Revision as of 00:44, 30 October 2008

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Synthetic Chromatin Bit (Part II)

Why use Chromatin as a Tool for Synthetic Biology?

Transcriptional activators and repressors, the mainstays of current synthetic genetic circuits, work at the level of the promoter by affecting the recruitment of the basal transcription machinery. The eukaryotic cell, however, has a more potent mechanism to regulate gene expression: chromatin (gene silencing). Chromatin-based gene regulation has a number of interesting properties in vivo. We reasoned that it might be a powerful tool for synthetic biology. Therefore, we spent the summer developing a synthetic chromatin system, and demonstrating its unique features .

Analysis of our Data

Single-cell analysis was done using flow-cytometry. Example cells positive for GFP are seen in the microscope images on the lower right panel and their predicted population distribution is shown in the green curve in the ficticious graph in the left (below).

The Properties of Our Synthetic Chromatin Bit

We tested our synthetic chromatin bit in a number of ways.

1. Targeting of Sir2 leads to complete silencing of reporter

In the eukaryotic cell, heterochromatin blocks gene expression completely. We targeted the silencing machinery to a transgenic locus and monitored reporter expression.

RESULT 1:

After the addition of galactose (inducing LexA-Sir2 expression), we observed complete silencing of the GFP reporter. In this case, a medium constitutive promoter (Cyc1P) was used, but similar results were obtained for other promoters (e.g. Fig1 P, see below).

2. Dominant over Transcription Factors

Heterochromatin plays a primary role in the differentiation of the cells of higher eukaryotes, and therefore must be resistant to activation by transcription factors. We tested whether our synthetic chromatin bit, once closed (heterochromatin), could be activated. The GFP reporter in this case was driven by the pheromone-inducible Fig1 promoter.

RESULT 2:

Transcriptional ctivation of the reporter gene was completely blocked in cells that were grown in galactose to induce silencing. Indeed, even the basal activity of the Fig1 promoter (compare the red and blue traces)was blocked by silencing.

3. Regional Silencing

Unlike typical transcriptional activators or repressors, silencing should be promoter independent, and thus should function equally well when initiated from upstream or downstream of a gene (i.e. silencing should be bi-directional). We used a dual reporter construct to test this possibility in our synthetic system.

RESULT 3:

The upstream mCherry and downstream GFP reporter were both silenced completely, indicating that silencing spreads (and functions) bi-directionally.

4. Spread of Silencing

But how far does silencing spread? In S. cerevisiae, spreading from endogenous sites of initation at the telomere ends occurs to ~3 Kb. We tested the range of our synthetic system using a series of spacer constructs, where the LexA operators (initiation of silencing) were 250-3,000 bp downstream of the GFP reporter.

RESULT 4:

Silencing was robust to 2,000 bp. At 3,000 bp distance, the cells were bi-modal for GFP expression, indicating that the limit is between 2,000 and 3000 bp.

5. Binary

Unlike standard transcriptional repressors which yield graded, or moderately cooperative repression, we noticed a distinct lack of intermediate GFP expression levels in our silencing experiments. To determine how cooperative the transition between the ON and OFF state is, we assayed silencing over a wide range of steady-state LexA-Sir2-mCherry expression levels. To achieve graded output from the Gal-inducible promoter driving the LexA-Sir2-mCherry construct, we used a delta Gal2 strain (

RESULT 5:

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6. Memory

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RESULT 6:

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Higher-Order Systems (next)