and the catalytic subunits of the SWI/SNF chromatin remodeling complex, Brg1 and Brahma. Activities for Geminin in coordinating cellular events at the transition from proliferation to differentiation have recently emerged in multiple developmental contexts. This review will summarize Geminin's increasingly diverse roles as a developmental regulatory molecule.

Amino Acid Sequence↗

PubMed2007

Hepatic differentiation and transcriptional profile of the mouse liver epithelial progenitor cells (LEPCs) under the induction of sodium butyrate.

The liver regenerates by progenitor cells when it is damaged in chronic liver diseases and extensive damage. The progenitor cells, also termed "oval cells" according to their morphological traits, can differentiate into hepatocytes and bile duct cells in vivo. To better understand the transcriptional pattern that accompanies the hepatic differentiation of oval cells, we applied cDNA microarray to analyze the oval cell-derived liver epithelial progenitor cells (LEPCs) during in vitro induced differentiation. Upon exposure to sodium butyrate, a histone deacetylase inhibitor, cultured LEPCs differentiate and express functional hepatocyte markers albumin, tryptophan 2, 3-dioxygenase and alcohol dehydrogenase. For expression profiling, cells were harvested at 6 h, 12 h, 1 d, 3 d and 7 d after exposure to sodium butyrate. After analyzing the microarray data by SOM clustering, total of 796 differentially regulated genes were grouped into 48 clusters. Consistent with the phenotype change of LEPCs after sodium butyrate treatment, many hepatocyte functional genes are revealed by analyzing the clusters containing genes up-regulated through all the time points. The clusters, containing down-regulated genes immediately after the induction, are also analyzed. The microarray data was validated by analyzing the expression of selected genes by quantitative real-time PCR. A set of genes expressed synergistically in these clusters may play a central role during the process of differentiation. Sodium butyrate decreases cyclin B1 and Cdk4 expression, which would be associated with LEPCs growth arrest shortly after treatment. Bmi1, a polycomb group protein, is also down-regulated immediately after treatment and remains at a low level during the induction. These findings highlight the key molecular mechanisms by which sodium butyrate, mediates its effects on cell growth arrest and induction of differentiation. In conclusion, our data reflect a global view of gene expression during hepatic differentiation of LEPCs induced by sodium butyrate.

Animals↗

PubMed2026

Biomolecular Condensates Integrate Transcriptional and Epigenetic Responses to Hypoxia.

Hypoxia is a defining feature of physiological stress and the core of solid tumors, where aberrant vascularization limits oxygen delivery; cells respond through mechanisms that extend beyond the canonical stabilization of hypoxia-inducible factors (HIFs). Recent studies suggest that hypoxia can promote the formation of specific biomolecular conden-sates, membraneless compartments generated through liquid-liquid phase separation in which regulatory proteins and RNAs become locally enriched at genomic regions, while chromatin mainly serves as an organizational scaffold. Transcription factors, the coacti-vators p300/CBP, Mediator, and BRD4, chromatin-modifying enzymes, and architectural RNAs such as NEAT1 and MALAT1 partition into these compartments, and their con-densation can help reorganize local chromatin structure and enhancer-promoter interac-tions. Because molecular oxygen is a shared co-substrate for the Jumonji-C histone demethylases and the ten-eleven translocation (TET) DNA dioxygenases, hypoxia reshapes histone methylation and DNA methylation in parallel, and readers that bridge these marks, including UHRF1, may participate in condensate-associated chromatin regulation. Hypoxia-driven condensation of ZHX2 rewires enhancer-promoter contacts and higher-order genome architecture, influencing cell identity, stemness, and metastatic potential, and Polycomb condensates represent another candidate epigenetic compartment that may be influenced by hypoxic signaling. These processes may be particularly important in cancer, where chronic hypoxia provides a sustained stimulus for condensate formation and epigenetic remodeling. Together, these findings support a model in which phase separation and epigenetic reprogramming are not separate layers but one integrated response to low oxygen, offering opportunities to target maladaptive condensates in disease.

Epigenesis, Genetic↗

PubMed2005

Effector proteins for methylated histones: an expanding family.

Methylation of histone lysine residues in eukaryotic chromatin has been an exciting area of research ever since the first histone methyltransferase enzyme, Suv39h, was found to methylate lysine 9 of histone H3 in 2000. Only a year later, the HP1 chromodomain polypeptide was identified as a recognition module for this histone modification. Similar to bromodomain-containing proteins that recognize histone acetylation sites and subsequently stabilize large complexes to chromatin, effector proteins can also be recruited and stabilized by histone methylation. Although histone acetylation generally correlates with active transcription, histone methylation is associated with both the activation and silencing of transcription, depending on which lysine residue is modified. The list of proteins that may in fact directly associate with specific methylated histone lysines is expanding. Since the finding of HP1, many additional proteins have been shown to bind methylated histone residues. For instance, Polycomb, Chd1, 53BP1, and Crb2/Rad9 proteins all associate with methylated chromatin in a unique manner governed by their respective recognition motifs. Here we highlight recent data on the recognition specificity and biological significance of proteins that associate with methylated histone lysines.

Carrier Proteins↗

PubMed1999

The 35UZ transposon of Drosophila melanogaster reveals differences in maintenance of transcriptional control between embryonic and larval stages.

The D. melanogaster transposon P[35UZ] contains a lacZ reporter gene fused to 35 kb of Ubx upstream sequences which drive a Ubx-like expression in embryos and in metathoracic imaginal discs. Transposition of P[35UZ] followed by analysis of different lines in wild-type and mutant backgrounds allowed us to analyze the interplay between Ubx regulatory elements, including the Polycomb response element (PRE), located inside the transposon and cis-acting regulatory elements, located outside. We found that all lines show a Ubx-like beta-galactosidase expression pattern in the embryo, but proximity to strong imaginal enhancers can change this pattern drastically. These data illustrate how maintenance of gene expression depends on the chromosomal environment and on dynamic interactions between all developmentally regulated enhancers located close to a promoter under PcG control.

Animals↗

PubMed1999

Initiation, establishment and maintenance of Hox gene expression patterns in the mouse.

Spatially and temporally restricted expression of the Hox genes along the main and appendicular axes is essential for correct patterning of vertebrate embryos. In this overview we discuss the latest data that shed light on the mechanisms underlying the generation of the expression domains of the Hox genes. The molecular genetic interactions governing initial transcription of the Hox genes in the posterior part of the primitive streak during mouse and chick gastrulation remain enigmatic. But the recent discovery by Kondo and Duboule (Cell, 97, 1999, 407-417) of a "cluster repressive regulation", will undoubtedly lead to a better understanding of the molecular genetic mechanism underlying colinear and sequential initiation of Hox gene transcription. Recently progress has been booked in characterizing the basal processes driving progression of the Hox expression domains during their establishment. Hox expression is still labile while being established. The transcriptional state of Hox genes in anterior tissues can be reprogrammed under the influence of more posterior locations. Posteriorizing activity may involve RA and FGF signaling. It is only when these interactions and, in some cases at least, regulatory interactions with Hox and cdx gene products occur appropriately, that the Hox expression domains would be correctly established. After the Hox expression domains have been established, regulatory processes involving the products of Polycomb and trithorax- Group genes start operating, perpetuating the transcriptional state of the Hox genes within and outside the expression domains. Whether control at the level of chromatin structure, believed to operate during the late maintenance phase of Hox gene expression, is also involved in regulating concerted initial expression of these genes, is a possibility that has been suggested.

Animals↗

PubMed2000

The Drosophila brahma complex is an essential coactivator for the trithorax group protein zeste.

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. Here, we have sought to dissect the requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes. Reconstituted transcription reactions established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex revealed a number of novel subunits. We found that Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and OSA. The leucine zipper of Zeste mediates binding to MOR. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting.

Animals↗

PubMed2001

A gene expression profile of embryonic stem cells and embryonic stem cell-derived neurons.

Embryonic stem (ES) cells have the ability to differentiate into a variety of cell lineages. We are examining ES cell differentiation in vitro by using cDNA microarrays to generate a molecular phenotype for each cell type. El4 ES cells induced by retinoic acid after forming embryoid bodies differentiate almost exclusively to neurons. We obtained expression patterns for about 8500 gene sequences by comparing mRNAs from undifferentiated ES cells and their differentiated derivatives in a competitive hybridization. Our results indicate that the genes expressed by ES cells change dramatically as they differentiate (58 gene sequences up-regulated, 34 down-regulated). Most notably, totipotent ES cells expressed high levels of a repressor of Hox expression (the polycomb homolog Mphl) and a co-repressor (CTBP2). Expression of these genes was undetectable in differentiated cells; the ES cell-derived neurons expressed a different set of transcriptional regulators, as weil as markers of neurogenesis. The gene expression profiles indicate that ES cells actively suppress differentiation by transcriptional repression; cell-cell contact in embryoid bodies and retinoic acid treatment may overcome this suppression, allowing expression of Hox genes and inducing a suite of neuronal genes. Gene expression profiles will be a useful outcome measure for comparing in vitro treatments of differentiating ES cells and other stem cells. Also, knowing the molecule phenotype of transplantable cells will allow correlation of phenotype with the success of the transplant.

Cell Differentiation↗