Molecular and evolutionary analysis of mussel histone genes ("Mytilus" spp.): possible evidence of an "orphon origin" for H1 histone genes

[Abstract:] Linker histones are a divergent group of histone proteins with an independent evolutionary history in which, besides somatic subtypes, tissue- and differentiation-specific subtypes are included. In the present work H1 histone coding and noncoding segments from five Mytilus mussel species (Mollusca: Bivalvia) widely distributed throughout the world have been determined and characterized. Analysis of promoter regions shows clear homologies among Mytilus H1 genes, sea urchin H1 genes, and vertebrate differentiation-specific H1 subtypes (H5 and H10), all having an H4 box motif in common. The amino acid sequence of the H1 protein central conserved domain is also closely related to that previously defined for the vertebrate divergent subtypes. A phylogenetic tree reconstructed from different H1 genes from several species strengthens the hypothesis of an “orphon” origin for the Mytilus H1 genes, as well as for the H10/H5 genes from vertebrates and the H1D gene from the sea urchin Strongylocentrotus purpuratus, is suggested. As additional data, the average copy number of the H1 genes in the species analyzed was estimated as being 100 to 110 copies per haploid genome, where FISH revealed telomeric chromosomal location for several H1 copies in M. galloprovincialis. The contribution of such proximity to heterochromatic regions over the amount of codon bias detected for H1 genes is discussed.Ministerio de Ciencia e Innovación; IFD97-129

PCR cloning of a histone H1 gene from Anopheles stephensi mosquito cells: comparison of the protein sequence with histone H1-like, C-terminal extensions on mosquito ribosomal protein S6

BACKGROUND: In Aedes and Anopheles mosquitoes, ribosomal protein RPS6 has an unusual C-terminal extension that resembles histone H1 proteins. To explore homology between a mosquito H1 histone and the RPS6 tail, we took advantage of the Anopheles gambiae genome database to clone a histone H1 gene from an Anopheles stephensi mosquito cell line. RESULTS: We designed specific primers based on RPS6 and histone H1 alignments to recover an Anopheles stephensi histone H1 corresponding to a conceptual An. gambiae protein, with 92% identity. Southern blots suggested that Anopheles stephensi histone H1 gene has multiple variants, as is also the case for histone H1 proteins in Chironomid flies. CONCLUSIONS: Histone H1 proteins from Anopheles stephensi and Anopheles gambiae mosquitoes share 92% identity to each other, but only 50% identity to a Drosophila homolog. In a phylogenetic analysis, Anopheles, Chironomus and Drosophila histone H1 proteins cluster separately from the histone H1-like, C-terminal tails on RPS6 in Aedes and Anopheles mosquitoes. These observations suggest that the resemblance between histone H1 and the C-terminal extensions on mosquito RPS6 has been maintained by convergent evolution

The RNA Polymerase II Core Promoter in Drosophila.

Transcription by RNA polymerase II initiates at the core promoter, which is sometimes referred to as the "gateway to transcription." Here, we describe the properties of the RNA polymerase II core promoter in Drosophila The core promoter is at a strategic position in the expression of genes, as it is the site of convergence of the signals that lead to transcriptional activation. Importantly, core promoters are diverse in terms of their structure and function. They are composed of various combinations of sequence motifs such as the TATA box, initiator (Inr), and downstream core promoter element (DPE). Different types of core promoters are transcribed via distinct mechanisms. Moreover, some transcriptional enhancers exhibit specificity for particular types of core promoters. These findings indicate that the core promoter is a central component of the transcriptional apparatus that regulates gene expression

Birth-and-death evolution with strong purifying selection in the histone H1 multigene family and the origin of "orphon" H1 genes

[Abstract:] Histones are small basic nuclear proteins with critical structural and functional roles in eukaryotic genomes. The H1 multigene family constitutes a very interesting histone class gathering the greatest number of isoforms, with many different arrangements in the genome, including clustered and solitary genes, and showing replication-dependent (RD) or replication-independent (RI) expression patterns. The evolution of H1 histones has been classically explained by concerted evolution through a rapid process of interlocus recombination or gene conversion. Given such intriguing features, we have analyzed the long-term evolutionary pattern of the H1 multigene family through the evaluation of the relative importance of gene conversion, point mutation, and selection in generating and maintaining the different H1 subtypes. We have found the presence of an extensive silent nucleotide divergence, both within and between species, which is always significantly greater than the nonsilent variation, indicating that purifying selection is the major factor maintaining H1 protein homogeneity. The results obtained from phylogenetic analysis reveal that different H1 subtypes are no more closely related within than between species, as they cluster by type in the topologies, and that both RD and RI H1 variants follow the same evolutionary pattern. These findings suggest that H1 histones have not been subject to any significant effect of interlocus recombination or concerted evolution. However, the diversification of the H1 isoforms seems to be enhanced primarily by mutation and selection, where genes are subject to birth-and-death evolution with strong purifying selection at the protein level. This model is able to explain not only the generation and diversification of RD H1 isoforms but also the origin and long-term persistence of orphon RI H1 subtypes in the genome, something that is still unclear, assuming concerted evolution.Xunta de Galicia; PGIDT (10PX110304

Examining a Functional Interaction Between Chromatin Remodeler CHD1 and Histone H1 in D. melanogaster

Chromodomain-helicase-DNA-binding-protein-1 (CHD1) is a highly conserved ATP- dependent remodeling protein. It is localized to active genes and directs nucleosome spacing, while its loss has been linked to various human diseases, such as human prostate cancer. In Drosophila, CHD1 is important for fertility and wing development, and overexpression of CHD1 leads to severe wing vein defect phenotypes. The Linker Histone H1, which is known for maintaining heterochromatin and is associated with inactive genes, had been previously identified as a possible functional partner of CHD1, though the exact nature of their interaction is unclear. I undertook a genetic approach to examining the interaction between CHD1 and H1, making use of a novel genetic assay that had been previously developed in the Armstrong lab. This genetic assay uses the wing vein defects caused by CHD1 overexpression to identify factors that influence CHD1 function. I observed that CHD1 overexpression with the simultaneous knockdown of H1 resulted in an increase in the severity of wing vein defects, leading me to refine our working model for CHD1 and H1 interactions. Our working model suggests that CHD1 and H1 work competitively towards each other, with the absence of H1 allowing for increased CHD1 binding

Analysis of Properties of Heterochromatin Relative to Meiotic Recombination and Heterochromatic Gene Expression

To better understand the genetic properties of heterochromatin, I have pursued two avenues: meiotic recombination around the border of euchromatin and heterochromatin, and position effects at the heterochromatic light locus. Heterochromatin lacks recombination, and using meiotic recombination frequencies, I show that the recombination inhibition border concurs with the previously defined molecular border based on changes in histone proteins, specifically histone 3 methylation, characteristic of heterochromatin. I also show that the heterochromatic gene light behaves in a similar fashion to a previously studied heterochromatic gene, in that its function is impaired when moved out of the heterochromatic environment, but can be restored when brought near to large blocks of heterochromatin. These findings support the idea that gene function and recombination can be tightly controlled by the molecular environment of heterochromatin

Nucleus-specific linker histones Hho1 and Mlh1 form distinct protein interactions during growth, starvation and development in Tetrahymena thermophila

Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.York University Librarie

The Ribosomal Protein RpL22 Interacts In Vitro with 5′-UTR Sequences Found in Some Drosophila melanogaster Transposons

Mobility of eukaryotic transposable elements (TEs) are finely regulated to avoid an excessive mutational load caused by their movement. The transposition of retrotransposons is usually regulated through the interaction of host- and TE-encoded proteins, with non-coding regions (LTR and 5′-UTR) of the transposon. Examples of new potent cis-acting sequences, identified and characterized in the non-coding regions of retrotransposons, include the insulator of gypsy and Idefix, and the enhancer of ZAM of Drosophila melanogaster. Recently we have shown that in the 5′-UTR of the LTR-retrotransposon ZAM there is a sequence structured in tandem-repeat capable of operating as an insulator both in Drosophila (S2R+) and human cells (HEK293). Here, we test the hypothesis that tandem repeated 5′-UTR of a different LTR-retrotransposon could accommodate similar regulatory elements. The comparison of the 5′-UTR of some LTR-transposons allowed us to identify a shared motif of 13bp, called Transposable Element Redundant Motif (TERM). Surprisingly, we demonstrated, by Yeast One-Hybrid assay, that TERM interacts with the D. melanogaster ribosomal protein RpL22. The Drosophila RpL22 has additional Ala-, Lys- and Prorich sequences at the amino terminus, which resembles the carboxy-terminal portion of histone H1 and histone H5. For this reason, it has been hypothesized that RpL22 might have two functions, namely the role in organizing the ribosome, and a potential regulatory role involving DNA-binding similar to histone H1, which represses transcription in Drosophila. In this paper, we show, by two independent sets of experiments, that DmRpL22 is able to directly and specifically bind DNA of Drosophila melanogaster

Characterization of the Ribosomal Protein L22e Family in Drosophila melanogaster: Evidence for Functional Diversification of Duplicated Ribosomal Protein Genes

Gene duplication is a contributing factor to genome evolution in eukaryotes. With an additional copy, selective pressure is relieved, allowing for accumulation of genetic variation and possible development of new or altered functions. Ribosomal protein (Rp) genes are a common class of duplicated genes found throughout eukaryotes. Typically encoding highly similar or identical proteins at separate loci, duplicated Rps were originally thought to be redundant and to relieve the high demand for translation. However, recent reports in yeast have shown phenotypic differences between Rp paralogue knockouts, suggesting functional non-redundancy. Little effort has been devoted toward elucidating the function of Rp paralogues in eukaryotes other than in yeast. Furthermore, in yeast, paralogous Rps are typically highly identical, making studying gene function difficult without protein tagging. To explore whether duplicated Rp genes have redundant roles, we focused on the eukaryotic-specific RpL22e family in Drosophila melanogaster. The Drosophila RpL22e family consists of two members, the ancestral rpL22e and its duplicate rpL22e-like, which are 37% identical. Divergence is evident in the genomic sequence, codon usage, and protein sequence, but whether this results in novel functions has not been previously addressed and is the focus of this dissertation.It is widely known that the ancestral RpL22e is ubiquitous, but our data show that RpL22e-like expression is primarily restricted to the male germline and is a true ribosomal component. Further investigation shows that in testis tissue, RpL22e is primarily SUMOylated and phosphorylated. Only unmodified RpL22e co-sediments with the translation machinery in Drosophila S2 cells, leading to the interpretation that the majority of testis RpL22e is not part of the translation machinery and that paralogue functions are non-redundant. Immunohistochemical analysis further supports non-redundant paralogue roles, as RpL22e is primarily restricted to the nucleoplasm in the maturing meiotic germline; RpL22e-like is cytoplasmic in these cells. Additionally, there is an unequal requirement for RpL22e members in vivo, as only rpL22e is essential in the fly.Taking the data in this dissertation together, it is evident that the Drosophila RpL22e paralogues have diverged in function within the male germline. RpL22e assumes an additional and unique role compared to RpL22e-like

Chromosomal mapping reveals a dynamic organization of the histone genes in aphids (Hemiptera: Aphididae)

Despite their involvement in different processes, histone genes have been analysed in few insects. In order to improve the knowledge about this important gene family, genes coding for histones have been analysed in the aphid Acyrthosiphon pisum showing that at the amino acid level, aphid histones are highly conserved. In particular, data from A. pisum confirm that H1 is the most variable of the five histones, whereas histones H3 and H4 are highly conserved with the H3 almost identical from insects to vertebrates. A. pisumhistone genes are organized in a quintet with the H1 gene followed by H2A and H2B genes that are adjacent and transcribed in same directions, in the opposite strand in respect to the H1 gene. At the 3’ end of the histone cluster, genes H3 and H4 constitute an oppositely transcribed pair. The span of the aphid histone genes (more than 7 kb) is greater than the average length of the histone cluster till now reported in insects (about 5 kb). Furthermore, spacers that separate the aphid histone genes vary in length. The histone genes have been mapped in A. pisum and successively in the aphids Myzus persicae and Rhopalosiphum padi showing that they are present in a single large cluster located in an interstitial position of autosomes 1, differently from what reported in the Russian wheat aphid Diuraphis noxia,where histone genes have been localized in a telomere of the two X chromosomes suggesting a dynamic organization of this multigene family in aphids