Figure 6.
Enforced Notch signaling in CLL cells induces increased expression of cell cycle associated genes through direct and indirect epigenetic mechanisms. Splenic CD19+B220− CLL cells were sorted from 3- to 5-month-old IgH.TEμ;control (CTRL) and IgH.TEμ;RNICΔ/+ mb1-Cre (NIC) stage-matched littermates and analyzed for chromatin accessibility (ATAC-seq) and genome-wide binding of RBP-J and H3K27ac (ChIP-seq). (A) Venn diagram representing regions significantly (false discovery rate < 0.05) changed between CTRL and NIC for peaks called in ATAC-seq (fold > 1 for NIC and > −1 for CTRL), ChIP-seq for α-RBP-J and α-H3K27ac. (B) Binned orientation and distance to a transcription start site (TSS) for peaks called specifically in NIC, but not in CTRL. Distance to TSS is depicted in kilobases (kb). (C) GSEA for top Hallmark gene sets (MSigDB) affected by significant peaks (false discovery rate < 0.05) associated to the most proximal gene specifically in NIC and with regulated expression induced by enhanced Notch signaling (adjusted P value < 0.05). Panels from left to right depict Hallmark gene sets from ATAC-seq analysis, ChIP-seq for RBP-J, and ChIP-seq for H3K27ac. (D) Proposed mechanism of direct regulation by NIC at the proximal promoter regions of HeyL, Hes1, and Mybl2. Right panels represent genomic regions of regulated genes from Integrative Genomics Viewer (version 2.7.2) with peaks called from α-RBP-J ChIP-seq in CTRL and NIC, then α -H3K27ac ChIP-seq and ATAC-seq. Gene structure is shown below the tracks. (E) Proposed mechanism of indirect regulation by NIC at the proximal promoter region of cell-cycle gene Ccnb2 (left). Center panel represents genomic region of regulated gene from IGV with peaks called from α-RBP-J ChIP-seq in CTRL and NIC, then α-H3K27ac ChIP-seq and ATAC-seq. Gene structure is shown below the tracks. Gene expression from RNA-seq analysis represented as transcript per kilobase million (TPM) in CTRL (n = 7) and NIC (n = 6) is shown on the right. P value was calculated using unpaired Student t test: **P < .01. (F) Schematic depicts superphysiological NIC indirectly suppressing Foxp2 promoter by epigenetic mechanisms resulting in loss of Cdk1 repression. Bottom left panel represents IGV tracks of epigenetically regulated Foxp2 promoter, with peaks called in α-RBP-J ChIP-seq, α-H3K27ac ChIP-seq, and ATAC-seq shown for CTRL and NIC. Centered panel depicts a zoomed view of ATAC-seq footprinting analysis (TOBIAS version 11) upstream from Cdk1 promoter (Cdk1 gene structure is schematically indicated on the top left of the zoomed footprint). Tracks at the Foxp2 binding motif show the corrected ATAC-seq signal for CTRL and NIC, translated by TOBIAS into CTRL and NIC footprint score (CTRL bound site with score 1.6, NIC unbound site with score 0.8). Right panels show gene expression from RNA-seq for Foxp2 and Cdk1 expression between CTRL (n = 6) and NIC (n = 7) CLL cells. Total Cdk1 protein level was assessed by western blot analysis on ex vivo CLL cells from IgH.TEμ:control (n = 4) and IgH.TEμ;RNICΔ/+ mb1-Cre (n = 4) mice with antibodies α-Cdk1 (35 kDa) and α-β-Actin (42 kDa). P values were calculated using paired Student t test: **P < .01; ****P < .0001.

Enforced Notch signaling in CLL cells induces increased expression of cell cycle associated genes through direct and indirect epigenetic mechanisms. Splenic CD19+B220− CLL cells were sorted from 3- to 5-month-old IgH.TEμ;control (CTRL) and IgH.TEμ;RNICΔ/+ mb1-Cre (NIC) stage-matched littermates and analyzed for chromatin accessibility (ATAC-seq) and genome-wide binding of RBP-J and H3K27ac (ChIP-seq). (A) Venn diagram representing regions significantly (false discovery rate < 0.05) changed between CTRL and NIC for peaks called in ATAC-seq (fold > 1 for NIC and > −1 for CTRL), ChIP-seq for α-RBP-J and α-H3K27ac. (B) Binned orientation and distance to a transcription start site (TSS) for peaks called specifically in NIC, but not in CTRL. Distance to TSS is depicted in kilobases (kb). (C) GSEA for top Hallmark gene sets (MSigDB) affected by significant peaks (false discovery rate < 0.05) associated to the most proximal gene specifically in NIC and with regulated expression induced by enhanced Notch signaling (adjusted P value < 0.05). Panels from left to right depict Hallmark gene sets from ATAC-seq analysis, ChIP-seq for RBP-J, and ChIP-seq for H3K27ac. (D) Proposed mechanism of direct regulation by NIC at the proximal promoter regions of HeyL, Hes1, and Mybl2. Right panels represent genomic regions of regulated genes from Integrative Genomics Viewer (version 2.7.2) with peaks called from α-RBP-J ChIP-seq in CTRL and NIC, then α -H3K27ac ChIP-seq and ATAC-seq. Gene structure is shown below the tracks. (E) Proposed mechanism of indirect regulation by NIC at the proximal promoter region of cell-cycle gene Ccnb2 (left). Center panel represents genomic region of regulated gene from IGV with peaks called from α-RBP-J ChIP-seq in CTRL and NIC, then α-H3K27ac ChIP-seq and ATAC-seq. Gene structure is shown below the tracks. Gene expression from RNA-seq analysis represented as transcript per kilobase million (TPM) in CTRL (n = 7) and NIC (n = 6) is shown on the right. P value was calculated using unpaired Student t test: **P < .01. (F) Schematic depicts superphysiological NIC indirectly suppressing Foxp2 promoter by epigenetic mechanisms resulting in loss of Cdk1 repression. Bottom left panel represents IGV tracks of epigenetically regulated Foxp2 promoter, with peaks called in α-RBP-J ChIP-seq, α-H3K27ac ChIP-seq, and ATAC-seq shown for CTRL and NIC. Centered panel depicts a zoomed view of ATAC-seq footprinting analysis (TOBIAS version 11) upstream from Cdk1 promoter (Cdk1 gene structure is schematically indicated on the top left of the zoomed footprint). Tracks at the Foxp2 binding motif show the corrected ATAC-seq signal for CTRL and NIC, translated by TOBIAS into CTRL and NIC footprint score (CTRL bound site with score 1.6, NIC unbound site with score 0.8). Right panels show gene expression from RNA-seq for Foxp2 and Cdk1 expression between CTRL (n = 6) and NIC (n = 7) CLL cells. Total Cdk1 protein level was assessed by western blot analysis on ex vivo CLL cells from IgH.TEμ:control (n = 4) and IgH.TEμ;RNICΔ/+ mb1-Cre (n = 4) mice with antibodies α-Cdk1 (35 kDa) and α-β-Actin (42 kDa). P values were calculated using paired Student t test: **P < .01; ****P < .0001.

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