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PubMed2002

Long-range chromatin regulatory interactions in vivo.

Communication between distal chromosomal elements is essential for control of many nuclear processes. For example, genes in higher eukaryotes often require distant enhancer sequences for high-level expression. The mechanisms proposed for long-range enhancer action fall into two basic categories. Non-contact models propose that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately communicate with the gene. Contact models propose that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that 'loop out' the intervening sequences. Although much attention has focused on contact models, the existence and nature of long-range interactions is still controversial and speculative, as there is no direct evidence that distant sequences physically interact in vivo. Here, we report the development of a widely applicable in situ technique to tag and recover chromatin in the immediate vicinity of an actively transcribed gene. We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction. The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication.

Animals↗
PubMed1997

Distinct mechanisms direct SCL/tal-1 expression in erythroid cells and CD34 positive primitive myeloid cells.

The SCL/tal-1 gene (hereafter designated SCL) encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. The molecular basis for expression of SCL in stem cells and its subsequent modulation during lineage commitment is of fundamental importance for understanding how early "decisions" are made during hematopoiesis. We now compare the activity of SCL promoters 1a and 1b in erythroid cells and in CD34 positive primitive myeloid cells. SCL mRNA expression in CD34 positive myeloid cells did not require GATA-1. Promoter 1a activity was weak or absent in CD34 positive myeloid cells and appeared to correlate with the presence or absence of low levels of GATA-1. However, promoter 1b, which was silent in committed erythroid cells, was strongly active in transient assays using CD34 positive myeloid cells, and functioned in a GATA-independent manner. Interestingly, RNase protection assays demonstrated that endogenous promoter 1b was active in both erythroid and CD34 positive myeloid cells. These results demonstrate that fundamentally different mechanisms regulate the SCL promoter region in committed erythroid cells and in CD34 positive myeloid cells. Moreover these observations suggest that in erythroid, but not in CD34 positive myeloid cells, promoter 1b required integration in chromatin and/or additional sequences for its activity. Stable transfection experiments showed that both core promoters were silent following integration in erythroid or CD34 positive myeloid cells. Our data therefore indicate that additional regulatory elements were necessary for both SCL promoters to overcome chromatin-mediated repression.

Animals↗
PubMed1998

Repression by the Mad(Mxi1)-Sin3 complex.

The functions of Myc in transformation and transactivation are countered by the suppressive actions of the Mad(Mxi1) family. Mad(Mxi1) proteins not only compete with Myc for dimerization to Max and binding to Myc/Max consensus sites but also recruit powerful repressors of gene expression. A prediction of the yin-yang relationship between Myc and Mad(Mxi1) families would be that the latter constitutes a new class of tumor suppressors. Here, we review the current literature on the Mad(Mxi1) family, with particular attention paid to the molecular mechanisms by which these proteins antagonize the actions of Myc in normal and neoplastic cells.

Animals↗
PubMed2002

DNA binding and protein interactions of the AHR/ARNT heterodimer that facilitate gene activation.

Gene activation by the aryl hydrocarbon receptor (AHR) and its DNA binding partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) requires a number of sequential steps that occur following the binding of ligand and entry of the AHR into the nuclear compartment. This includes heterodimerization of the AHR and ARNT, formation of the appropriate amino acid/nucleotide contacts at the GCGTG recognition site and interactions between either the AHR or ARNT with proteins that facilitate changes in chromatin structure. The majority of these steps are likely modulated by changes in both phosphorylation and oxidation status of the AHR, ARNT and associated proteins. Studies of both the basic helix-loop-helix transcription factors and the nuclear hormone receptor family can provide significant insights into how this unique signaling pathway activates its target genes.

Animals↗
PubMed1998

Biotin-labeled hairpin oligonucleotides: probes to detect double-strand breaks in DNA in apoptotic cells.

Hairpin oligonucleotides were synthesized with stems ending in a double-stranded structure, which can be ligated to double-strand breaks in DNA, and with loops that contain nucleotides modified by the attachment of biotin. These probes specifically and sensitively detect double-strand breaks in apoptotic cells. Localization of these probes is restricted to areas of chromatin characteristic of apoptosis, whereas much more diffuse labeling was obtained when all available 3' DNA ends were labeled by terminal transferase. In principle, hairpin oligonucleotide probes can be designed with any type of 3' or 5' overhang complementary to double-strand DNA termini being detected.

Animals↗
PubMed2006

Mapping of binding regions for the circadian regulators BMAL1 and CLOCK within the mouse Rev-erbalpha gene.

Two major interconnected feedback loops govern circadian rhythmicity in mammals. The transcriptional activators BMAL1/MOP3 and CLOCK drive one loop by binding to E-box motifs in the regulatory regions of circadian target genes. Time-of-day-dependent inhibition of the activity of BMAL1 and CLOCK by PERIOD (PER) and CRYPTOCHROME (CRY) proteins provokes consequent rhythmic gene expression. In this fashion, the Per and Cry genes can feedback onto their own synthesis. The orphan nuclear receptor Rev-erbalpha manifests the second feedback loop. Genetically linked to the molecular oscillator, it was identified as a repressor of the Bmal1 and Clock genes. In an attempt to understand the circadian regulation of the mouse Rev-erbalpha gene, we developed a screening technique for BMAL1 and CLOCK binding regions consisting of chromatin-immunoprecipitation. Here we show that the mouse Rev-erbalpha gene contains at least two binding regions for the transcriptional activators close to its previously identified promoter region and in its first intron. However, our data also indicate that not all potential E-box motifs in vitro are occupied in vivo.

ARNTL Transcription Factors↗
PubMed1999

Silencing of the Epstein-Barr virus latent membrane protein 1 gene by the Max-Mad1-mSin3A modulator of chromatin structure.

The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells.

Base Sequence↗
PubMed1986

Differential chromosomal distribution of ribonucleoprotein antigens in nuclei of Drosophila spermatocytes.

The ribonucleoprotein (RNP) composition of the active Y chromosomal structures in spermatocyte nuclei of Drosophila hydei has been investigated using the anti-RNP antibodies Dm 28K2 and pp60 as a probe. Antibody Dm 28K2 was raised against an RNP protein of cytoplasmic RNP particles in D. melanogaster cells, while antibody pp60 was raised against a pre-messenger RNP fraction from oocytes of Xenopus laevis. Both antibodies detect nuclear RNP (nRNP) antigens of D. hydei. This is shown by CsCl density centrifugation of nRNP from D. hydei cells and immunoblotting across the density gradient. Dm 28K2 and pp60 recognize antigens of nRNP complexes which band at a characteristic buoyant density of approximately 1.4 g/cm3 in CsCl. By indirect immunofluorescence we observe that the nRNP complexes identified by Dm 28K2 are localized at only two of the five Y chromosomal loop structures which are named according to their distinct morphology. Dm 28K2 decorates RNPs within the "clubs," within the cones, and within the matrix of the "pseudonucleolus." Ultrastructural bodies that are candidates for this immunoreaction are RNP granules that resemble the so-called perichromatin granules. Antibody pp60 recognizes RNP complexes close to the axes of the active Y chromatin. In the "pseudonucleolus" it can be shown that the structures recognized by pp60 are quite distinct from those detected by Dm 28K2. Thus, the "pseudonucleolus" is a striking example for the presence of different RNP populations within a same defined nuclear compartment. Together with previous results (Glätzer, K. H., 1984, Mol. Gen. Genet., 196:236-243), our data represent evidence that the morphological and apparently functional differences between the active Y chromosomal loops, which are involved in male fertility, are caused by the presence of qualitatively and possibly also functionally different RNP populations within these nuclear compartments. Because both RNP antigens are discussed in the literature in connection with repressed mRNP the observed cross-reaction of the respective antibodies in D. hydei suggests a more general and important function of these proteins in the RNA metabolism of eukaryotic cells.

Animals↗
PubMed1988

Hypoxic injury to medullary thick ascending limbs in perfused rat kidneys: reversible and irreversible phases.

The tubular epithelial cells located in the renal medulla are normally working in a hypoxic milieu. In isolated rat kidneys perfused with a cell-free medium, the medullary thick ascending limbs of Henle's loop are selectively and reproducibly injured by the imbalance between oxygen demand and supply in this area. Hypoxic lesions rapidly progress from reversible to irreversible forms of cell damage. Reversible injury consists of chromatin margination and mitochondrial swelling, which can disappear upon restoration of an adequate balance of oxygenation. Irreversible injury consists of nuclear pyknosis and cytoplasmic fragmentation, lesions which persist after re-oxygenation or even progress to cell death. Reversible and irreversible phases of hypoxic injury in this distal tubule segment are comparable to, but different from, those previously defined for the proximal tubule.

Animals↗
PubMed1981

In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus.

An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of alpha-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 microgram/ml of alpha-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatin. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15-20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli.

Amanitins↗
PubMed1989

Ultrastructure of somatic and meiotic nucleoids.

In this review emphasis is placed on the contribution of transmission electron microscopy to the analysis of spread chromosomes and nucleoids. Support is advanced for the DNA loop and rosette organization of meiotic and metaphase chromosomes and nucleoids. Extensive discussion is given to the biochemical treatments used for producing nucleoids and the effect of divalent cations and chelating agents on chromatin compactization (supercoiling). Detailed studies on nucleoids from hepatocytes are presented, with emphasis on the significance of DNA attachment to the internal nuclear matrix and to the nuclear lamina. It is firmly predicted that from the increasing knowledge of the structural organization of eukaryotic chromatin and the genome, a greater understanding of the functional roles of the various intranuclear structures will ultimately follow.

Animals↗
PubMed2026

Developmental roles of LSD1/KDM1A-like (LDL) proteins in plants.

LYSINE-SPECIFIC DEMETHYLASE 1-like (LDL) proteins are conserved FAD-dependent amine oxidases that serve as pivotal regulators in plants. While animal systems typically rely on a single LSD1/KDM1A enzyme, the Arabidopsis thaliana genome encodes an expanded family of LDL homologues (FLD, LDL1, LDL2, and LDL3), resulting in substantial subfunctionalization and specialized recruitment mechanisms. This review explores the diverse developmental roles of plant LDLs, ranging from flowering time and circadian clock regulation to heterochromatin maintenance and epigenetic regulation. We discuss the redundant roles of FLD, LDL1, and LDL2 in repressing the floral repressor FLC and their nonredundant specialized function within the CCA1/LHY-TOC1 circadian feedback loop. A central focus of our review is the emerging mechanism of transcription-coupled demethylation, in which LDLs associate with the phosphorylated C-terminal domain of RNA polymerase II to modify chromatin cotranscriptionally within gene bodies. By integrating findings from Arabidopsis thaliana and crops such as tomato and soybean, we illustrate how the diversified LDL-mediated regulatory toolkit facilitates precise, gene-specific regulation. Ultimately, the LDL family represents a cornerstone of the sophisticated epigenetic strategies that regulate plant phenotypic plasticity in response to developmental and environmental cues.

Circadian clock↗
PubMed1996

Cytochromes P450 5: induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription.

The induction of microsomal cytochrome P4501A1 by polycyclic aromatic hydrocarbons represents an interesting response by which mammalian cells adapt to xenobiotic exposure. Enzyme induction reflects increased transcription of the corresponding CYP1A1 gene. Analyses of the induction mechanism using genetic, biochemical, and molecular biological approaches have revealed a novel transcriptional regulatory pathway that involves ligand-dependent heterodimerization between two basic helix-loop-helix proteins (the Ah receptor and Arnt), interaction of the heterodimer with a xenobiotic-responsive enhancer, transmission of the induction signal from the enhancer to the CYP1A1 promoter, and alterations in chromatin structure. Current techniques permit examination of the induction mechanism in intact cells and analyses of the CYP1A1 gene in its native chromosomal configuration. Such experiments generate new insights into the control of mammalian transcription that are of relatively broad interest.

Animals↗
PubMed1988

The matrix attachment regions of the chicken lysozyme gene co-map with the boundaries of the chromatin domain.

The matrix attachment regions of the chicken lysozyme domain were studied in an in vitro DNA binding assay by incubating oviduct nuclear matrices with labeled restriction fragments. A strong attachment region was localized between 11.1 and 8.85 kb upstream of the transcription start site and a weaker one between 1.3 and 5.0 kb downstream of the poly(A)+ addition site. Both attachment regions co-map with the previously established boundaries of the chromatin domain. The upstream matrix attachment region is distinguishable from known enhancers and is composed of multiple binding sites. We find specific but weaker binding of the same restriction fragments to matrix preparations from transcriptionally inactive chicken erythrocytes indicating a cell-type and transcription-independent conservation of the sites for specific binding of matrix attachment sequences. We also demonstrate that the matrix attachment regions are located at the base of a chromosomal loop in histone-extracted nuclei. Thus, the lysozyme domain represents a topologically-sequestered functional unit containing the coding region and all known lysozyme-specific, cis-acting regulatory elements.

Animals↗
PubMed2006

Spreading of silent chromatin: inaction at a distance.

One of the oldest unsolved problems in genetics is the observation that gene silencing can 'spread' along a chromosome. Although spreading has been widely perceived as a process of long-range assembly of heterochromatin proteins, such 'oozing' might not apply in most cases. Rather, long-range silencing seems to be a dynamic process, involving local diffusion of histone-modifying enzymes from source binding sites to low-affinity sites nearby. Discontinuous silencing might reflect looping interactions, whereas the spreading of continuous silencing might be driven by the processive movement of RNA or DNA polymerases. We review the evidence for the spreading of silencing in many contexts and organisms and conclude that multiple mechanisms have evolved that silence genes at a distance.

Animals↗
PubMed1980

Transcription units of chicken ovalbumin gene observed after injection of cloned complete genes into Xenopus oocyte nuclei.

The organization of transcription of a well-characterized protein encoding gene was studied by microinjection and electron microscopy. Circular recombinant DNA molecules containing the complete chicken ovalbumin sequences (7.7 kilobases, contained in 11.5 kilobases of chicken DNA) were microinjected into germinal vesicles of living oocytes of the clawed toad Xenopus laevis, and their transcription was studied in nuclear spread preparations. Evaluation of spread chromatin showed a limited number of observed molecules transcribed in "specific" patterns--i.e., circular chromatin molecules containing transcription units approximately 2.3 micrometer long, consisting of regular series of densely packed lateral ribonucleoprotein fibrils gradually increasing in length. The appearance of these fibril gradients was similar to that of actively transcribed endogenous protein encoding genes contained in lampbrush-chromosome loops of the same nuclei and to the putative Bombyx silk fibroin transcription units. In addition, less-regular arrays of transcript fibrils were seen in some circles, including fully fibril-covered molecules, indicative of the occurrence of irregular transcriptional events. The results of this heterologous transcription experiment indicate that the transcriptional machinery of the amphibian oocyte nucleus is capable of transcribing protein encoding genes from an avian species in typical regular arrays of transcription units.

Animals↗
PubMed2002

Plzf mediates transcriptional repression of HoxD gene expression through chromatin remodeling.

The molecular mechanisms that regulate coordinated and colinear activation of Hox gene expression in space and time remain poorly understood. Here we demonstrate that Plzf regulates the spatial expression of the AbdB HoxD gene complex by binding to regulatory elements required for restricted Hox gene expression and can recruit histone deacetylases to these sites. We show by scanning forced microscopy that Plzf, via homodimerization, can form DNA loops and bridge distant Plzf binding sites located within HoxD gene regulatory elements. Furthermore, we demonstrate that Plzf physically interacts with Polycomb proteins on DNA. We propose a model by which the balance between activating morphogenic signals and transcriptional repressors such as Plzf establishes proper Hox gene expression boundaries in the limb bud.

Animals↗