DOI: 10.5061/dryad.9zw3r22n8
title: A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency

description: 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.
description: Immunofluorescence microscopy images used in the manuscript for
 quantitation
description: # Research data 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.