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Additional file 7. Material and Methods.

Additional file 2. Supplementary Figures.

Additional file 1. Supplementary Material: REMSA analysis.

Figure S1. Schematic representation of the exons belonging to the different variants of human BRAF reported in NCBI and Ensembl. Figure S2. Scan of exon 18 of BRAF in 8 cancer types. Figure S3. Count of the reads mapping to all BRAF exons, E19 included. Figure S4. Count of reads mapping to all BRAF exons (w/o E19 on the left and w E19 on the right). Figure S5. Count of E13-E16 exon-spanning reads. Figure S6. BRAF-004 transcript variant is expressed but it is truncated. Figure S7. Position of the primers/siRNAs used to detect/downregulate BRAF-ref, BRAF-X1, and BRAF-X2. Figure S8. Real-time PCR detection of BRAF-ref, BRAF-X1 plus X2, BRAF-X1, and BRAF-X2. Figure S9. Box plot of the reads that span E17-E18.2, E18.2-E18b, E18.2-E19, and E17-E19. Figure S10. Stability of reference and X1/X2 BRAF mRNA. Figure S11. Correlation among the expression levels of the different BRAF isoforms in breast cancer, head and neck cancer, lung SCC, AML, and DLBCL. Figure S12. Lack of association of reference, X1, and X2 BRAF levels with age (left panels), gender (middle panels), and stage at diagnosis in primary and metastatic melanoma patients. Figure S13. Lack of association of age (left panels), gender (middle panels), and stage at diagnosis (right panels) with the ratio between BRAF-X1 and BRAF-ref levels (upper panels, red) and with the ratio between BRAF-X2 and BRAF-ref levels (lower panels, blue) in primary and metastatic melanoma patients. Figure S14. Tools for the detection of BRAF CDS plus the E19-derived 3’UTR. Figure S15. Length of BRAF-X1 and BRAF-X2 3’UTR in melanoma. Figure S16. siRNA-mediated downregulation of BRAF isoforms in melanoma cells. Figure S17. Cartoon summarizing the position of the primers and the siRNAs used to determine the levels and the identity of the Δ[3–10] splicing variant of BRAF. Figure S18. Real-time PCR detection of BRAF-ref (grey) and BRAF-X1 plus X2 (black) in vemurafenib-resistant clones and clonal populations. Figure S19. The 3’UTR of X1 and X2 Δ[3–10]BRAF splicing variant is up to 7 kb long. Figure S20. Sequence of reference, X1, and X2 BRAF proteins. Figure S21. Alignment between the sequence of human BRAF-ref (NP_004324.2, left) or BRAF-X1 (XP_005250102.1, right) and mouse Braf-ref (NP_647455.3). Figure S22. Alignment between the sequence of human BRAF-ref (NP_004324.2, left) or BRAF-X1 (XP_005250102.1, right) and rat Braf-X1 (XP_001070228.2). Figure S23. Alignment between the sequence of human BRAF-ref (NP_004324.2, left) or BRAF-X1 (XP_005250102.1, right) and pig Braf-X1 (XP_005654324.1). Figure S24. Dot plot of the secondary structure of BRAF-X1 (blue) and BRAF-X2 (green) mRNA sequences. Figure S25. Enlargement of box1 as reported in Figure S24. Figure S26. BRAF peptides identified by an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, a) and by a 5600 TripleTOF mass spectrometer (ABSciex, b) in A375 melanoma cells. Figure S27. BRAF peptides identified by LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) in WM793B melanoma cells. Figure S28. Translation efficiency of reference, X1 and X2 BRAF mRNAs in A375 cells. Figure S29. BRAF-X2 protein displays a faster decay due to increased proteosomal-mediated degradation. (PDF 14500 kb)

Primers and siRNAs location along BRAF-ref, BRAF-X1, and BRAF-X2 mRNAs. (PDF 67Â kb)

Table S1. Coordinates of the exons of human BRAF analyzed in this study. Table S2. List of the human tumor samples used in this study. Table S3. Real-time PCR primers. Table S4. PCR primers. Table S5. siRNA sequences. (PDF 172Â kb)

In metazoan cells the DNA replication origins are not well defined. Differently from what observed for bacteria cells and for budding yeast, in metazoan the origins does not show a conserved sequence and they appear to be specified by many factors. In order to better understand the mechanisms involved in the origin specification, many studies have been done to identify the proteins involved in the recognition and activation of the origins. From these kind of analysis is emerging that, beside the wellknown proteins of the pre replicative complex, also other factors might be involved. Between these, the HOX proteins seem to be able to play a role in the origin activity. One of the first studies of this involvement was done by our group and leads to the identification of three homeotic proteins able to specifically bind in vitro the human lamin B2 origin. Thus, in the study conducted during this PhD program, was investigated the involvement of one of these homeotic proteins, namely HOXC13, with human DNA replication origins and with replicative complexes. We found an interaction of HOXC13 with two crucial factors of the pre Replication Complex (pre-RC), ORC1 and Cdc6 and that HOXC13 binds a good fraction of the origins, in particular the early replicating ones, like the lamin B2 origin and other known human origins. The HOXC13 protein is bound to origin chromatin, at least for the lamin B2 origin, at a precise site within the pre-RC at specific moments of the cell cycle. Interaction with the origin occurs within the area protected by the pre-RC in G1, very close to the start sites of leading strand synthesis and to the binding sites of ORC1, ORC2, Cdc6, topoisomerase (topo) I and topo II. The protein is absent from the origin in M and appears on it at the beginning of G1, reach a peak at G1/S and as synthesis starts, the interaction of HOXC13 with the origin fades, in parallel with the transition from this large pre-RC to a smaller and differently organized post-RC. Recently also other HOX proteins have been identify as proteins involved in regulation processes of DNA replication, suggesting that the interaction of HOXC13 with the origins might occur in a multi-homeotic proteins complex. Depletion of one of these proteins however is compatible with the continuation of the cell cycle and, according with what observed for the other homeotic proteins, we found that also the depletion of HOXC13 does not alter cell cycle progression or S phase entry. This is probably due to the redundancy of homeotic proteins and indicates a relatively generic function for the HOX proteins. Among the identified elements influencing the choice and the activity of a sequence as DNA replication origin, much relevance is assumed by the chromatin structure and topology of DNA. Therefore, we analysed the effects of chromatin structure disruption using Tricostatin A, a histone deacetylase inhibitor. The alteration of chromatin caused by this treatment not only sharply reduces origin function, but also disturbs the binding of replication complex members like HOXC13 and the well known Cdc6 to the DNA replication origins, while does not affect the binding of other unrelated proteins like USF1. On the basis of this finding, we infer that an appropriate chromatin organization and DNA topology strongly influence the binding between factors of the pre Replication Complex and DNA replication origins. This influence could be a key element in origin specification. The described interactions are not restricted to a single origin nor to a single homeotic protein, leading us to conclude that HOX proteins, probably in the context of a multi-protein homeotic effectors, contribute to recruit and stabilize the replicative complexes onto early replicating origins, in presence of specific chromatin and topological configurations. The relevance of HOXC13 in DNA replication is also underlined by its involvement in oncogenesis, clearly demonstrated in acute myeloid leukaemia when HOXC13 is fused with NUP98 protein.

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.