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. 2014 Jun;42(11):7436-49.
doi: 10.1093/nar/gku329. Epub 2014 May 3.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

Orlando de Lange  1 , Christina Wolf  1 , Jörn Dietze  1 , Janett Elsaesser  1 , Robert Morbitzer  1 , Thomas Lahaye  2
Affiliations

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

Orlando de Lange et al. Nucleic Acids Res. 2014 Jun.

Abstract

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

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Figures

Figure 1.
Figure 1.
Sequence-based comparison of TALE-like proteins. (A) Comparison of TALE (AvrBs3) and Bat architecture. The lengths of all domains are drawn to the indicated scale, except the dashes representing core repeats. TALE domains are shown in blue and Bat domains in purple. Rectangles indicate the N-terminal non-repetitive domain of each while a triangle indicates the non-repetitive C-terminal domain of TALEs including the transcriptional AD. Ovals represent core repeats, hexagons represent cryptic repeats (repeat number is indicated above). (B) Alignment of Bat1 core repeats, generated with Clustal Omega and Boxshade. Repeats are shown in order of appearance in the polypeptide. Repeat numbers are given on the left and positions within the repeat, including the RVD (indicated by an orange bar) above. (C) A consensus repeat generated from this alignment is compared to similarly generated consensus repeats from Bat2, Bat3, Brg11 (RipTAL) and AvrBs3 (TALE). From these a set of 10 hyper-conserved residues termed the consensus TALE-like repeat (CTR) was generated. The RVD positions are excluded from this. Repeat residues previously identified as involved in stabilising intra-molecular interactions from structural studies in TALEs (4) are highlighted with red lettering in the AvrBs3 consensus repeat, while the residues forming the first and second alpha helices (4) are underlined.
Figure 2.
Figure 2.
In vitro interaction studies of Bat proteins with predicted DNA targets. (A) Electrophoretic mobility shift assays were carried out for Bat1, 2 and 3 using 5’Cy5 labelled double-stranded DNA, bearing target sequences deduced from the TALE code. Each protein (100 nM) was tested against each target DNA (10 nM). Cy5 fluorescence was visualized after running through a native polyacrylamide gel. A shifted band, running slower on the gel, indicates the protein–DNA complex. (B) The interaction between Bat1 and its target (BEBat1) was quantified using microscale thermophoresis. The fluorescence ratio over the thermophoretic jump is shown on the y-axis against DNA concentration. Standard deviation for four repetitions is indicated. Measurements were made with 40% LED and 20% laser power. The dark grey line indicates the Kd fit. (C) This was repeated for BE Bat1 derivatives bearing A (grey bar), C (filled stripes) or G (spotted) at the zero position. The Kd was calculated in each case and is shown compared to that with BEBat1 (T0, empty bar).
Figure 3.
Figure 3.
A Bat1 derived transcriptional activator (acBat1) is functional in a human cell reporter assay. (A) Schematic drawing showing the domain composition of acBat1. NLSs (yellow bars), a 3xFLAG tag (red crescent line) and a VP64 AD (green triangle) were fused onto Bat1 (purple) via flexible linkers (orange). This was introduced into HEK293T cells via transfection alongside a DNA reporter (grey) bearing BE Bat1 (purple) upstream of a dsEGFP coding sequence (green). Transcriptional activation of the reporter (green arrow) follows binding to BE Bat1, leading to production of dsEGFP protein (green star). acBat1 is detected via the 3xFLAG epitope with use of an Alexa Fluor 594 labelled secondary antibody. (B) Alexa Fluor 594, dsEGFP and DAPI fluorescence are shown for transfected cells. acBat1 is compared to derivatives lacking AD (acBat1ΔAD) or NLSs (acBat1ΔNLSs) and to a dTALE created with the same NLSs and AD and with the same core repeat number and RVD composition as Bat1 (dTALEBat1mimic). The scale bar indicates 10 μm. (C) FACS analysis was used to quantify dsEGFP fluorescence for transfected cells expressing acBat1, ΔAD derivative or dTALEBat1mimic as well as cells transfected with the reporter only. dsEGFP values are shown for the whole population (curves) as well as boxplots showing fold changes in fluorescence intensity compared to the reporter control. Boxplot whiskers represent the 2.5% and 97.5% data limits. Median values are written next to or inside each box plot and shown graphically with thick black lines.
Figure 4.
Figure 4.
In vitro assessment of Bat1-FokI nuclease activity. Bat1- and TALE-FokI fusion proteins were expressed in vitro and equal volumes of transcription-translation product were incubated with a purified PCR product bearing two copies of BEBat1 in reverse complement, separated by 5–19 base pairs. A target with a control sequence replacing the Bat1 target boxes was also used. After 3 h incubation at 37°C DNA was purified from the nuclease reactions and run on a 2% agarose gel to discriminate cleaved and uncleaved DNA (indicated with arrows and illustrations on left side). Cleavage efficacy was calculated from the ratio of cleaved to uncleaved DNA band intensities in each lane with ImageJ (14). Full and striped bars indicate activities of the Bat1-FokI and TALEN constructs respectively. ND = none detected.
Figure 5.
Figure 5.
Functional analysis of acBat1 repeat truncations. Tests were carried out as described (Figure 3). Flow cytometry measurements of dsEGFP fluorescence are displayed as population distributions (top) or box plots (centre). Distinct colour codes are used throughout the whole figure and correspond to indicated constructs. Boxplots show fold changes in fluorescence intensity compared to the reporter control with whiskers representing the 2.5% and 97.5% data limits. Median values are written next to or inside each box plot and shown graphically as thick black lines. Cartoon representations of the tested truncations are shown below. Dashed lines with scissors indicate fixed (black) and variable (coloured) truncation points. Bat repeats and fused domains of acBat1 are represented as in Figure 3A. (A) Within the repeats grey or purple indicate truncated or retained regions, respectively. (B) N- (ΔNTD) or C- (ΔCTD) terminal truncations were tested. NND is the short non-repetitive N-terminal domain at the N-terminus of Bat1.
Figure 6.
Figure 6.
Functional analysis of designer (d)Bat constructs generated by RVD (A) or repeat switch (B). dBats were tested using flow cytometry with a transcriptional activation reporter as described (Figure 3). dsEGFP fluorescence values are displayed as population distributions (top) or boxplots (centre). dsEGFP values are normalized to the reporter only control (Supplementary Figure S13), which was BEBat1 for all constructs except RVD switch 1 and 2 (Supplementary Figure S6). Boxplots show fold changes in fluorescence intensity compared to the reporter control with whiskers representing the 2.5% and 97.5% data limits. Median values are written next to or inside each box plot and shown graphically as thick black lines. dBat design is outlined below in each case. Coloured boxes indicate the repeats (ovals) modified in a given dBat. In the case of the RVD switch (A) modified repeats are highlighted with darker grey. RVDs are shown and colour coded by type. Arrows indicate the rearrangement of RVDs between repeats. In the case of the repeat switch (B) repeats are coloured to indicate that each has a unique set of non-RVD residues. Arrows indicate movement of whole repeats within the array.
Figure 7.
Figure 7.
Functional analysis of designer (d)Bat constructs targeting the human SOX2 promoter. dBats were tested using flow cytometry with a transcriptional activation reporter as described (Figure 3). Population curves for dsGFP fluorescence are shown (top) as well as boxplots of fluorescence intensities (bottom) compared to the reporter control (logarithmic scale). Boxplots show fold changes in fluorescence intensity compared to the reporter control with whiskers representing the 2.5% and 97.5% data limits. Median values are written next to each box plot and shown graphically as thick black lines. Two dBats, designed based on the RVD (dBatSOX2 RVD switch) or repeat switch (dBatSOX2 repeat switch), and an equivalent dTALE were tested.
Figure 8.
Figure 8.
Functional analysis of Bat1 repeats within the context of a TALE repeat array. Trimers of identical Bat1 repeats or TALE repeats with the same RVDs as the Bat1 repeats were embedded into the repeat domain of the 17-repeat TALE AvrBs3 that targets the pepper Bs3 promoter (Bs3p). Repeats 5–7 (3xRVD NI in AvrBs3) where replaced either by TALE repeat trimers with the RVDs NN or NG or by trimers of Bat1 repeats 2, 6, 8 and 17. This is shown in cartoon form with dTALE regions shown in light grey with the trimer of Bat1 repeats or dTALE repeats shown as white ovals. The grey rectangle and triangle indicate the native N- and C-terminal regions of AvrBs3, respectively. RVDs are given in each case and the matching bases in the target box underneath. The resulting chimeras (striped bars) were tested for their ability to activate a Bs3p derivative bearing the matching binding site upstream of a uidA (GUS) reporter gene and compared to non-chimeric dTALEs (filled bars) with the same RVDs. Dashed lines separate groups of constructs all with the same RVDs and tested against the same reporter. Barred lines indicate standard deviation. Two-tailed t-tests were used to compare chimeric and non-chimeric dTALEs for each reporter. A double asterisk indicates a P-value of below 0.02 and n.s. indicates a P-value of above 0.05.
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