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PubMed2002

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1.

The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. We therefore conclude that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, our observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells.

3T3 Cells↗
PubMed1963

THE BASE COMPOSITION OF RIBONUCLEIC ACID IN LAMPBRUSH CHROMOSOMES, NUCLEOLI, NUCLEAR SAP, AND CYTOPLASM OF TRITURUS OOCYTES.

The base composition of RNA's extracted from chromosomes, nucleoli, nuclear sap, and cytoplasm of Triturus oocytes has been determined by microelectrophoresis. The chromosomal RNA has a content of guanine+cytosine equal to that of DNA, but there is no complementarity in the composition as for DNA. Nuclear sap contains a highly variable RNA with a tendency towards high uracil values. Nucleolar and cytoplasmic RNA's are similar in composition and both are of the guanine-cytosine rich type. The chromosomes and nucleoli contain roughly equivalent amounts of RNA, somewhat less than is present in the nuclear sap. The RNA/DNA ratio of the whole chromosomes is about 10. However, the ratio in the synthetically active regions, the loops, is much higher, since the loops contain all the chromosomal RNA but only a small fraction of the DNA.

Adenine↗
PubMed2002

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene.

Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho-1. Here we show that a heme-binding factor, Bach1, is a critical physiological repressor of ho-1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho-1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho-1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho-1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound-deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho-1. Thus, regulation of ho-1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor-activator antagonism.

3T3 Cells↗
PubMed1980

Ribonucleoprotein structure in nascent hnRNA is nonrandom and sequence-dependent.

We have studied the ribonucleoprotein morphology of nascent hnRNA using chromatin spreading methods. This approach allows visualization of multiple transcripts of the same DNA sequence. Thus the RNP structure of hnRNA molecules with the same nucleotide sequence can be compared. We find that RNP particles averaging 240 A in diameter occur on the majority of hnRNA transcription units, but that RNA fibrils are generally not completely covered with particles. Furthermore, the RNP particle location is nonrandom for the transcripts of a given gene. Particle number and arrangement on RNP fibrils vary from one transcription unit to the next, and are not obviously related to transcription unit size or activity. Analysis of particle location with respect to the 5' RNA terminus allows RNP particle localization to homologous sequences of less than 500 ribonucleotides on the different fibrils of a single transcription unit. RNP particles assemble very soon after synthesis of the particular sequence. Sites of particle formation are distinct from sites of double-stranded hairpin loops on the RNA fibrils. A differential, or differentially stable, RNP structure with respect to RNA sequence may bear on mechanisms of specific hnRNA processing.

Animals↗
PubMed1996

Interrelationships between nuclear structure and ligand-activated intracellular receptors.

The role of ligand-activated intracellular receptors in activation of gene expression most probably involves a multistep process that requires alteration in chromatin structure. Results of studies with members of the steroid hormone receptor superfamily as well as the ligand-activated member of the basic region/helix-loop-helix family of transcriptional activators suggests that two different nuclear structures and their associated nuclear proteins are important in the initial stages of gene activation: the nuclear matrix and nucleosomes. Cell- and tissue-specific nuclear matrix proteins and the variant and modified histones appear to be important for tissue and species specificity of ligand-induced responses. Because the function of a receptor may be limited in vivo by promoter and transcription factor accessibility, the various roles of nuclear ligand-receptor complexes may involve interaction with nuclear matrix proteins and/or nucleosomes. Tissue-specific structural nuclear proteins could control the conformation (looping through matrix attachment regions) of the DNA and unwinding or rearrangement of nucleosomes, thus providing specificity to the expression of certain genes. Modulation of cooperative elements required for gene activation may involve association of the gene promoter with the nuclear matrix together with the presence of nucleosomes. Thus, the series of events involved in ligand-receptor activation of genes requires alterations in chromatin structure, which allow access of the receptor complex to elements within the gene.

Animals↗
PubMed2000

A sense of the end.

How a cell distinguishes a double-strand break from the end of a chromosome has long fascinated cell biologists. It was thought that the protection of chromosomal ends required either a telomere-specific complex or the looping back of the 3' TG-rich overhang to anneal with a homologous double-stranded repeat. These models must now accommodate the findings that complexes involved in nonhomologous end joining play important roles in normal telomere length maintenance, and that subtelomeric chromatin changes in response to the DNA damage checkpoint. A hypothetical chromatin assembly checkpoint may help to explain why telomeres and the double-strand break repair machinery share essential components.

Animals↗
PubMed1997

Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

Antigens, Nuclear↗
PubMed1989

An analog of Xenopus N1N2 protein in Pleurodeles waltl.

The oocyte nucleus of Pleurodeles waltl contains a major 185-kDa protein analog of Xenopus N1N2. Rabbit antibodies were raised against the 185-kDa protein. Affinity-purified antibody directed against the acid part (pI 4.7) of the protein was prepared using antigens separated by two-dimensional electrophoresis. Specificity of the antibody was controlled on two-dimensional gels of nuclear proteins. It was shown that the 185-kDa protein was separated into 2 forms: an acid form of pI 4.7-5.3, and a base form of pI 7. Peptide maps of the 2 spots revealed that they were closely related. The antibody was tested on: a) spread nuclear content, b) on sections of embryonic stages from stage 2 to stage 34, and c) the sections of adult tissues. The 185-kDa protein appeared to be associated with the RNP matrix of a particular type of lampbrush chromosome loop, the granular loop. This protein was present in the nuclei of all embryonic cells. In adult tissues, it was present only in the nuclei of cells which presented high mitotic activity. These results confirm that, like N1N2, the 185-kDa protein interacts with the constitution of chromatin; furthermore, they provide evidence for the role of this protein in transcriptional activity.

Animals↗
PubMed1996

Developmental regulation of the vertebrate globin multigene family.

"Hemoglobin switching," or the sequential expression of globin genes in erythroid cells during development, has provided an important paradigm for tissue- and stage-specific gene regulation. Over the past decade, regulatory DNA sequences and transcription factors involved in controlling the expression of individual globin genes in erythroid cells have been identified. The picture that has emerged indicates that gene proximal control elements collaborate with a "locus control region" located far upstream - probably via a DNA looping mechanism - to ensure that each gene is turned on only in erythroid cells and at the appropriate time during development. Interactions among the various regulatory sequences are thought to be mediated and stabilized by an array of tissue-specific and ubiquitous proteins. Chromatin structure plays a critical but still poorly understood role in this process.

Chromatin↗
PubMed1981

Correlation of hnRNP structure and nascent transcript cleavage.

Using electron microscopy of spread chromatin, we have observed nonnucleolar transcription units from Drosophila melanogaster and Calliphora erythrocephala that display specific cleavage of nascent transcripts. We have quantitatively analyzed 20 of these relatively long transcription units. The primary RNP structure of homologous transcripts is nonrandom with respect to both RNA sequence and the cleavage event. In general, released RNA fragments have a smooth fibrillar RNP morphology (approximately 50 A wide) and retained segments have a thicker particulate morphology (approximately 250 A diameter). A characteristic secondary structure formation also accompanies cleavage--that is, RNP fibril loops form by association of noncontiguous transcript sequences that correspond to the terminal regions of the segment to be released. RNP particles form at the loop base sequences prior to their association and apparently coalesce upon loop formation. These loops, and thus the released segments, range in length from 1 and 25 kb on the examples we have analyzed. Cleavage of nascent hnRNA transcripts appears to be a fairly common event in these organisms and occurs within 0.3-3 min after transcription of the cleavage site.

Animals↗
PubMed2006

Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome.

Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.

Active Transport, Cell Nucleus↗
PubMed1999

Detection of chromatin-bound PCNA in mammalian cells and its use to study DNA excision repair.

Compelling evidence indicates that proliferating cell nuclear antigen (PCNA) is an indispensable factor not only in DNA replication but in nucleotide excision repair (NER), alternative pathway of base excision repair (BER), and mismatch repair. The common function of PCNA in each of these is to assist in the initiation of DNA synthesis by providing a scaffolding clamp as a trimer catalyzed by RF-C at the 3'-OH terminus of a nascent DNA strand, to which DNA polymerase delta or epsilon can bind. Interestingly, DNA synthesis is reported to be ingeniously inhibited in replication, but not in NER owing to the interaction with CDKN1A (formerly known as p21/WAF1/CIP1). Furthermore, several proteins, XPG, FEN1, and DNA ligase I, recently were shown to competitively bind to the same region of PCNA, the interdomain connector loop, to which DNA polymerase delta or epsilon also binds. PCNA therefore seems to have a regulatory role in these DNA transactions. The in vitro reconstituted experimental system has been a powerful tool to obtain these lines of evidence, but another approach, immunofluorescence studies, also has been a contributor. In fact, the involvement of PCNA in DNA replication, NER, and BER has for the first time been indicated by a unique method that makes visible only in vivo chromatin-bound PCNA. The usefulness of this method and the importance of cooperative studies done with in vitro and in vivo experimental systems is discussed in terms of DNA excision repair.

Animals↗
PubMed1993

The centromere of budding yeast.

Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in the cell cycle.

Base Sequence↗
PubMed2000

MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension.

All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three alpha-helices separated by two beta-strand loop regions which together constitute a histone fold. In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly. In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647delta) the C-terminal extension. The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)4 tetramer, were apparently less stable than those formed by (rMJ1647delta)4. The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647delta in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1. Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E. coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones.

Amino Acid Sequence↗
PubMed2005

Euchromatic and heterochromatic domains at Drosophila telomeres.

Noncoding repetitive sequences make up a large portion of eukaryotic genomes, but their function is not well understood. Large blocks of repetitive DNA-forming heterochromatin around the centromeres are required for this region to function properly, but are difficult to analyze. The smaller regions of heterochromatin at the telomeres provide an opportunity to study their DNA and protein composition. Drosophila telomere length is maintained through the targeted transposition of specific non-long terminal repeat retrotransposons to chromosome ends, where they form long tandem arrays. A subterminal telomere-associated sequence (TAS) lies immediately proximal to the terminal-retrotransposon array. Here, we review the experimental support for the heterochromatic features of Drosophila telomeres, and provide evidence that telomeric regions contain 2 distinct chromatin subdomains: TAS, which exhibits features that resemble beta heterochromatin; and the terminal array of retrotransposons, which appears euchromatic. This organization is significantly different from the telomeric organization of other eukaryotes, where the terminal telomerase-generated repeats are often folded in a t-loop structure and become part of the heterochromatin protein complex.

Animals↗
PubMed1978

Ultrastructure of anaplasmal inclusions (Pawhuska isolate) and their appendages in intact and hemolyzed erythrocytes and in complement-fixation antigen.

Hemolysis of parasitized erythrocytes augmented visualization of the anaplasmal inclusion, including its initial bodies, inclusion membrane, and inclusion appendage ("tail" or "band"). A dense attachment complex joined the appendage to the inclusion membrane (wall of the inclusion vacuole). The inclusion appendage consisted of tightly packed, interconnected laminae and assumed loop, dumbbell, and comet configurations described by other workers. The erythrocytic plasmalemma and the inclusion membrane had a thickness of 9.0 +/- 0.8 nm and similar structures. The initial bodies were covered by a thin inner organismic membrane (7.0 +/- 0.7 nm thick) attached to the organismic chromatin, an intermembranous matrix, and by an outer membranous sheath or pellicle (12.5 +/- 1.2 nm thick). Dense granular aggregates (24 to 40 nm in diameter) within chromatin clumps were the only structures in the initial body remotely similar to ribosomes, yet they were too large, were never free of chromatin, and appeared to disappear upon hemolysis. Complement-fixation antigen prepared by fractionation contained initial bodies, inclusion appendages, a few mitochondria, vesicularized membranes, and stromal debris. The preparatory treatment caused segregation of the organismic chromatin into independent dense particles 103 +/- 12 nm in diameter still bound by inner organismic membrane. Similar particles were seen also in the plasma and inclusion vacuoles of hemolyzed erythrocytes.

Anaplasma↗
PubMedDate not supplied

The role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment.

Biological experiments were combined with molecular dynamics simulations to understand the importance of amino acidic residues present in the bromodomain of the yeast histone acetyltransferase Gcn5p. It was found that residue Pro371 plays an important role in the molecular recognition of the acetylated histone H4 tail by Gcn5p bromodomain. Crystallographic analysis of the complex showed that this residue does not directly interact with the histone substrate. It has been demonstrated that a double mutation Pro371Thr and Met372Ala in the Gcn5p bromodomain impairs chromatin remodeling activity. It is demonstrated here that, in this double mutant and in the fully deleted bromodomain strain, there is lower growth under amino acid deprivation conditions. By in vitro surface plasmon resonance (Biacore) experiments it is shown that the binding affinity of the double mutation to acetyl lysine 16 histone H4 peptide decreases. Molecular dynamics simulations were used to explain this loss in acetyl lysine-Gcn5p bromodomain affinity, in the double mutant. By comparing nanosecond molecular dynamics trajectories of the native as well as the single and doubly mutated bromodomain, it is concluded that the presence of Pro371 is important to the functionality of the Gcn5p bromodomain. In the simulation a point mutation involving this highly conserved residue induced an increase in the flexibility of the ZA loop, which in turn modulated the exposure of the binding pocket to the acetyl lysine. The combined double mutations (Pro371Thr-Met372Ala) not only markedly perturb the motion of the ZA loop but also destabilize the entire structure of the bromodomain.

Acetylation↗
PubMed2004

Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.

The Ca2+ sensor protein calmodulin can interact with the DNA binding basic helix-loop-helix (bHLH) domain of E12, E47, and SEF2-1 (E2-2), which belong to the E-protein subclass of bHLH transcription factors. This interaction inhibits the DNA binding of these bHLH proteins in vitro, and an ionophore that increases intracellular Ca2+ can inhibit transcriptional activation by the E-proteins. Here we have attempted to determine if these phenomena reflect a direct calmodulin-dependent inhibition of DNA binding by E-proteins in vivo. We show that calmodulin overexpression inhibits the transcriptional activity of E12, E47, and SEF2-1. We have compared calmodulin effects on DNA binding in vitro and on activation of transcription in vivo using a series of E12 mutants harboring defined alterations within the basic sequence of the bHLH domain that reduce their ability to bind calmodulin to varying degrees. We find a striking direct correlation between the ability of calmodulin to inhibit their DNA binding in vitro and the ability of overexpressed calmodulin or cellular Ca2+ mobilization to inhibit their transcriptional activity in vivo. Furthermore, E12 and overexpressed calmodulin were co-localized in the nucleus, and calmodulin pull-down experiments with cell extracts showed a Ca2+-dependent interaction between calmodulin and E12 but not with a calmodulin inhibition-deficient E12 mutant. Chromatin immunoprecipitation showed that calmodulin overexpression leads to decreased binding of E12 and E47, but not a calmodulin inhibition-deficient E12 mutant, to the DNA recognition sequence in vivo. The data suggest that Ca2+ signaling can inhibit the transcriptional activities of E-proteins through direct binding of Ca2+/calmodulin to the basic sequence of E-proteins, resulting in inhibition of their DNA binding.

Amino Acid Sequence↗