Enrichment for histone H3 lysine 9 methylation at Alu repeats in human cells

The aim of this study was to identify in human cells common targets of histone H3 lysine 9 (H3-Lys9) methylation, a modification that is generally associated with gene silencing. After chromatin immunoprecipitation using an H3-Lys9 methylated antibody, we cloned the recovered DNA and sequenced 47 independent clones. Of these, 38 clones (81%) contained repetitive elements, either short interspersed transposable element (SINE or Alu elements), long terminal repeat (LTR), long interspersed transposable element (LINE), or satellite region (ALR/Alpha) DNA, and three additional clones were near Alu elements. Further characterization of these repetitive elements revealed that 32 clones (68%) were Alu repeats, corresponding to both old Alu (23 clones) and young Alu (9 clones) subfamilies. Association of H3-Lys9 methylation was confirmed by chromatin immunoprecipitation-PCR using conserved Alu primers. In addition, we randomly selected 5 Alu repeats from the recovered clones and confirmed association with H3-Lys9 by PCR using primer sets flanking the Alu elements. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly decreased the level of H3-Lys9 methylation in the Alu elements, suggesting that H3-Lys9 methylation may be related to the suppression of Alu elements through DNA methylation. Thus H3-Lys9 methylation is enriched at human repetitive elements, particularly Alu elements, and may play a role in the suppression of recombination by these elements.     

The structure of HSAG-1, a middle repetitive genetic element which elicits a leukemia-related cellular surface antigen.

HSAG-1 is a cloned member of a heterogeneous middle repetitive family of genetic elements which is capable of eliciting a leukemia-related surface antigen detected with a monoclonal antibody after DNA transformation of mouse cells. HSAG-1 was originally isolated from a Chinese hamster-human leukemia hybrid cell gene library both by sib-selection for antigen producing activity and by hybridization with labelled human genomic human DNA. We show here that the human labelled site is at the right hand end of the insert, while the antigen-eliciting portion is included in a 1450 bp fragment at the left hand end of the insert. We also present the complete nucleotide sequence of the 3369 bp insert. The sequence contains 12 elements which bear a significant resemblance to accepted consensus sequences for Alu repetitive elements. The right hand end contains adjacent elements with close sequence similarity to portions of the human and hamster type I and type II Alu consensus sequences. All of the other Alu-related elements have diverged relative to the Alu consensus sequences by additions, long deletions and substitutions. The left hand portion of the insert which has the antigen-producing activity contains four of these diverged elements representing a relatively high proportion (26%) of the nucleotide sequence. The sequence is thus consistent with our previous observations of a repetitive family with biological function.     

Birth of a gene: locus of neuronal BC200 snmRNA in three prosimians and human BC200 pseudogenes as archives of change in the Anthropoidea lineage

The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.     

Structure and inducible regulation of the human c-erb B2/neu promoter

Overexpression of the p185 product of the c-erb B2/neu gene is correlated with cell transformation and tumorigenesis. To study expression of this gene, a 1548-base pair fragment (-1571 to -24 bp relative to the ATG initiator codon) of the human c-erb B2/neu 5'-noncoding region was isolated, sequenced, and analyzed with respect to basal and inducible activity using the luciferase expression vector pSVOAL delta 5'. This fragment contained an Alu repetitive element which was confirmed in two independent clones. Basal activity of the 1548-base pair fragment was equivalent to the epidermal growth factor receptor promoter region and 32 and 16% as active as the Herpes simplex thymidine kinase and Rous sarcoma virus promoters, respectively. Induction of luciferase activity was observed in response to epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, dibutyryl cAMP, and retinoic acid. Additive and synergistic responses with more than 30-fold increases were observed after treatment with combinations of inducing agents, indicating complex regulation of this gene. These results show that the promoter region of the c-erb B2/neu gene contains sequences that dictate regulatory responses to several environmental signals.     

A test of the master gene hypothesis for interspersed repetitive DNA sequences

Many families of interspersed repetitive DNA elements, including human Alu and LINE (Long Interspersed Element) elements, have been proposed to have accumulated through repeated copying from a single source locus: the "master gene." The extent to which a master gene model is applicable has implications for the origin, evolution, and function of such sequences. One repetitive element family for which a convincing case for a master gene has been made is the rodent ID (identifier) elements. Here we devise a new test of the master gene model and use it to show that mouse ID element sequences are not compatible with a strict master gene model. We suggest that a single master gene is rarely, if ever, likely to be responsible for the accumulation of any repeat family.     

Xenobiotic responsive element in the 5'-upstream region of the human P-450c gene.

The nucleotide sequence of the 5'-upstream region up to about -4.1 kb of the human P-450c gene was determined. Two kinds of repetitive sequences were located; one was the Alu sequence which was inserted at three positions (-3127 to -3038, -3017 to -2770, and -2167 to -1851), and the other was the SINE-R element located just upstream of the most distal Alu sequences. The region other than the two repeated sequences showed an overall similarity of 70% to that of the rat P-450c gene. Survey of XRE or its homologues, responsible for the inducible expression of the rat P-450c gene, revealed eight XRE core sequences in this region of the human P-450c gene. Three of them were carried in the Alu sequences. A fusion gene which was constructed by ligating the upstream region of the human P-450c gene to the chloramphenicol acetyltransferase (CAT) gene expressed the CAT activity in response to the inducer, methylcholanthrene, when transfected into Hepa-1 cells. Stepwise decrease in CAT activity in three regions was observed as the 5'-upstream sequence containing XRE motifs was removed. However, the XRE core sequence in the Alu sequences seemed inactive, because elimination of the three elements in the Alu sequences did not affect the expressed CAT activity. In accordance with this observation, competition experiments using gel mobility shift assay showed that XRE core sequences in the Alu sequences could not compete with the XRE sequence for the inducer-bound receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution.

The Alu repetitive family of short interspersed elements (SINEs) in primates can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. The older subfamilies are generally very abundant, while the younger subfamilies have fewer copies. Some of the youngest Alu elements are absent in the orthologous loci of nonhuman primates, indicative of recent retroposition events, the primary mode of SINE evolution. PCR analysis of one young Alu subfamily (Sb2) member found in the low-density lipoprotein receptor gene apparently revealed the presence of this element in the green monkey, orangutan, gorilla, and chimpanzee genomes, as well as the human genome. However, sequence analysis of these genomes revealed a highly mutated, older, primate-specific Alu element was present at this position in the nonhuman primates. Comparison of the flanking DNA sequences upstream of this Alu insertion corresponded to evolution expected for standard primate phylogeny, but comparison of the Alu repeat sequences revealed that the human element departed from this phylogeny. The change in the human sequence apparently occurred by a gene conversion event only within the Alu element itself, converting it from one of the oldest to one of the youngest Alu subfamilies. Although gene conversions of Alu elements are clearly very rare, this finding shows that such events can occur and contribute to specific cases of SINE subfamily evolution.