A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency
Data files
Dec 18, 2023 version files 1.58 GB
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IF_compilation_-_FINAL.zip
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README.md
Abstract
As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (∆N LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ∆N LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.
README: Research data
https://doi.org/10.5061/dryad.9zw3r22n8
The experimental procedures are to examine the effect of lymphoid enhancer factor 1 on Epstein-Barr virus reactivation. Knockdown and over expression approaches were used. Two cell types were used (EBV+NOK, epithelial cells) and Akata BX-1 (Burrito's lymphoma cell line).knockdown or over expression analysis were use to evaluate EBV reactivation using BZLF1 and E-AD viral lytic proteins as markers. In addition, LEF1 effects on differentiation were also investigated.
Description of the data and file structure
The files are raw immunofluorescence images used for quantitation in various assays. Images were captured on the Axis Observer Apothem microscope. The files are raw images used in the companion manuscript and for quantitation. The images are located in 1 main directory [IF compilation] that contains two subfolders [Images in Paper] and [Images used for quantitation]. These subfolders are subsequently divided into 5 folders organized by the figure that they relate to in the paper in a subfolder that indicates sample and then the target protein visualized.
The folder [Figure 4] contains images of EBV+NOK analyzing EBV reactivation in cells where LEF1 isoforms were siRNA depleted or over expressed. The readouts were the immediate early protein, Z (BZLF1), or the early protein, EA-D. The folders are include various treatment types that include vector controls: [vector for 3], [vector for 4]; LEF1 overexpression of isoform 3 and 4 [Isoform 3], [Isoform 4] and siRNA non target controls [NT] and isoform dependent knockdown [siLEF1 FL], [siLEF1 FL+ dN].
The folder [Figure 5] contains images for the analysis of epithelial differentiation following siRNA LEF1 depletion (NT, siLEF1 FL, siLEF1 FL+ dN). Cells were treated with calcium and serum to stimulate epithelial differentiation (induced). I1 refers to the parental EBV+NOK, induced is with calcium; uninduced is not treated. Differentiation markers analyzed where KLF4, BLIMP1/PRDM1, and involucrin.
The folder [Figure 8] contains images EBV+ Akata BXLF1 BL analyzing viral reactivation induced by B cell receptor cross linking at 2 and 4 hours post treatment with anti-human Ig. uninduced refers to no treatment. The viral protein marker Z (BZLF1) was used to measure EBV reactivation.
The folder [Spontaneous reactivation, S2_Fig] contains images to analyze if depletion of LEF1 isoforms spontaneously induces EBV reactivation. Each treatment group is indicated by its folder name (NT is non-target control, siLEF1 FL +dN, siLEF1 FL). BZLF1 was used as the marker for EBV reactivation.
The folder [transient isoform reactivation, S3-Fig] contains images to analyze the effect of LEF1 over expression on EBV reactivation. The folder subdivided in the treatment type (vector control or forced expression of LEF1 isoform 1 (iso1) or 4 (iso4)). Z (BZLF1) was used as a marker for EBV reactivation.
In total there were 6 images captured per sample type/treatment. The images are assembled from 3 separate channels pseudo-colored as follows: BLUE =Hoechst in channel 1; RED= channel 3 (546nm); GREEN=channel 4 647nm, and the overlay pf the 3 channels. In figures where LEF1 and LEF1 myc tag was measured, LEF1 is pseudo-colored in green measured in the (Channel 4).The figures in the manuscript indicate the pseudo-color used for each of the targets analyzed.
Organization of file folders:
Images in paper and Images for quantitation/Figure #/sample type/Target/ 4 TIF files
Sharing/Access information
Data was derived from the following sources:
- Zeiss AxioObserver Z1 inverted fluorescent microscope with Apotome and Zen software
Code/Software
Data was analyzed with Image J.
Methods
Immunofluorescence microscopy images used in the manuscript for quantitation