Figure 6. D-cyclins Repress Expression of Fas and FasL via E2F1.

(A) Relative expression of E2F1, E2F2 and E2F3 transcripts in bone marrow cells 48 hours following an acute ablation of D-cyclins (MxTKO).

(B) Relative expression of E2F1 transcripts in hematopoietic stem cells (HSC) 24 hours after ablation of D-cyclins (MxTKO). N=3 mice for each genotype.

(C) Protein levels of E2F1, E2F2 and E2F3 in bone marrow cells 48 hours following acute ablation of D-cyclins (MxTKO).

(D-G) Bone marrow cells were infected with viruses encoding anti-E2F1 shRNA (shE2F1), or with control, scrambled shRNA (shCtrl). Please note that bone marrow cells did not undergo any selection after viral transduction. Therefore, E2F1 knockdown took place only in a fraction of cells and hence the observed changes likely underestimate the effects of E2F1 depletion. See also Figure S5A.

(D) Results of RT-PCR analysis for the levels of the endogenous Fas and FasL transcripts.

(E to G) Mean percentages of cleaved caspase-3 positive cells (E), trypan blue-positive dead cells (F), and mean total number of remaining bone marrow cells (G), following knock-down of E2F1.

(H) Fas and FasL promoter-luciferase constructs were co-transfected with vectors encoding E2F1, E2F2, E2F3, or with an empty vector (EV). Note that E2F1 expression significantly reduced transcription from both Fas and FasL promoters (see also Figures S5E and S5G).

(I) Chromatin immunoprecipitation (ChIP) studies of E2F1 binding to the Fas promoter in HSPC. Binding of E2F1 to a region of the Fas promoter was observed with both anti-E2F1 antibodies used (Santa Cruz, bright red; Millipore, dark red). Data represent mean ± SEM of three replicates. Negative control region (NCR) is a control region away from the Fas promoter. Below, a schematic representation of the Fas promoter.

In (A), (B), (D-I), error bars denote SD.

See also Figures S4 and S5.