Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression.

HP1 enrichment at pericentric heterochromatin is considered important for centromere function. Although HP1 binding to H3K9me3 can explain its accumulation at pericentric heterochromatin, how it is initially targeted there remains unclear. Here, in mouse cells, we reveal the presence of long nuclear noncoding transcripts corresponding to major satellite repeats at the periphery of pericentric heterochromatin. Furthermore, we find that major transcripts in the forward orientation specifically associate with SUMO-modified HP1 proteins. We identified this modification as SUMO-1 and mapped it in the hinge domain of HP1α. Notably, the hinge domain and its SUMOylation proved critical to promote the initial targeting of HP1α to pericentric domains using de novo localization assays, whereas they are dispensable for maintenance of HP1 domains. We propose that SUMO-HP1, through a specific association with major forward transcript, is guided at the pericentric heterochromatin domain to seed further HP1 localization.

IntroductionFanconi anemia (FA) is a rare autosomal recessive cancer susceptibility syndrome characterized by developmental abnormalities, progressive bone marrow failure, and cellular hypersensitivity to DNA cross-linking agents. 1 Eleven FA complementation groups have been identified (A, B, C, D1, D2, E, F, G, I, J, and L) 2,3 and 8 FA genes have been cloned. 2,4,5 The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. 6 The 8 encoded FA proteins (A, C, D1, D2, E, F, G, L) cooperate in a common cellular pathway, the FA/BRCA pathway. 7 In this pathway, 6 of the FA proteins (A, C, E, F, G, L) 8,9 bind in a constitutive nuclear protein complex (the FA complex). In response to DNA damage 10 or during the S phase of the cell cycle, 11 the FA complex promotes the monoubiquitination of the downstream FANCD2 protein. This event requires a molecular interaction between the FANCE and FANCD2 proteins. 12,13 Monoubiquitination of FANCD2 is required for targeting of FANCD2 into nuclear foci containing BRCA1, FANCD1/ BRCA2, and RAD51. 11 These subnuclear foci may be sites of homologous recombination-mediated DNA repair, given the known roles of BRCA1, BRCA2, and RAD51 in this process. 14,15 Disruption of the FA/BRCA pathway results in the characteristic cellular and clinical features of FA, including hypersensitivity to DNA cross-linking agents. 16 A critical regulatory event in the FA/BRCA pathway is the monoubiquitination of FANCD2 on Lysine 561. 10 Analysis of FANCD2 monoubiquitination provides a rapid diagnostic screen for the integrity of the FA/BRCA pathway. 17 In addition, FANCD2 undergoes an ionizing radiation (IR)-inducible, ataxia telangiectasia (ATM)-dependent phosphorylation on Serine 222. 18 Phosphorylation of this serine is required for the establishment of an intra-S-phase checkpoint response but is not required for FANCD2 monoubiquitination, FANCD2 targeting to foci, or FANCD2-mediated DNA repair.Little is known about the regulation or functional outcome of FANCD2 monoubiquitination. First, the newly cloned FANCL protein has a plant homeodomain (PHD) domain with E3 ubiquitin ligase activity, although its ubiquitination of FANCD2 has not been demonstrated in vitro. 2 For personal use only. on May 12, 2018. by guest www.bloodjournal.org From monoubiquitinated isoform of FANCD2 (FANCD2-L) accumulates in discrete nuclear foci in damaged cells, 10 suggesting that it is actively transported to these structures. Accordingly, these foci may contain a specific receptor for FANCD2-L or its ubiquitin moiety. Third, following DNA repair or during the mitotic phase of the cell cycle, FANCD2-L is deubiquitinated, suggesting a reversible and more complex mechanism of regulation.The nucleus is organized into an integrated structure in which chromatin is associated with a nonhistone scaffold termed the nuclear matrix. 22 Various aspects of nucleic acid metabolism, including DNA replication, transcription, and the repair of UVinduced thymidine dimers, require an interaction between chromatin and the nu...