A novel disrupter of telomere silencing 1-like (DOT1L) interaction is required for signal transducer and activator of transcription 1 (STAT1)-activated gene expression

Abstract

JAK-STAT-activated gene expression is both rapid and transient and requires dynamic post-translational modification of the chromatin template. Previously, we showed that following IFN-γ treatment, trimethylation of histone H3 at lysine 79 (H3K79me3) is rapidly and highly induced in the 5'-end of the STAT1-dependent gene interferon regulatory factor 1 (IRF1), but the role of this histone modification was unexplored. Here we report that DOT1L, the non-SET domain containing methyltransferase that modifies Lys-79, is localized across IRF1 in the uninduced state and is not further recruited by IFN-γ induction. RNAi-mediated depletion of DOT1L prevents the induction of H3K79me3 and lowers the transcription of IRF1 2-fold, as expected. Surprisingly, STAT1 binding to its DNA recognition element near the IRF1 promoter is diminished 2-fold in the DOT1L-depleted cell line. In vivo and in vitro protein interaction assays reveal a DOT1L-STAT1 interaction. Domain mapping identifies the middle region of DOT1L (amino acids 580-1183) as the STAT1 interaction domain. Overexpression of the DOT1L STAT1 interaction domain represses IRF1 transcription (2-fold) and interferes with STAT1 DNA binding at IRF1 and endogenous DOT1L histone methyltransferase activity. Collectively, our findings reveal a novel STAT1-DOT1L interaction that is required for the regulation JAK-STAT-inducible gene expression.

FIGURE 1.

Profile of H3K79me3 and DOT1L at the IRF1 gene. A and B, ChIP using antibodies to H3K79me3 (A) and DOT1L (B) in 2fTGH cells treated with IFN-γ for the indicated times or left untreated. qPCR, using primers spanning the IRF1 gene locus, quantified the precipitate yield reported as the percentage of input. IgG served as the negative control. C, graphic depiction of the IRF1 gene (∼9 kb), showing the location of the qPCR primers (black dashes) used in this study.

FIGURE 2.

RNAi-mediated depletion of DOT1L decreases IRF1 gene expression. A, DOT1L Western blot of whole cell extracts collected from 2fTGH cells stably expressing the pTRIPZ shRNAmir non-silencing (shRNA-NS) or shRNAmir-DOT1L (shRNA-DOT1L) vectors with or without IFN-γ. Dynamin served as a loading control. B and C, qRT-PCR to quantitate IRF1 mRNA and heteronuclear RNA (hnRNA) levels in the shRNAmir non-silencing and shRNAmir-DOT1L cell lines treated with IFN-γ for the times indicated or left untreated (Un). IRF1 was normalized to GAPDH, and expression is presented as -fold change relative to the uninduced, shRNAmir-NS condition. Error bars indicate S.E. (n = 3). Student's t test determined significance; **, p ≤ 0.01, *, p ≤ 0.05. D, IRF1 Western blot of whole cell extracts of shRNAmir non-silencing or shRNAmir-DOT1L cells treated with IFN-γ for the indicated times or left untreated. GAPDH served as a loading control. E, H3K79me3 Western blot of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines. Pan H3 served as a loading control. F, STAT1 and phospho-STAT1 Western blots of whole cell extracts collected from 2fTGH cells stably expressing the shRNAmir non-silencing or shRNAmir-DOT1L vectors. The ratio of phospho-STAT1 (pSTAT) to total STAT1 is the same in both cell lines. Western blot bands were quantified with ImageJ.

FIGURE 3.

RNA polymerase II and STAT1 localization to the IRF1 promoter is reduced in shRNAmir-DOT1L cells. A–H, ChIP of shRNAmir non-silencing (shRNA-NS) or shRNAmir-DOT1L (shRNA-DOT1L) cells treated with IFN-γ for 30 min (right panels) or left untreated (left panels). ChIP was performed with the antibodies indicated, and qPCR, using primers spanning the IRF1 gene locus, quantified the precipitate yield reported as the percentage of input. IgG served as the negative control. p ≤ 0.05 for panels A, B, D, F, and H for solid lines with black diamonds versus dotted lines with black diamonds.

FIGURE 4.

STAT1 co-immunoprecipitates with DOT1L. A–C, whole cell extracts prepared from 2fTGH (A), shRNAmir-DOT1L (shRNA-DOT1L) (B), or shRNAmir non-silencing (shRNA-NS) (C) cells were immunoprecipitated (IP) with α-DOT1L, α-STAT1, or IgG (negative control) and then immunoblotted (IB) with the indicated antibodies. A 5% input aliquot of the extracts was included for reference.

FIGURE 5.

Mapping of the DOT1L region that interacts with STAT1 in GST pulldown assays. A, graphic depiction of the DOT1L fragments and their relative STAT1 binding affinities. The presence (+) or absence (−) of an interaction is shown, with +, ++, and +++ indicating weak, modest, and strong interactions, respectively. N-term, N-terminal; C-term, C-terminal. B, GST pulldown assays using whole extracts prepared from 2fTGH cells transiently transfected with pcDNA3β FLAG-tagged DOT1L or FLAG-tagged DOT1L fragment vectors and then immunoblotted (IB) with α-FLAG. A 5% input aliquot of the extracts was included for reference.

FIGURE 6.

Overexpression of the SID of DOT1L represses IRF1 gene expression and alters STAT1 binding. A, qRT-PCR measured IRF1 mRNA expression in 2fTGH, shRNAmir non-silencing (shRNA-NS), and shRNAmir-DOT1L (shRNA-DOT1L) cell lines that were transiently transfected with pcDNA3.0 DOT1L SID, ΔC, ΔN, or empty vector. IFN-γ induction was for 30 min. IRF1 was normalized to GAPDH, and expression is presented as -fold change relative to the uninduced, shRNAmir-NS cell line transfected with empty vector. Error bars indicate S.E. (n = 2). Student's t test determined significance, **, p ≤ 0.01, *, p ≤ 0.05. B, Western blots of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines that were transiently transfected as in panel A. PanH3 served as a loading control. C–H, ChIP, using the indicated antibodies in shRNAmir-DOT1L (right panels) or shRNAmir non-silencing (left panels) cells transiently transfected with pcDNA3.0 DOTL1 SID, ΔC, ΔN, or empty vector and treated with IFN-γ for 30 min. p ≤ 0.05 for black lines with an X or gray squares versus black lines with black squares or white squares in panel C; black lines with gray squares versus others in panel D; black lines with an X or white squares versus black lines with black squares or gray squares in panel E; black lines with white squares versus others in panel F.