Fig 2: LIF and BMP4 Directly Regulate Transcription of Lif-r and Gp130(A) Time-course qRT-PCR spanning 48 hr of Lif-r along with pluripotency markers Oct4 and Nanog after addition of either BMP4 alone or LIF and BMP4. Controls were addition of nothing (AF) or addition of LIF for 48 hr.(B) Transcription of Oct4, Nanog, and Lif-r was assayed in the presence of LIF and BMP4 and cycloheximide (chx, 30ng/ml) for 3 hr.(C) Chromatin immunoprecipitation (ChIP) was performed on three regions upstream of the Lif-r gene, Lif-r-enh, Lif-r-pro1, and Lif-r-pro2. These regions were determined based on data from Chen et al. (Lif-r-enh) and Yamashita et al. (Lif-r-pro1 and 2) (Chen et al., 2008; Yamashita et al., 2010). The amplified regions are denoted by the red box. Gels were run after ChIP-PCR on DNA samples collected from total cell input (Input), a negative control with just addition of rabbit immunoglobulin G (IgG), and pull-downs of total STAT3, P300, and SMAD1. Quantification of band intensities is shown below gel images.(D) Approximately 2 kb regions flanking the binding sites (yellow boxes) were cloned upstream of a luciferase reporter, assayed for activity and relative activity quantified.Error bars represent SD n = 3 technical replicates for (A) and (B), SE n = 4 (two replicates per two independent experiments) for (D). *p < 0.05, one sample two-tailed t test.

Fig 3: Small molecule inhibition of p300 downregulates ACE2 and DPP4 expression and blocks viral infection.(A-B) Vero E6 cells were treated with 1.25 µM GSK-J1 (A) or A485 (B) for the indicated times, ACE2 mRNA and protein levels were measured by RT-qPCR and immunoblot, respectively. (C-D) Vero E6 cells were pretreated with GSK-J1 (C) or A485 (D) inhibitors for 2 days and then infected with SARS-CoV-2 at an MOI of 0.2. Cell viability was measured at 3 dpi. (E) Vero E6 cells were pretreated with Comp12 for 2 days and then infected with the indicated SARS-CoV-2 variants at an MOI of 0.2. Cell viability was measured at 3 dpi. (F) Primary human bronchial epithelial cells (HBECs) were treated with A485 for 3 days and then measured the ACE2 and DPP4 expression by qPCR. (G-H) HBECs were pretreated A485 for 3 days then infected with SARS-CoV-2 and MERS at an MOI of 0.5 for 2 dpi. Virus replication was measured by qPCR (G) and plaque assays (H). Dash line, limit of detection. (I) Influenza virus replication at 1 dpi by qPCR in HBECs pretreated with A485 for 3 days. (J) Human intestinal enteroids (HIEs) were treated with 2.5 µM A485 for 3 days then measured the ACE2 and DPP4 expression by qPCR. (K) HIEs were pretreated A485 for 3 days then infected with SARS-CoV-2 and MERS at 105 PFU/ml for 2 dpi. Virus replication was measured and plaque assays. Dash line, limit of detection. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. Shown are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: The KDM6A-KMT2D-p300 axis is essential for murine coronavirus infection.(A-B) Kdm6a and Kmt2d KO BV2 cells were infected with mouse hepatitis virus strain A59 (MHV-A59) (A) and MHV-3 (B) at 0.05 and 5 MOI for 24 hpi and 48 hpi. (C) Kdm6a and Kmt2d KO BV2 cells were infected with VSV peudovirus (VSVpp): VSV-G and MHV-A59-S. Luciferase relative to the VSVpp-VSV-G control was measured 1 dpi. (D) Ceacam1 expression in Kdm6a and Kmt2d KO BV2 cells. (E) BV2 cells were pretreated with A485 at 1 µM and 5 mM for 2 days and then infected with MHV-A59 at a MOI of 0.05. Virus production was measured by plaque assays. (F) BV2 cells were pretreated with A485 for 2 days and then infected with VSV peudovirus (VSVpp): VSV-G and MHV-A59-S. Luciferase relative to the VSVpp-VSV-G control was measured 1 dpi. (G) Ceacam1 expression in A485 treated BV2 cells. Dash line, limit of detection. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. Shown are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: KDM6A recruits KMT2D and p300 through the TPR domain for receptor expression and viral entry.(A-B) ACE2 and DPP4 expression in KMT2D and EP300 polyclonal KO Huh7.5 cells at mRNA level (A) and protein level (B). (C) Co-immunoprecipitation assays in KDM6A KO Vero cells reconstituted with WT KDM6A, ?TPR or empty vector (EV). (D) Co-immunoprecipitation assays in KMT2D polyclonal KO Huh7.5 cells. (E) ChIP-qPCR assays were performed using KMT2D and p300 antibody in WT and KDM6A KO Vero E6 cells to detect the binding sites in ACE2 proximal and distal enhancer regions. (F) KMT2D and EP300 polyclonal KO cells were infected with icSARS-CoV-2-mNG at an MOI of 1. Infected cells were imaged via fluorescence microscopy (left) and mNeonGreen expressing cell frequency was measured 2 dpi (right). Scale bar: 300 µm. (G) KMT2D and EP300 polyclonal Huh7.5 cells were infected with SARS-CoV-2 (left), HKU5-SARS-CoV-S (middle) and MERS-CoV (right) at an MOI of 0.2. Cell viability relative to a mock infected control was measured 3 dpi with CellTiter Glo. (H) KMT2D and EP300 polyclonal cells were infected with VSV pseudovirus (VSVpp): VSV-G, SARS-CoV-2-S (left), SARS-CoV-S (middle), and MERS-CoV-S (right). Luciferase relative to the VSVpp-VSV-G control was measured 1 dpi. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. Shown are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.