Fig 2: Quantitative mRNA and protein expression analysis of the TTI2 mutations. (A) Quantitative RT-PCR (qRT-PCR) analysis of TTI2 mRNA in peripheral blood leukocytes from patients (II-1 and II-3), unaffected parents (I-1 and I-2), and a control individual (C1). The results of the quantification of TTI2 transcripts were first normalized to the ACTB housekeeping gene. The values presented are the means of triplicate determinations ± standard deviation (SD). The C1 was set to 1.0. (B) TTI1, TELO2, and TTI2 protein expression in lymphocytes obtained from the family members and C1, with β-actin serving as a loading control. The results represent one of three similar experiments.

Fig 3: Schematic map and expression profile of two alternatively spliced TTI2 isoforms. (A) Schematic map of TTI2 showing the normal (upper) or variable spliced isoforms (lower), as well as the positions of mutations. Boxed regions denote exons, and connecting solid lines indicate introns. The mutation above the horizontal axis marked in blue is the novel frameshift mutation identified; those below are missense mutations reported to date. Primers E3F and E7R were used in RT-PCR. (B) Expression profile of the two TTI2 isoforms in 16 types of normal human tissues. (C) SH-SY5Y cells were transfected with empty vector, GFP-tagged TTI2 isoform 1 or isoform 2. Expressed GFP-TTI2 (green) and stained DAPI (blue) were observed.

Fig 4: The transcriptional control exercised by E4F1 on the TTT complex component TTI2, strongly impacts on ATM and ATR protein levels in the TNBC cell line, SUM159. (A)—E4F1 depletion impacts on TTI2, ATM and ATR protein levels. Immunoblot analysis of indicated proteins in total extracts prepared from SUM159 cells treated with either shCtrl or shE4F1. Extracts were prepared 4 days after cell infection with lentiviral vectors expressing shRNAs; (B)—E4F1 depletion impacts on TTI2 mRNA level, but not on ATM and ATR mRNA levels, nor on the mRNA level of TTI1, another component of the TTT complex. Measured by RT-qPCR, 5 days after cell infection with lentiviral vectors expressing either shCtrl or shE4F1. Data are means ± SD, n= 3 independent experiments. ** p < 0.01, as determined by a one way ANOVA test performed on GraphPad Prism; (C)—siRNA-mediated depletion of TTI2 impacts on ATR and ATM protein levels in SUM159. Immunoblot analysis of indicated proteins in total extracts prepared from SUM159 cells treated for 48 h with either a control siRNA (siCtrl) or an siRNA directed against human TTI2 mRNA; (D)—Immunoblot analysis of indicated proteins in total extracts prepared from SUM159 cells treated with either shCtrl or shE4F1 and with the lysosomal inhibitor E64 (10 μM).

Fig 5: E4F1 directly controls a limited set of genes in the TNBC cell line SUM159, including a sub-program involved in the ATM/ATR-CHEK signaling pathway. (A)—Fold change plot of differentially expressed genes, assessed by RNA-Seq, in SUM159 cells four days after the initial exposure to either shCtrl or shE4F1. The expression of a large set of genes is moderately deregulated in E4F1-depleted cells compared to control; (B)—Identification of E4F1 target genes by ChIP-Seq. E4F1 ChIP-Seq read densities in the 3 kb regions surrounding the Transcription Start Sites (TSS) of the closest genes located nearby E4F1 binding sites, as identified in SUM159 cells. Out of the 116 ChIP-Seq peaks identified by ChIP-Seq in SUM159 cells (n = 2; Tables S1–S3, Supplementary Materials), 102 were in these 3kb regions and 94 in promoters, indicating that E4F1 is mainly located nearby or on the TSS of its target genes; (C)—MEME logo sequence analysis of the DNA fragments bound by E4F1 in SUM159 cells reveals a human E4F1 consensus motif that is similar to the Mouse E4F1 consensus motif previously identified by our laboratory [17]; (D)—Venn diagram of the overlap between all of the E4F1 bound genes identified by ChIP-Seq in SUM159 cells (GSE128104 (in red)) and genes differentially expressed (p < 0.05) between SUM159 cells treated with either shCtrl or shE4F1 (GSE128099 (in blue)). These genome-wide analyses were performed at a time where all of the cells treated with shRNAs were still viable (annexin-V negative) and still actively growing. The overlap defines a set of 57 genes directly bound and regulated by E4F1 in SUM159; (E)—List of the 57 E4F1 direct target genes as defined in panel D. Table provides information about the differential expression of transcripts in SUM159 cells treated with either shCtrl or shE4F1 (Ratio KO/WT), the distance of the E4F1 consensus motif to the transcription start site (TSS) of the gene, and whether these genes code for factors involved in cellular stress response and checkpoints (red+) including proteins involved in the ATM/ATR-CHK kinases axis (CHK1, PPP5C and TTI2).