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...

Nuclear lamin filaments and associated proteins form a nucleoskeletal (“lamina”) network required for transcription, replication, chromatin organization and epigenetic regulation in metazoans. Lamina defects cause human disease (“laminopathies”) and are linked to aging. Barrier-to-autointegration factor (BAF) is a mobile and essential component of the nuclear lamina that binds directly to histones, lamins and LEM-domain proteins, including the inner nuclear membrane protein emerin, and has roles in chromatin structure, mitosis and gene regulation. To understand BAF's mechanisms of action, BAF associated proteins were affinity-purified from HeLa cell nuclear lysates using BAF-conjugated beads, and identified by tandem mass spectrometry or independently identified and quantified using the iTRAQ method. We recovered A- and B-type lamins and core histones, all known to bind BAF directly, plus four human transcription factors (Requiem, NonO, p15, LEDGF), disease-linked proteins (e.g., Huntingtin, Treacle) and several proteins and enzymes that regulate chromatin. Association with endogenous BAF was independently validated by co-immunoprecipitation from HeLa cells for seven candidates including Requiem, poly(ADP-ribose) polymerase 1 (PARP1), retinoblastoma binding protein 4 (RBBP4), damage-specific DNA binding protein 1 (DDB1) and DDB2. Interestingly, endogenous BAF and emerin each associated with DDB2 and CUL4A in a UV- and time-dependent manner, suggesting BAF and emerin have dynamic roles in genome integrity and might help couple DNA damage responses to the nuclear lamina network. We conclude this proteome is a rich source of candidate partners for BAF and potentially also A- and B-type lamins, which may reveal how chromatin regulation and genome integrity are linked to nuclear structure.

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.

Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.

IntroductionFanconi anemia (FA) is an autosomal recessive cancer susceptibility disorder characterized by diverse clinical features such as skeletal or skin abnormalities, progressive bone marrow failure, and increased risk of malignancies. 1-5 FA has been reported in diverse ethnic groups, with an estimated heterozygous carrier frequency of 1 in 300, 6 although this estimate may run higher in certain ethnic groups. 7 Early and accurate diagnosis of Fanconi anemia is important, because it profoundly affects patient monitoring and treatment decisions and permits early genetic counseling of family members. Given the striking sensitivity of patients with FA to DNA-damaging agents, [8][9][10][11] timely diagnosis is critical prior to the use of chemotherapy or radiation therapy of these patients in the bone marrow transplant setting. Diagnosis based on clinical manifestations alone can be difficult because 30% to 40% of patients lack developmental malformations or a positive family history. 12,13 Patients previously not known to carry the diagnosis of FA may present with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) as the initial manifestation of their disease. Patients with acute leukemia have been diagnosed with FA after developing toxicity from their bone marrow transplant conditioning regimen. 14 The currently used diagnostic test for FA relies on the increased chromosomal breakage and radial formation of FA cells in response to diepoxybutane (DEB) compared with cells from healthy control subjects, 15,16 or from patients with other chromosomal instability disorders 17 or genetic syndromes. The DEB test has excellent sensitivity, specificity, and reproducibility, 16 and it is effective in prenatal diagnosis of FA. 18,19 The DEB test is labor intensive and time consuming, however, and does not easily lend itself to broad screening studies. Furthermore, in some cases of Fanconi mosaicism, false-negative results have been reported with DEB screening of patient lymphocytes.Studies of FA proteins have yielded insights into the molecular pathogenesis of FA. Fanconi anemia is a genetically heterogeneous disorder comprised of at least 8 complementation groups. 20,21 The genes corresponding to each of these groups (A, 22 B, 23 C, 24 D1, 23 D2, 21 E, 25 F, 26 and G 27 ) have been cloned. The similar clinical presentation of patients of different subtypes suggests that these genes function in a common pathway. Indeed, interactions between FANCA, FANCC, FANCE, FANCF, and FANCG have been described. [28][29][30][31][32] Activation of the FA protein complex by DNA damage or cell cycle progression results in monoubiquitination of the downstream FANCD2 protein. 33 Monoubiquitination is a newly recognized class of posttranslational protein modification. 34 Following monoubiquitination, FANCD2 localizes to nuclear foci where it colocalizes with other DNA-repair proteins such as BRCA1 (Figure 1). 33 A point mutation of FANCD2 at lysine 561, the site of monoubiquitination, abrogates its ability to correct the mitomy...

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common cellular pathway. To further understand the in vivo role of one of these human genes (FANCG), we generated a targeted disruption of murine Fancg and bred mice homozygous for the targeted allele. Similar to the phenotype of the previously described Fancc ؊/؊ and Fanca ؊/؊ mice, the Fancg ؊/؊ mice had normal viability and no gross developmental abnormalities. Primary splenic lymphocytes, bone marrow progenitor cells, and murine embryo fibroblasts from the Fancg ؊/؊ mice demonstrated spontaneous chromosome breakage and increased sensitivity to mitomycin C and, to a lesser extent, ionizing radiation. IntroductionFanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome characterized by multiple congenital anomalies and progressive bone marrow failure. 1,2 FA patients develop several types of cancers, including acute myeloid leukemias and squamous cell carcinomas of the head and neck. 3 FA cells are sensitive to DNA cross-linking agents, such as mitomycin C (MMC) and, to a lesser extent, ionizing radiation (IR). 4,5 Based on somatic cell fusion studies, FA is composed of 8 distinct complementation groups. 6,7 Six of the human FA genes, including the genes for FANCA, 8,9 FANCC, 10 FANCD2, 11 FANCE, 12 FANCF,13 and FANCG, 14 have been cloned.Recent studies have demonstrated that the 6 cloned FA proteins interact in a common cellular pathway. 15 The FANCA, FANCC, FANCE, FANCF, and FANCG proteins assemble in a multisubunit nuclear complex. [16][17][18][19][20][21] The FA protein complex regulates the monoubiquitination of the downstream FANCD2 protein, suggesting that the complex is a multisubunit monoubiquitin ligase or regulates a ligase activity. When normal (non-FA) cells are exposed to DNA-damaging agents, such as IR, MMC, or UV light, FANCD2 is monoubiquitinated and targeted to nuclear foci containing the BRCA1 protein. 15 Disruption of this pathway leads to the characteristic cellular and clinical abnormalities observed in FA.In an attempt to understand the in vivo function of FA genes, targeted disruptions of FA genes have been generated. Two murine models, containing disruptions of the murine homolog of FANCC, have been developed. Chen et al 22 generated a disruption of exon 8 of Fancc, while Whitney et al 23 used homologous recombination to create a disruption of exon 9. In both models, spontaneous chromosome breakage was observed. In addition, an increase in chromosome breaks in splenic lymphocytes in response to bifunctional alkylating agents was observed. In both models, Fancc Ϫ/Ϫ mice had germ cell defects and decreased fertility. Unlike human FA patients, the Fancc Ϫ/Ϫ mice had no obvious gross developmental abnormalities or cancer susceptibility. To date, the Fancc murine knockout model has been useful...