Cytogenetic Mapping of 5S and 18S rRNAs and H3 Histone Genes in 4 Ancient Proscopiidae Grasshopper Species: Contribution to Understanding the Evolutionary Dynamics of Multigene Families

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phosphorylation of Histone H3S10 in Animal Chromosomes: Is There a Uniform Pattern?

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Chromosomal mapping of repetitive DNAs in the beetle Dichotomius geminatus provides the first evidence for an association of 5S rRNA and histone H3 genes in insects, and repetitive DNA similarity between the B chromosome and A complement

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location

We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers. © 2011 Springer Science+Business Media B.V.

Cytogenetic mapping of H1 histone and ribosomal RNA genes in hybrids between catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum

A physical chromosome mapping of the H1 histone and 5S and 18S ribosomal RNA (rRNA) genes was performed in interspecific hybrids of Pseudoplatystoma corruscans and P. reticulatum. The results showed that 5S rRNA clusters were located in the terminal region of 2 chromosomes. H1 histone and 18S ribosomal genes were co-localized in the terminal portion of 2 chromosomes (distinct from the chromosomes bearing 5S clusters). These results represent the first report of association between H1 histone and 18S genes in fish genomes. The chromosome clustering of ribosomal and histone genes was already reported for different organisms and suggests a possible selective pressure for the maintenance of this association. © 2012 S. Karger AG, Basel.

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C0t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes. © 2013 Bueno et al.

New ligands of the cellular retinoic acid-binding protein 2 (CRABP2) suggest a role for this protein in chromatin remodeling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oxygen tension affects histone remodeling of in vitro-produced embryos in a bovine model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Changes in tri-methylation profile of lysines 4 and 27 of histone H3 in bovine blastocysts after cryopreservation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Holocarboxylase Synthetase-dependent Biotinylation of Histone H4

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysine (K) residues in histones H3 and H4. Histone biotinylation marks play important roles in the repression of genes and retrotransposons. Preliminary studies suggested that K16 in histone H4 is a target for biotinylation by HCS. Here we demonstrated that H4K16bio is overrepresented in repeat regions {pericentromeric alpha satellite repeats; long terminal repeats (LTR)} compared with euchromatin promoters. H4K16bio was also enriched in the repressed interleukin-2 gene promoter. The enrichment at LTR22 and promoter 1 of the sodium-dependent multivitamin transporter (SMVT) depended on biotin supply; and was significantly lower in fibroblasts from an HCS-deficient patient compared with an HCS wild-type control. We conclude that H4K16bio is a real phenomenon and plays a role in the transcriptional repression of repeats and genes. HCS catalyzes the covalent binding of biotin to carboxylases, in addition to its role as a histone biotinyl ligase. HCS null individuals are not viable whereas HCS deficiency is linked to developmental delays and phenotypes such as short life span and low stress resistance. Here, we developed a 96-well plate assay for high-throughput analysis of HCS based on the detection of biotinylated p67 using IRDye-streptavidin and infrared spectroscopy. We demonstrated that the catalytic activity of rHCS depends on temperature and time, and proposed optimal substrate and enzyme concentrations to ensure ideal measurement of rHCS activity and its kinetics. Additionally, we demonstrated that this assay is sensitive enough to detect biotinylation of p67 by endogenous HCS from Jurkat lymphoid cells.

Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

Subcellular distribution of the insulin-like growth factor (IGF) binding proteins (IGFBPs) 2 and 3 in articular chondrocytes

The insulin-like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF-binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP-2 and IGFBP-3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP-2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP-2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP-2 were in the cytoplasmic fraction. Immunoreactive IGFBP-3 was found in the cytoplasmic and peri-nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP-2 and IGFBP-3 but only IGFBP-3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action.

Structure of a mouse histone-encoding gene cluster

In addition to a previously described histone (H)-encoding H4 gene [Meier et al., Nucleic Acids Res. 17 (1989) 795], the mouse genomic DNA clone 53 contains two H3 genes, one functional and one partially deleted H2A gene, and one H2B gene. Clone 53 overlaps for 3 kb with MH143, another previously isolated mouse H-encoding clone [Yang et al., J. Biol. Chem. 262 (1987) 17118-17125], thus defining a 32-kb region of mouse chromosome 13 with a total of seven H-encoding genes. We have determined the nucleotide sequences and transcription start points of two genes coding for the H2A.1 and H3.2 proteins.

Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors.

Histone RNA 3' end formation occurs through a specific cleavage reaction that requires, among other things, base-pairing interactions between a conserved spacer element in the pre-mRNA and the minor U7 snRNA present as U7 snRNP. An oligonucleotide complementary to the first 16 nucleotides of U7 RNA can be used to characterize U7 snRNPs from nuclear extracts by native gel electrophoresis. Using similar native gel techniques, we present direct biochemical evidence for a stable association between histone pre-mRNA and U7 snRNPs. Other complexes formed in the nuclear extract are dependent on the 5' cap structure and on the conserved hairpin element of histone pre-mRNA, respectively. However, in contrast to the U7-specific complex, their formation is not required for processing. Comparison of several authentic and mutant histone pre-mRNAs with different spacer sequences demonstrates that the formation and stability of the U7-specific complex closely follows the predicted stability of the potential RNA-RNA hybrid. However, this does not exclude a stabilization of the complex by U7 snRNP structural proteins.