21ENGBIO - Engineering Nucleosome Positioning in Plants

Plant biotechnology has been enormously successful with biotech crops grown in 29 countries worldwide and associated with improved welfare of smallholder farmers and reduced use of pesticides. In addition, the low cost of plant cultivation together with the abundance and diversity of natural products that they accumulate has led to their development as photosynthetic platforms for producing valuable molecules used in health and industry. For both crop improvement and to improve the yields of useful molecules, we require a detailed understanding of how regulatory function is encoded into DNA sequences together with methods to control gene expression.

To fit their chromosomes into the nucleus, the DNA of eukaryotes is packaged into chromatin. The basic structural unit of chromatin is the nucleosome, consisting of a segment of DNA wound around eight histone proteins. The position and stability of nucleosomes can dictate the accessibility of DNA to proteins and other factors and, therefore, have important roles in DNA replication and repair as well as in gene expression. There is evidence that DNA sequences can be coded to influence the position and stability of nucleosomes. Demonstrations in yeast have shown that recoding DNA to influence nucleosome position or stability can be used to modulate gene expression but similar work has not been done in plants.

In preliminary studies, we found that plant regulatory sequences designed to specify fragile nucleosomes and increase the accessibility of the DNA gave increased expression. In this proposal, we will investigate this further with the aim of obtaining direct evidence that nucleosome positioning can be used to tune the level of plant synthetic promoters. Previously, we and others have also found that expression levels from synthetic genes are often significantly affected by the expression of closely located genes. Here, we will also investigate the use of nucleosome disfavouring sequences to mitigate these undesired context effects.

Many applications of plant biotechnology, including crop improvement and the use of plant cells as photosynthetic platforms for producing valuable molecules, require a detailed understanding of how regulatory function is encoded into DNA sequences together with methods to control or modulate the expression of both inserted and endogenous genes. To fit their chromosomes into the nucleus, eukaryotic DNA is wrapped around octamers of proteins known as histones to form structures called nucleosomes. In addition to DNA packaging, nucleosomes play roles in DNA replication, repair and gene expression. The position and stability of nucleosomes can dictate the accessibility of DNA to transcription factors and transcriptional machinery. There is evidence that DNA sequences can be coded to influence the position and stability of nucleosomes and demonstrations in yeast have shown that recoding DNA to influence nucleosome position or stability can be used to modulate gene expression. However, similar work has not been done in plants. In preliminary studies, we found that plant regulatory sequences designed to specify fragile nucleosomes and increase the accessibility of the DNA gave increased expression. In this proposal, we will investigate this further by testing a wider range of synthetic sequences and, subsequently, using MNase sequencing to determine the position and stability of nucleosomes with the aim of obtaining direct evidence that nucleosome positioning can be used to tune the level of plant synthetic promoters. In addition, we will investigate the use of nucleosome disfavouring sequences to prevent undesired context effects within plant genetic circuits by inserting such sequences between synthetic genes that we have previously shown to affect each other's expression levels. The findings will assist our work to develop plants as biomanufacturing platforms and to engineer plant regulatory networks that coordinate responses to nutrients.