Steven Spiker

Use of matrix attachment regions (MARs) to minimize transgene silencing.

Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix in vitro. It is possible, although unproven, that they also mediate binding of chromatin to the nuclear matrix in vivo and alter the topology of the genome in interphase nuclei. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable expression in transgenic plants or cell lines, most likely by minimizing gene silencing. Our review explores current data and presents several plausible models to explain MAR effects on transgene expression.

Scaffold attachment regions increase reporter gene expression in stably transformed plant cells.

The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.

Nuclear Matrix Attachment Regions and Transgene Expression in Plants

DNA sequences called matrix attachment regions (MARs) or scaffold attachment regions (SARs) have recently attracted much attention because of their perceived capacity to increase levels of transgene expression and reduce transformant-to-transformant variation of transgene expression in both plants and animals. Work with these sequences is in its early stages and data that seem to be contradictory have been presented. We do not intend to resolve these controversies here (this will be accomplished by further research). Rather, we will discuss the hypothesized role of MARs in chromatin structure, how MARs are isolated and characterized, what effects MARs have had on the expression of transgenes and the models that have been evoked to explain those effects.

High mobility group chromosomal proteins bind to AT-rich tracts flanking plant genes.

AT-rich sequences in the 5′ flanking regions of several plant genes have been shown to bind nuclear proteins, but the nature of these proteins has remained largely unknown. We report here that certain plant high mobility group (HMG) chromosomal proteins can interact specifically (in the presence of excess non-specific competitor) with AT-rich sequences located upstream of the pea ferredoxin 1 gene (Fed-1) and a member of the wheat Em gene family. Binding was observed with highly purified preparations of HMGa or HMGb, but not with HMGc or HMGd. HMG-DNA complexes were similar to one of the two types of Fed-1 complexes we observed previously using pea nuclear extracts [7]. HMG binding to the Fed-1 DNA was localized to a region containing AT-rich sequences; very similar sequences are present 5′ to Em and several other plants genes. Such sequences have been shown to bind unidentified nuclear proteins in a number of these systems. Binding experiments with a synthetic oligo (dA) • oligo (dT) probe and competition experiments with synthetic DNA polymers suggest that HMG binding may depend upon structural features of AT-rich DNA rather than being sequence-specific. We discuss the implications of these findings and suggest a role for HMG binding which is consistent with previous evidence linking HMGs with transcriptionally competent chromatin.

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

Many studies in both plant and animal systems have shown that matrix attachment regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MAR-containing vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression.

An auxin-inducible gene from loblolly pine (Pinus taeda L.) is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein.

We have isolated a gene from loblolly pine, 5NG4, that is highly and specifically induced by auxin in juvenile loblolly pine shoots prior to adventitious root formation, but substantially down-regulated in physiologically mature shoots that are adventitious rooting incompetent. 5NG4 was highly auxin-induced in roots, stems and hypocotyls, organs that can form either lateral or adventitious roots following an auxin treatment, but was not induced to the same level in needles and cotyledons, organs that do not form roots. The deduced amino acid sequence shows homology to the MtN21 nodulin gene from Medicago truncatula. The expression pattern of 5NG4 and its homology to a protein from Medicago involved in a root-related process suggest a possible role for this gene in adventitious root formation. Homology searches also identified similar proteins in Arabidopsis thaliana and Oryza sativa. High conservation across these evolutionarily distant species suggests essential functions in plant growth and development. A 38-member family of genes homologous to 5NG4 was identified in the A. thaliana genome. The physiological significance of this redundancy is most likely associated with functional divergence and/or expression specificity of the different family members. The exact biochemical function of the gene is still unknown, but sequence and structure predictions and 5NG4::GFP fusion protein localizations indicate it is a transmembrane protein with a possible transport function.

Chromosomal proteins of Arabidopsis thaliana.

In plants with large genomes, each of the classes of the histones (H1, H2A, H2B, H3 and H4) are not unique polypeptides, but rather families of closely related proteins that are called histone variants. The small genome and preponderance of single-copy DNA in Arabidopsis thaliana has led us to ask if this plant has such families of histone variants. We have thus isolated histones from Arabidopsis and analyzed them on four polyacrylamide gel electrophoretic systems: an SDS system; an acetic acid-urea system; a Triton transverse gradient system; and a two-dimensional system combining SDS and Triton-acetic acid-urea systems. This approach has allowed us to identify all four of the nucleosomal core histones in Arabidopsis and to establish the existence of a set of H2A and H2B variants. Arabidopsis has at least four H2A variants and three H2B variants of distinct molecular weights as assessed by electrophoretic mobility on SDS-polyacrylamide gels. Thus, Arabidopsis displays a diversity in these histones similar to the diversity displayed by plants with larger genomes such as wheat.The high mobility group (HMG) non-histone chromatin proteins have attracted considerable attention because of the evidence implicating them as structural proteins of transcriptionally active chromatin. We have isolated a group of non-histone chromatin proteins from Arabidopsis that meet the operational criteria to be classed as HMG proteins and that cross-react with antisera to HMG proteins of wheat.

Elevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3' MAR.

We have analyzed effects of a matrix attachment region (MAR) from the tobacco RB7 gene on transgene expression from six different promoters in stably transformed tobacco cell cultures. The presence of MARs flanking the transgene increased expression of constructs based on the constitutive CaMV 35S, NOS, and OCS promoters. Expression from an induced heat shock promoter was also increased and MARs did not cause expression in the absence of heat shock. There was also no effect of MARs on the pea ferredoxin promoter, which is not normally expressed in this cell line. Importantly, most transgenes flanked by RB7 MAR elements showed a large reduction in the number of low expressing GUS transformants relative to control constructs without MARs.

Chromatin Structure And Gene Regulation In Higher Plants

Publisher Summary Chromatin is the material of which chromosomes are composed. When chromatin is removed from the cell, fixed, or spread in order to study it biochemically or microscopically, undoubtedly some of its inherent properties are altered. The exact composition of chromatin then depends not only upon the organism and cell type from which it is isolated but also upon the method of isolation. In general terms, chromatin consists mainly of about equal parts by weight of DNA and a group of low-molecular weight basic proteins, the histones, and varying proportions of a diverse group of other proteins that for convenience are called “nonhistone chromatin proteins.” Furthermore, the studies suggest that there is more than one type of active chromatin. Active chromatin is based on higher order structure and on the structure of individual nucleosomes. In addition, there are variations of the active structure within these two general categories, and upon occasion changes of chromatin structure from inactive to active involve both higher order changes and changes at the nucleosome level.