Histone Gene Loci Research Articles

Actin-dependent intranuclear repositioning of an active gene locus in vivo

Although bulk chromatin is thought to have limited mobility within the interphase eukaryotic nucleus, directed long-distance chromosome movements are not unknown. Cajal bodies (CBs) are nuclear suborganelles that nonrandomly associate with small nuclear RNA (snRNA) and histone gene loci in human cells during interphase. However, the mechanism responsible for this association is uncertain. In this study, we present an experimental system to probe the dynamic interplay of CBs with a U2 snRNA target gene locus during transcriptional activation in living cells. Simultaneous four-dimensional tracking of CBs and U2 genes reveals that target loci are recruited toward relatively stably positioned CBs by long-range chromosomal motion. In the presence of a dominant-negative mutant of β-actin, the repositioning of activated U2 genes is markedly inhibited. This supports a model in which nuclear actin is required for these rapid, long-range chromosomal movements.

NELF Interacts with CBC and Participates in 3′ End Processing of Replication-Dependent Histone mRNAs

Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been implicated in numerous diseases ranging from neurological disorders to cancer. Here we show that NELF interacts with the nuclear cap binding complex (CBC), a multifunctional factor that plays important roles in several mRNA processing steps, and the two factors together participate in the 3' end processing of replication-dependent histone mRNAs, most likely through association with the histone stem-loop binding protein (SLBP). Strikingly, absence of NELF and CBC causes aberrant production of polyadenylated histone mRNAs. Moreover, NELF is physically associated with histone gene loci and forms distinct intranuclear foci that we call NELF bodies, which often overlap with Cajal bodies and cleavage bodies. Our results point to a surprising role of NELF in the 3' end processing of histone mRNAs and also suggest that NELF is a new factor that coordinates different mRNA processing steps during transcription.

An architectural perspective of cell‐cycle control at the G1/S phase cell‐cycle transition

A prominent role for the execution of cell cycle and growth regulatory mechanisms within the three-dimensional context of nuclear architecture is becoming increasingly evident. Signaling pathways and regulatory networks that govern activation and suppression of genes controlling proliferation are functionally integrated for the organization and assembly of transcriptional machinery in nuclear microenvironments. The transcriptional activation of histone genes at the G1/S phase transition (S-point) is temporarily, functionally, and spatially distinct from transcriptional mechanisms at the restriction point (R-point). The spatial distinction in R-point versus S-point control is the localization of clustered histone gene loci at cajal bodies, which is modulated during the cell cycle. Histone nuclear factor P (HiNF-P), the principal factor mediating H4 histone gene transcription, is the final link in the signaling cascade that is initiated with growth factor dependent induction of cyclin E/CDK2 kinase activity at the R-point and culminates in the NPAT-mediated activation of histone H4 genes through HiNF-P at the G1/S phase cell-cycle transition.

The Drosophila melanogaster Cajal body

Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB–specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.

The HIR corepressor complex binds to nucleosomes generating a distinct protein/DNA complex resistant to remodeling by SWI/SNF

The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR repressor complex. The HIR complex promotes histone deposition onto DNA in vitro and constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA and nucleosomes. Furthermore, HIR complex binding to nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Thus, the HIR complex is a novel nucleosome assembly complex which functions with SWI/SNF to regulate transcription.

Reverse transcriptase-related proteins in telomeres and in certain chromosomal loci of Rhynchosciara (Diptera: Sciaridae).

The localization of reverse transcriptase-related proteins in polytene chromosomes of dipterans was investigated using previously characterized antibodies to a recombinant polypeptide containing conserved motifs of insect reverse transcriptases. The immunoreactions were carried out with polytene chromosome squashes of eight sciarids, one chironomid and three Drosophila species. Telomeric staining was regularly observed on chromosomes of the sciarid Rhynchosciara americana under normal growth conditions. Five of eight chromosomal tips were labelled except for the heterochromatic ends that are occasionally found associated forming a chromocentre in the salivary gland. Reverse transcriptase-related proteins were detected at chromosomal tips of young larvae and remained bound to the telomeres throughout larval development. As in salivary gland chromosomes, five non-telocentric ends of the chromosomes from Malpighian tubules of R. americana appeared clearly stained with anti-reverse transcriptase. The occurrence of telomeric reverse transcriptase in R. americana correlates with the presence of RNA in addition to an unusual enrichment with homopolymeric dA/dT DNA associated with the telomeric heterochromatin. The antibodies also reacted with a few interstitial sites in chromosomes of four Rhynchosciara species, one band overlapping the histone gene locus of three species in the americana -like group. The results provide evidence for a reverse transcriptase-related protein as a constitutive component in telomeres of R. americana and also in certain interstitial loci of Rhynchosciara species in which RNA was immunologically detected in the form of RNA:DNA hybrids.

NPAT links cyclin E–Cdk2 to the regulation of replication-dependent histone gene transcription

In eukaryotic cells, histone gene expression is one of the major events that mark entry into S phase. While this process is tightly linked to cell cycle position, how it is regulated by the cell cycle machinery is not known. Here we show that NPAT, a substrate of the cyclin E-Cdk2 complex, is associated with human replication-dependent histone gene clusters on both chromosomes 1 and 6 in S phase. We demonstrate that NPAT activates histone gene transcription and that this activation is dependent on the promoter elements (SSCSs) previously proposed to mediate cell cycle-dependent transcription. Cyclin E is also associated with the histone gene loci, and cyclin E-Cdk2 stimulates the NPAT-mediated activation of histone gene transcription. Thus, our results both show that NPAT is involved in a key S phase event and provide a link between the cell cycle machinery and activation of histone gene transcription.

Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: evidence for PreU2 within Cajal bodies.

The Cajal (coiled) body (CB) is a structure enriched in proteins involved in mRNA, rRNA, and snRNA metabolism. CBs have been shown to interact with specific histone and snRNA gene loci. To examine the potential role of CBs in U2 snRNA metabolism, we used a variety of genomic and oligonucleotide probes to visualize in situ newly synthesized U2 snRNA relative to U2 loci and CBs. Results demonstrate that long spacer sequences between U2 coding repeats are transcribed, supporting other recent evidence that U2 transcription proceeds past the 3' box. The presence of bright foci of this U2 locus RNA differed between alleles within the same nucleus; however, this did not correlate with the loci's association with a CB. Experiments with specific oligonucleotide probes revealed signal for preU2 RNA within CBs. PreU2 was also detected in the locus-associated RNA foci, whereas sequences 3' of preU2 were found only in these foci, not in CBs. This suggests that a longer primary transcript is processed before entry into CBs. Although this work shows that direct contact of a U2 locus with a CB is not simply correlated with RNA at that locus, it provides the first evidence of new preU2 transcripts within CBs. We also show that, in contrast to CBs, SMN gems do not associate with U2 gene loci and do not contain preU2. Because other evidence indicates that preU2 is processed in the cytoplasm before assembly into snRNPs, results point to an involvement of CBs in modification or transport of preU2 RNA.

Induction of coiled body-like structures in Xenopus oocytes by U7 snRNA

The coiled bodies, or "sphere organelles," of amphibian oocytes, first identified by their unique morphology, are large structures of up to 10 microm in diameter. There are 40 to 120 coiled bodies per oocyte nucleus. Most of the coiled bodies are in the nucleoplasm but a few are attached to specific chromosomal loci containing the histone gene repeats. Like the coiled bodies of somatic cells, they contain high concentrations of U7 snRNA, a small nuclear RNA required for 3' end formation of histone transcripts, and of a unique protein called coilin/SPH-1. We show here that increasing the nuclear concentration of U7 snRNA, by injection of either in vitro synthesized U7 snRNA or functional U7 snRNA genes, induces the formation of coiled body-like structures in vivo. In contrast, the formation of these structures is not induced when either a promoterless U7 snRNA gene construct is injected or when functional U7 snRNA genes are co-injected with alpha-amanitin. Increasing the concentration of U1 snRNA or histone mRNA does not induce the formation of these structures, indicating that the formation of these coiled body-like structures is specifically induced by the U7 snRNA. These results suggest that U7 snRNA may be a central nucleating factor of coiled bodies and that the appearance of coiled bodies at histone gene loci results from an increased local concentration of U7 snRNA near the nascent histone pre-mRNAs.

The stem-loop binding protein (SLBP1) is present in coiled bodies of the Xenopus germinal vesicle.

The stem-loop binding protein (SLBP1) binds the 3' stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3' end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Coilin can form a complex with the U7 small nuclear ribonucleoprotein.

Coiled bodies (CBs) in the amphibian oocyte nucleus are spherical structures up to 10 microm or more in diameter, much larger than their somatic counterparts, which rarely exceed 1 microm. Oocyte CBs may have smaller granules attached to their surface or embedded within them, which are identical in structure and composition to the many hundreds of B-snurposomes found free in the nucleoplasm. The matrix of the CBs contains the diagnostic protein p80-coilin, which is colocalized with the U7 small nuclear ribonucleoprotein (snRNP), whereas the attached and embedded B-snurposomes contain splicing snRNPs. A few of the 50-100 CBs in the oocyte nucleus are attached to lampbrush chromosomes at the histone gene loci. By coimmunoprecipitation we show that coilin and the U7 snRNP can form a weak but specific complex in the nucleoplasm, which is dependent on the special U7 Sm-binding site. Under the same conditions coilin does not associate with the U1 and U2 snRNPs. Coilin is a nucleic acid-binding protein, as shown by its interaction with single-stranded DNA and with poly r(U) and poly r(G). We suggest that an important function of coilin is to form a transient complex with the U7 snRNP and accompany it to the CBs. In the case of CBs attached to chromosomes at the histone gene loci, the U7 snRNP is thus brought close to the actual site of histone pre-mRNA transcription.

Functional dissection of yeast Hir1p, a WD repeat-containing transcriptional corepressor.

The HIR1 gene product is required to repress transcription of three of the four histone gene loci in Saccharomyces cerivisiae, and like its counterpart, the HIR2 protein, it functions as a transcriptional corepressor. Although Hir1p and Hir2p are physically associated in yeast, Hir1p is able to function independently of Hir2p when it is artificially recruited to the histone HTA1 promoter. A deletion analysis of HIR1 has revealed two separate repression domains: one in its N terminus, where seven copies of the beta-transducin or WD40 motif reside, and the second in the remaining C-terminal amino acids. Overexpression of the WD repeats in a hir1delta strain complemented its Hir- phenotype, while overexpression of the C terminus in a wild-type strain caused both Hir- and Spt- phenotypes. The Hir1p C terminus physically interacted in vivo with Hir2p, and both Hir1p repression domains interacted with full-length Hir1p. It was additionally found that the Hir1p WD repeats functionally interacted with the SPT4, SPT5, and SPT6 gene products, suggesting that these repeats may direct Hir1p to different protein complexes.

Human genes encoding U3 snRNA associate with coiled bodies in interphase cells and are clustered on chromosome 17p11.2 in a complex inverted repeat structure.

Coiled bodies (CBs) are nuclear organelles whose morphological structure and molecular composition have been conserved from plants to animals. Furthermore, CBs are often found to co-localize with specific DNA loci in both mammalian somatic nuclei and amphibian oocytes. Much as rDNA sequences are called nucleolus organizers, we term these coiled body-associated sequences 'coiled body organizers' (CBORs). The only sequences that have been shown to be CBORs in human cells are the U1, U2 and histone gene loci. We wanted to determine whether other snRNA genes might also act as CBORs. In this paper we show that human U3 genes (the RNU3 locus) preferentially associate with CBs in interphase cells. In addition, we have analyzed the genomic organization of the RNU3 locus by constructing a BAC and P1 clone contig. We found that, unlike the RNU1 and RNU2 loci, U3 genes are not tandemly repeated. Rather, U3 genes are clustered on human chromosome 17p11.2, with evidence for large inverted duplications within the cluster. Thus all of the CBORs identified to date are composed of either tandemly repeated or tightly clustered genes. The evolutionary and cell biological consequences of this type of organization are discussed.

The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II (66% identity). Organization of the core histone gene in pairs, and non-polyadenylation of mRNAs are features shared with animals, whereas peptide sequences and enhancer elements are shared with higher plants, assigning the volvocalean histone genes a position intermediate between animals and plants.

Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.

Evolution of histone gene loci in chironomid midges

In the present study we have localized the histone genes in the chromosomes of 16 different Chironomus species as well as in Prodiamesa olivacea, Glyptotendipes barbipes, and Acricotopus lucidus. In the genus of Chironomus we find four, five, or six different "major" chromosomal loci hybridizing with a histone gene cluster probe isolated from the genome of Chironomus thummi. These major histone gene loci probably contain clustered histone gene repeating units ("clustered" loci). They are located on one and the same chromosome arm in all but one of the species investigated. This shows that the histone gene clusters are rather conservative in their location over a long period of evolution. The comparison of the histone loci pattern from the chromosomes of the different chironomid species shows that there is good agreement with previously established chromosome maps and phylogenetic studies based on the chromosomal banding pattern. Stringent in situ hybridization with various histone gene containing clones suggest that the "clustered" histone gene loci are organized in a locus-specific way. In addition to the linked "clustered" histone gene loci, we found an isolated histone gene group ("orphon") present on chromosome IV in most Chironomus species. This gene group might be organized differently from the histone gene repeating unit described previously.

U7 small nuclear RNA in C snurposomes of the Xenopus germinal vesicle.

In the amphibian germinal vesicle small nuclear RNAs (snRNAs) occur in morphologically distinct structures called snurposomes. Three types (A, B, and C) have been distinguished on the basis of cytological appearance and snRNA composition. C snurposomes in Xenopus are spherical bodies ranging in diameter from < 1 microns to about 10 microns. They stain intensely with antibodies against trimethylguanosine and the small nuclear ribonucleoprotein-specific Sm antigen but give weak or negative in situ hybridization reactions for the snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6). We show here that C snurposomes in the Xenopus germinal vesicle contain U7 snRNA, an snRNA of low abundance involved in processing the 3' end of histone pre-mRNA. Xenopus U7 is 58 nucleotides long and is capped at the 5' end with trimethylguanosine. C snurposomes are often associated with B snurposomes, which contain the splicing snRNAs but not U7; B and C snurposomes together constitute a morphologically complex structure known as a sphere or sphere organelle. Although most spheres and C snurposomes are extrachromosomal, a few are attached at the histone gene loci on chromosomes 8, 9, and 16. Because they contain U7 snRNA and occur at the sites of histone pre-mRNA synthesis, C snurposomes presumably play a role in processing histone transcripts.

Characterization of HIR1 and HIR2, two genes required for regulation of histone gene transcription in Saccharomyces cerevisiae.

The products of the HIR1 and HIR2 genes have been defined genetically as repressors of histone gene transcription in S. cerevisiae. A mutation in either gene affects cell cycle regulation of three of the four histone gene loci; transcription of these loci occurs throughout the cell cycle and is no longer repressed in response to the inhibition of DNA replication. The same mutations also eliminate autogenous regulation of the HTA1-HTB1 locus by histones H2A and H2B. The HIR1 and HIR2 genes have been isolated, and their roles in the transcriptional regulation of the HTA1-HTB1 locus have been characterized. Neither gene encodes an essential protein, and null alleles derepress HTA1-HTB1 transcription. Both HIR genes are expressed constitutively under conditions that lead to repression or derepression of the HTA1 gene, and neither gene regulates the expression of the other. The sequence of the HIR1 gene predicts an 88-kDa protein with three repeats of a motif found in the G beta subunit of retinal transducin and in a yeast transcriptional repressor, Tup1. The sequence of the HIR2 gene predicts a protein of 98 kDa. Both gene products contain nuclear targeting signals, and the Hir2 protein is localized in the nucleus.

Characterization of HIR1 and HIR2, Two Genes Required for Regulation of Histone Gene Transcription in Saccharomyces cerevisiae

The products of the HIR1 and HIR2 genes have been defined genetically as repressors of histone gene transcription in S. cerevisiae. A mutation in either gene affects cell cycle regulation of three of the four histone gene loci; transcription of these loci occurs throughout the cell cycle and is no longer repressed in response to the inhibition of DNA replication. The same mutations also eliminate autogenous regulation of the HTA1-HTB1 locus by histones H2A and H2B. The HIR1 and HIR2 genes have been isolated, and their roles in the transcriptional regulation of the HTA1-HTB1 locus have been characterized. Neither gene encodes an essential protein, and null alleles derepress HTA1-HTB1 transcription. Both HIR genes are expressed constitutively under conditions that lead to repression or derepression of the HTA1 gene, and neither gene regulates the expression of the other. The sequence of the HIR1 gene predicts an 88-kDa protein with three repeats of a motif found in the G beta subunit of retinal transducin and in a yeast transcriptional repressor, Tup1. The sequence of the HIR2 gene predicts a protein of 98 kDa. Both gene products contain nuclear targeting signals, and the Hir2 protein is localized in the nucleus.

Snurposomes and Coiled Bodies

In the GV of amphibian oocytes, the splicing snRNPs (U1, U2, U4, U5, and U6) occur on most of the lampbrush chromosome loops in association with the nascent transcripts. They also occur in thousands of small extrachromosomal bodies (1-4 microns in diameter) designated B snurposomes. U7 snRNA, which is involved in processing the 3' end of histone pre-mRNAs, occurs in a few dozen extrachromosomal bodies (1-20 microns in diameter) called C snurposomes. C snurposomes often have B snurposomes attached to their surface and B-like inclusions; these compound structures are known as spheres or sphere organelles. One or two sphere organelles are attached to the lampbrush chromosomes at the histone gene loci. Two snRNAs (or snoRNAs) known to be involved in pre-rRNA processing (U3 and U8) occur in the 1000 or so extrachromosomal nucleoli of the GV. We looked for a snurposome that might contain U3 and U8 but not rDNA or rRNA. We were unable to find such a snurposome, but we did identify a hitherto unrecognized population of minute nucleoli in the size range of B snurposomes. Prenucleolar bodies in telophase/early interphase nuclei meet the definition of a pre-rRNA snurposome in that they contain U3 snoRNA and fibrillarin (and probably other processing components) but lack rDNA and do not synthesize rRNA. The structures previously identified as prenucleolar bodies in pronuclei formed in vitro in Xenopus egg extracts share many components with coiled bodies from HeLa nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)