Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors.

We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34(+) and CD34(+)38(-) clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34(+) and CD34(+)38(-) cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34(+), CD34(+)38(-) LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors.

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

Yeast histone H3 and H4 amino termini are important for nucleosome assembly in vivo and in vitro: redundant and position-independent functions in assembly but not in gene regulation.

The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core. Here we examine the importance of the amino termini and their position in the nucleosome with regard to both nucleosome assembly and gene regulation. Despite previous conclusions based on nonphysiological nucleosome reconstitution experiments, we find that the histone amino termini are important for nucleosome assembly in vivo and in vitro. Deletion of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts. The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability. Moreover, the tails need not be attached to their native carboxy-terminal core. Their exchange re-establishes both cellular viability and nucleosome assembly. In contrast, the regulation of GAL1 and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini.

Yeast histone H4 and H3 N-termini have different effects on the chromatin structure of the GAL1 promoter.

Deletion of the histone H4 N-terminal residues 4-23 decreases activation of the GAL1 promoter as much as 20-fold, while deletion of histone H3 N-terminal residues 4-15 hyperactivates GAL1 approximately 3-fold. In an attempt to understand the mechanisms by which these two different events take place, we have examined the effects of the H4 and H3 lesions on GAL1 chromatin structure. The bacterial dam methylase, which methylates adenine residues of GATC sequences, was used as an in vivo probe for chromatin structure and both indirect end-labeling and ligation mediated PCR (LMPCR) analysis of micrococcal nuclease digestions were used to analyze chromatin in isolated nuclei. We show that while deletions of the H4 and H3 N-termini have similar effects on dam methylase access in the GAL1 coding region, the H4 N-terminal deletion uniquely alters dam access at a region near the TATA element. This change is independent of the transcriptional state of GAL1. In addition, LMPCR analysis of micrococcal nuclease digests of yeast nuclei demonstrate that H4 N-terminal deletion has unique effects on nuclease accessibility in the nucleosomal region upstream of the TATA element. Our results are consistent with the H4 N-terminus mediating activation of GAL1 through its effect on the proximal promoter region near the TATA box. These data also suggest that the H3 N-terminus affects GAL1 hyperactivation through a different promoter element than that affected by H4.

The regulation of euchromatin and heterochromatin by histones in yeast.

Yeast chromosomes may lack the linker histone H1 (normally required to compact 10 nm beads-on-a-string fiber into the 30 nm fiber) and there is no cytological evidence for higher order fiber structure but they do contain regions which correspond to euchromatin and heterochromatin of higher eukaryotes. Both euchromatin and heterochromatin contain nucleosomal particles (composed of two molecules each of H2A, H2B, H3 and H4), however histones have been shown to regulate genes in these regions in quite different ways. The mechanisms by which such regulation occurs are the topic of this paper.

Identification of a non-basic domain in the histone H4 N-terminus required for repression of the yeast silent mating loci.

We have shown previously that a stretch of four charged residues (16-19) at the histone H4 N-terminus is involved in repression of the yeast silent mating loci. One of these residues, Lys16, is a site for acetylation, which may prevent repression of the silent mating loci. In this paper we ask whether other sequences in histone H4, possibly in conjunction with H3 residues, are required for repression. We find that even in combination, the other seven acetylatable lysines in H3 and H4 do not function in repression. In contrast, we have found that an adjacent relatively uncharged domain (residues 21-29) is required for repression and that single amino acid insertions and deletions in this region are extremely detrimental. We propose that the basic and non-basic domains together form a DNA (or protein) induced amphipathic alpha-helix required in the formation of a repressive chromatin structure.