IL-21 effects on newly EBV infected primary B-cell EBNA2 target gene CD23 expression.

(A) Immunoblot analysis of WCL from primary human B cells at 7 DPI, which were then mock treated or stimulated with IL-21 for six days. (B) Immunoblot analysis of WCL from primary B cells at 10 DPI, mock treated or treated with IL-15 or IL-21 for six days. (C) Plasma membrane CD23 abundances in primary human B-cell mock treated or treated with IL-21 (100 ng/ml) at Day 7 vs 18 post-infection by Akata EBV. IL-21 was refreshed every 2 days. (D) Mean ± SD CD23 abundances from n = 3 replicates of primary B-cells infected by Akata EBV in the absence or presence of IL-21, as in (C), ***p (TIFF)</p

STAT roles in IL-15 and IL-21 driven LMP1 and LMP2 promoter epigenetic remodeling.

(A-C) ChIP-qPCR analysis of LMP1 promoter H3K9me2 (A), H3K9me3 (B) or H3K27me3 (C) abundances from GM12878 expressing control or STAT3/5A/5B targeting sgRNAs, mock treated or treated with 100ng/ml IL-15 or IL-21 for six days. (D-F) ChIP-qPCR analysis of LMP2 promoter H3K9me2 (D), H3K9me3 (E) or H3K27me3 (F) abundances in GM12878 expressing control or STAT3/5A/5B targeting sgRNAs, mock treated or treated with IL-15 or IL-21 for six days. Mean ± SD input % of n = 3 replicates are shown, *p p (TIFF)</p

RNA-seq of Host Gene Expression.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.</div

STAT3 roles in LMP1 and LMP2 promoter IL-21 driven epigenetic remodeling in latency I B-cells.

(A-B) ChIP-qPCR analysis of LMP1 promoter H3K9me2 (A) or H3K9me3 and H3K27me3 (B) abundances from Mutu I expressing control or STAT3 targeting sgRNA, mock treated or treated with IL-21. (C-F) ChIP-qPCR analysis of LMP2 promoter H3K27Ac (C) or H2AK119Ub (D), H3K9me2 (E) or H3K9me3 and H3K27me3 (F) abundances in Mutu I expressing control or STAT3 targeting sgRNAs, mock treated or treated with IL-21. Cells were treated with 50 ng/ml IL-21 for one day. Mean ± SD input % of n = 3 replicates are shown, *p p (TIFF)</p

STAT3 and 5 role in GC cytokine mediated C promoter epigenetic remodeling.

(A) Schematic diagram of PROMO [59,60] predicted STAT binding sites on C promoter. (B-C) ChIP-qPCR analysis of STAT3 (B) or STAT5 (C) C promoter occupancy in GM12878 mock treated or treated with IL-15 or IL-21 for six days. (D-G) ChIP-qPCR analysis of Cp H3K27Ac (D), H2AK119Ub (E), H3K9me2 (F) and H3K9me3 (G) abundances in GM12878 expressing control versus STAT3/5A/5B targeting sgRNA, mock treated or treated with IL-15 or IL-21 for six days. All ChIP results are presented as % input mean ± SD from n = 3 replicates. Relative fold changes to Mock treated cells are labeled. *p p p < 0.001.</p

RNA-seq of EBV Gene Expression.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.</div

IL-15 and IL-21 remodeling of latency III gene expression in newly infected primary human B cells.

(A) Immunoblot analysis of WCL from primary human B cells at the indicated days post infection (DPI) by the Akata EBV strain. (B) Immunoblot analysis of WCL from primary human B cells at 7 DPI, which were then mock treated or treated with the indicated cytokines for six days. (C) Immunoblot analysis of WCL from primary B cells that were treated with DMSO or JAKi (200 ng/ml) for two days at 4, 7 or 10 DPI. GM12878 WCL was included as a control. (D) Primary human B-cell transformation assay characterizing effects of DMSO vs JAKi (200 ng/ml) treatment on primary human B-cell outgrowth following infection by Akata EBV. Fitted non-linear regression curves are presented as mean ± SD from n = 3 replicates, *p p < 0.01. Blots are representative of n = 3 replicates. Cytokines were used at 100 ng/ml.</p

Model of EBV latency promoter epigenetic remodeling by GC cytokine driven JAK/STAT signaling.

Cytokines secreted by germinal center FDC and Tfh cells trigger JAK/STAT signaling to drive STAT homodimer or heterodimer nuclear translocation. STAT dimers bind to the C promoter together with a co-repressor to increase levels of repressive 5-methylcytosine (5-mC), H3K9me2/3 and H2AK119Ub epigenetic marks, while decreasing activating H3K27Ac marks. C promoter inactivation decreases latency III EBNA expression. By contrast, STAT dimers bind to the LMP1 promoter together with a co-activator to drive epigenetic remodeling to increase levels of LMP1 promoter H3K27Ac and to decrease levels of H2AK119Ub, which supports LMP1 expression despite concurrently increased 5mC levels. By contrast, GC cytokines increase the abundances of LMP2 promoter repressive H2AK119Ub, H3K9me3 and 5mC marks to downmodulate LMP2 expression from levels observed in latency III. (BioRender was used to create the schematic models).</p

Reagents, Antibodies and Kits.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.</div

STAT 3 and 5 role in GC cytokine mediated latency III LMP1 promoter epigenetic remodeling.

(A) Schematic diagram of LMP1 promoter STAT binding sites, S1, S2 and S3 [58]. (B-C) Chromatin immunoprecipitation (ChIP) qPCR analysis of STAT3 (B) or STAT5 (C) LMP1 promoter occupancy in GM12878 mock treated or treated with IL-15 or IL-21 for six days. (D-E) ChIP-qPCR analysis of LMP1 promoter H3K27Ac (D) or H2AK119Ub (E) epigenetic mark abundances in GM12878 expressing control versus STAT3/5A/5B targeting sgRNA, mock treated or treated with IL-15 or IL-21 for six days. (F-G) ChIP-qPCR analysis of LMP2 promoter H3K27Ac (F) or H2AK119Ub (G) abundances in GM12878 expressing control versus STAT3/5A/5B targeting sgRNA, mock treated or treated with IL-15 or IL-21 for six days. (H-I) ChIP-qPCR analysis of LMP1 promoter H3K27Ac (H) or H2AK119Ub (I) abundances in Mutu I expressing control sgRNA versus STAT3 targeting sgRNA, mock treated or treated with IL-21 for one day. All ChIP results are presented as % input mean ± SD from n = 3 replicates. *p p p < 0.001.</p