Non-Coding, RNAPII-Dependent Transcription at the Promoters of rRNA Genes Regulates Their Chromatin State in S. cerevisiae.

Pervasive transcription is widespread in eukaryotes, generating large families of non-coding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production.

MCM8- and MCM9 Deficiencies Cause Lifelong Increased Hematopoietic DNA Damage Driving p53-Dependent Myeloid Tumors.

Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies.

DNA replication stress triggers rapid DNA replication fork breakage by Artemis and XPF.

DNA replication stress (DRS) leads to the accumulation of stalled DNA replication forks leaving a fraction of genomic loci incompletely replicated, a source of chromosomal rearrangements during their partition in mitosis. MUS81 is known to limit the occurrence of chromosomal instability by processing these unresolved loci during mitosis. Here, we unveil that the endonucleases ARTEMIS and XPF-ERCC1 can also induce stalled DNA replication forks cleavage through non-epistatic pathways all along S and G2 phases of the cell cycle. We also showed that both nucleases are recruited to chromatin to promote replication fork restart. Finally, we found that rapid chromosomal breakage controlled by ARTEMIS and XPF is important to prevent mitotic segregation defects. Collectively, these results reveal that Rapid Replication Fork Breakage (RRFB) mediated by ARTEMIS and XPF in response to DRS contributes to DNA replication efficiency and limit chromosomal instability.

DNA polymerase ν gene expression influences fludarabine resistance in chronic lymphocytic leukemia independently of p53 status.

Alteration in the DNA replication, repair or recombination processes is a highly relevant mechanism of genomic instability. Despite genomic aberrations manifested in hematologic malignancies, such a defect as a source of biomarkers has been underexplored. Here, we investigated the prognostic value of expression of 82 genes involved in DNA replication-repair-recombination in a series of 99 patients with chronic lymphocytic leukemia without detectable 17p deletion or TP53 mutation. We found that expression of the POLN gene, encoding the specialized DNA polymerase ν (Pol ν) correlates with time to relapse after first-line therapy with fludarabine. Moreover, we found that POLN was the only gene up-regulated in primary patients' lymphocytes when exposed in vitro to proliferative and pro-survival stimuli. By using two cell lines that were sequentially established from the same patient during the course of the disease and Pol ν knockout mouse embryonic fibroblasts, we reveal that high relative POLN expression is important for DNA synthesis and cell survival upon fludarabine treatment. These findings suggest that Pol ν could influence therapeutic resistance in chronic lymphocytic leukemia. (Patients' samples were obtained from the CLL 2007 FMP clinical trial registered at: clinicaltrials.gov identifer: 00564512).

Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes.

Chromosomal domains in Drosophila are marked by the insulator-binding proteins (IBPs) dCTCF/Beaf32 and cofactors that participate in regulating long-range interactions. Chromosomal borders are further enriched in specific histone modifications, yet the role of histone modifiers and nucleosome dynamics in this context remains largely unknown. Here, we show that IBP depletion impairs nucleosome dynamics specifically at the promoters and coding sequence of genes flanked by IBP binding sites. Biochemical purification identifies the H3K36 histone methyltransferase NSD/dMes-4 as a novel IBP cofactor, which specifically co-regulates the chromatin accessibility of hundreds of genes flanked by dCTCF/Beaf32. NSD/dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-dependent H3K36 trimethylation, nucleosome positioning, and RNA splicing. Our results unveil a model for how IBPs regulate nucleosome dynamics and gene expression through NSD/dMes-4, which may regulate H3K27me3 spreading. Our data uncover how IBPs dynamically regulate chromatin organization depending on distinct cofactors.

Chromatin immunoprecipitation indirect peaks highlight long-range interactions of insulator proteins and Pol II pausing.

Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify "indirect peaks" of multiple IBPs that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common cofactors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.

Identification of new RECQL4 mutations in Caucasian Rothmund-Thomson patients and analysis of sensitivity to a wide range of genotoxic agents.

Rothmund-Thomson syndrome (RTS), a rare recessive autosomal disorder, presents genome instability and clinical heterogeneity with growth deficiency, skin and bone defects, premature aging symptoms and cancer susceptibility. A subset of RTS patients presents mutations of the RECQL4 gene, member of the RecQ family of DNA helicases, including the RECQL2 (BLM) and RECQL3 (WRN) genes, defective in the cancer prone Bloom and Werner syndromes, respectively. Analysis of the RECQL4 gene in six clinically diagnosed RTS patients shows five patients, including two siblings, with eight mutations mainly located in the helicase domain, three patients presenting two mutations. The alterations include four missense mutations, one nonsense mutation and the same frameshift deletion, g.2881delG in exon 9 found in three patients. Seven RECQL4 polymorphisms, two being new, have also been identified. Primary RTS fibroblasts from these RTS patients show no sensitivity to a wide variety of genotoxic agents including ionizing or ultraviolet irradiation, nitrogen mustard, 4NQO, 8-MOP, Cis-Pt, MMC, H2O2, HU, or UV plus caffeine which could be related to the RECQL4 alterations identified here. This is in contrast with the DNA damage sensitive Bloom and Werner cells and highlights the complexity of the numerous RecQ protein functions implicated in the different cellular pathways required for maintaining genomic integrity.

Analysis of skin cancer risk factors in immunosuppressed renal transplant patients shows high levels of UV-specific tandem CC to TT mutations of the p53 gene.

Immunosuppressed renal transplant recipients (RTRs) are predisposed to non-melanoma skin cancers (NMSCs), predominantly squamous cell carcinomas (SCCs). We have analyzed skin lesions from RTRs with aggressive tumors for p53 gene modifications, the presence of Human Papillomas Virus (HPV) DNA in relation to the p53 codon 72 genotype and polymorphisms of the XPD repair gene. We detected 24 p53 mutations in 15/25 (60%) NMSCs, 1 deletion and 23 base substitutions, the majority (78%) being UV-specific C to T transitions at bipyrimidine sites. Importantly, 35% (6/17) are tandem mutations, including 4 UV signature CC to TT transitions possibly linked to modulated DNA repair caused by the immunosuppressive drug cyclosporin A (CsA). We found 8 p53 mutations in 7/17 (41%) precancerous actinic keratosis (AK), suggesting that p53 mutations are early events in RTR skin carcinogenesis. Immunohistochemical analysis shows a good correlation between p53 accumulation and mutations. HPV DNA was detected in 78% of skin lesions (60% Basal Cell Carcinomas, 82%AK and 79% SCCs). Thus, immunosuppression has increased the risk of infections by HPVs, predominantly epidermodysplasia verruciformis, speculated to play a role in skin cancer development. No association is found between HPV status and p53 mutation. Moreover, p53 codon 72 or frequencies of three XPD genotypes of RTRs are comparable with control populations. The p53 mutation spectrum, presenting a high level of CC to TT mutations, shows that the UV component of sunlight is the major risk factor and modulated DNA repair by immunosuppressive drug treatment may be significant in the skin carcinogenesis of RTRs.

Functional analysis of novel sonic hedgehog gene mutations identified in basal cell carcinomas from xeroderma pigmentosum patients.

Altered sonic hedgehog (SHH) signaling is crucial in the development of basal cell carcinomas (BCC), the most common human cancer. Mutations in SHH signal transducers, PATCHED and SMOOTHENED, have already been identified, but SHH mutations are extremely rare; only 1 was detected in 74 sporadic BCCs. We present data showing unique SHH mutations in BCCs from repair-deficient, skin cancer-prone xeroderma pigmentosum (XP) patients, which are characterized by high levels of UV-specific mutations in key genes involved in skin carcinogenesis, including PATCHED and SMOOTHENED. Thus, 6 UV-specific SHH mutations were detected in 5 of 33 XP BCCs. These missense SHH alterations are not activating mutations for its postulated proto-oncogene function, as the mutant SHH proteins do not show transforming activity and induce differentiation or stimulate proliferation to the same level as the wild-type protein. Structural modeling studies of the 4 proteins altered at the surface residues, G57S, G64K, D147N, and R155C, show that they do not effect the protein conformation. Interestingly, they are all located on one face of the compact SHH protein suggesting that they may have altered affinity for different partners, which may be important in altering other functions. Additional functional analysis of the SHH mutations found in vivo in XP BCCs will help shed light on the role of SHH in skin carcinogenesis. In conclusion, we report for the first time, significant levels of SHH mutations found only in XP BCCs and none in squamous cell carcinomas, indicating their importance in the specific development of BCCs.