Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

GCN2 phosphorylates HIV-1 integrase and decreases HIV-1 replication by limiting viral integration.

GCN2 is a serine/threonine kinase involved in cellular stress response related to amino acid starvation. Previously, we showed that GCN2 interacts with HIV-1 integrase and is activated during HIV-1 infection. Herein, we identified HIV-1 integrase as a previously unknown substrate of GCN2 in vitro with a major site of phosphorylation at residue S255 located in the C-terminal domain of HIV-1 integrase. The underlying mechanism was investigated and it appeared that the integrase active site was required in order for GCN2 to target the integrase residue S255. Moreover, various integrases from other retroviruses (e.g. MLV, ASV) were also recognized as a substrate by GCN2. In cells, HIV-1 lentiviral particles harboring mutation at integrase position 255 were affected in their replication. Preventing phosphorylation resulted in an increase in infectivity that correlated with an increase in viral DNA integration. Infectivity of MLV was also higher in cells knocked-out for GCN2 suggesting a conserved mechanism to control viral replication. Altogether, our data suggest that GCN2 may constitute a general guardian of genome stability by regulating foreign DNA integration and as such be part of the antiviral armamentarium of the cell.

Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

Protease-activated receptor genes are clustered on 5q13.

The serine protease, thrombin, is both a potent agonist for platelet aggregation and a mitogen inducing the proliferation of other cell types. Many cellular responses to thrombin are mediated by a G-protein-coupled thrombin receptor (protease-activated receptor-1, PAR-1). This represents the prototype of a new family of proteolytically cleaved receptors that includes PAR-2 and the recently identified PAR-3. Like PAR-1, PAR-3 is a potential thrombin receptor. Their similar gene structure, mechanism of activation, and colocalization to 5q13 raises the question of a common evolutionary origin and of their belonging to a clustered gene family. Construction of a physical map of the 5q13 region by pulsed-field gel electrophoresis (PFGE) has allowed us to identify six potential CpG islands and to establish a linkage of the PAR genes. Southern blot analysis showed that they were in a cluster on a 560-kb Asc I fragment, in the order PAR-2, PAR-1, and PAR-3. PAR-1 and PAR-2 genes were contained within the identical 240-kb Not I fragment, thus confirming a tight linkage between them. The localization of other CpG islands suggested that more PAR-family genes may be present.

Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family.

Human mucin gene MUC5B is mapped clustered with MUC6, MUC2, and MUC5AC on chromosome 11p15.5. We report here the isolation of three overlapping genomic clones of human MUC5B spanning approximately 40 kilobases. We have determined their partial restriction maps and the intron-exon boundaries of the central region encoding a single open reading frame. This coding region has been completely sequenced. Its length is 10,713 base pairs, and it encodes a 3570-amino acid peptide. Nineteen subdomains have been individualized. Some subdomains show similarity to each other, creating larger composite repeat units that we have called super-repeats. Four super-repeats of 528 amino acid residues are thus observed within the central exon. Each comprises (i) a subdomain composed of 11 repeats of the irregular repeat of 29 amino acid residues, (ii) a unique conserved subdomain with no typical repeat, and (iii) a cysteine-rich subdomain. This latter subdomain has high sequence similarity to the cysteine-rich domains described in MUC2 and MUC5AC. Sequence data of these three genes, together with their clustered organization, lead us to suggest that they may be a part of a multigene family. The super-repeat present in MUC5B is the largest ever determined in mucin genes and the central exon of this gene is, by far, the largest reported for a vertebrate gene.

Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes.

Four distinct genes that encode mucins have previously been mapped to chromosome 11p15.5. Three of these genes (MUC2, MUC5AC, and MUC6) show a high level of genetically determined polymorphism and were analyzed in the CEPH families. Linkage analysis placed all three genes on the genetic map in a cluster between HRAS and INS, and more detailed analysis of recombinant breakpoints revealed that MUC6 is telomeric to MUC2. Using these recombinants D11S150 was mapped close to MUC2. Ten of the 11 recombinant chromosomes studied in detail were paternal, and the recombinant events were distributed throughout the 11p15 region, suggesting that the high level of recombination observed in 11p15.5 is not due to a particular recombinational hot spot. Pulsed-field gel electrophoresis was used to make a detailed physical map of the MUC cluster and to integrate the physical and genetical maps. The gene order was determined to be HRAS-MUC6-MUC2-MUC5AC-MUC5B-IGF2. The MUC genes span a region of some 400 kb and the map extends 770 kb and contains 15 putative CpG islands. The order of the MUC genes on the map corresponds to the relative order of their expression along the anterior-posterior axis of the body, suggesting a possible functional significance to the gene order.

Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes?

To date five human mucin cDNAs (MUC2, 5A, 5B, 5C and 6) mapped to 11p15.3-15.5, so it appears that this chromosome region might contain several distinct gene loci for mucins. Three of these cDNAs, MUC5A, B and C, were cloned in our laboratory and previously published. A common number, 5, was recommended by the Human Gene Mapping Nomenclature Committee to designate them because of their common provenance from human tracheobronchial mucosa. In order to define whether they are products of the same gene locus or distinct loci, we describe in this paper physical mapping of these cDNAs using the strategy of analysis of CpG islands by pulse-field gel electrophoresis. The data suggest that MUC5A and MUC5C are part of the same gene (called MUC5AC) which is distinct from MUC5B. In the second part of this work, complete sequences of the inserts corresponding to previously described (JER47, JER58) and novel (JER62, JUL32, MAR2, MAR10 and MAR11) cDNAs of the so-called MUC5AC gene are presented and analysed. The data show that in this mucin gene, the tandem repeat domain is interrupted several times with a subdomain encoding a 130 amino acid cysteine-rich peptide in which the TR3A and TR3B peptides previously isolated by Rose et al. [Rose, Kaufman and Martin (1989) J. Biol. Chem., 264, 8193-8199] from airway mucins are found. A consensus peptide sequence for these subdomains involving invariant positions of most of the cysteines is proposed. The consensus nucleotide sequence of this subdomain is also found in the MUC2 gene and in the MUC5B gene, two other mucin genes mapped to 11p15. The functional significance for secreted mucins of these cysteine-rich subdomains and the modular organization of mucin peptides are discussed.

Degenerate 87-base-pair tandem repeats create hydrophilic/hydrophobic alternating domains in human mucin peptides mapped to 11p15.

A human tracheobronchial lambda gt 11 cDNA library was screened using antiserum prepared against the deglycosylated protein backbone of human tracheobronchial mucins. Two cDNAs, designated JER 28 and 57, obtained from this immunoscreening, were used to isolate two other cDNA clones, JUL 7 and JUL 10, from a human tracheobronchial lambda gt 10 cDNA library. These four clones (561, 1830, 1631 and 991 bp), which mapped to chromosome 11p15, were all found to contain degenerate 87-base-pair tandem repeats which encode non-repetitive peptides. Numerous deletions or insertions in an otherwise virtually perfect 87-base-pair tandem repeat create many shifts in reading frame which completely destroy the repetitive peptide structure. The peptide is composed of alternate hydrophobic and hydrophilic domains which probably differ in the extent to which they are glycosylated. The mRNAs are expressed both in the respiratory and in the digestive tracts. These human mucin probes may be important in assessing the abnormal mucins associated with inflammatory diseases or carcinoma from human mucosae.

Mucin 4 (MUC4) gene: regional assignment (3q29) and RFLP analysis.

The use of a probe (JER64) containing a mucin 4 (MUC4) cDNA insert of 1.83 kb allowed to assign by in situ hybridization, the MUC4 gene to 3q29. This probe detected RFLPs with all restriction enzymes used (BamHI, HindIII, PstI, EcoRI, and TaqI). Particularly numerous alleles were observed with PstI, EcoRI and TaqI, in a small sample of unrelated DNAs (25 digested with PstI, 8 with EcoRI and 8 with TaqI). The PIC values were 0.69, 0.63 and 0.70 for PstI, EcoRI and TaqI respectively. The polymorphisms observed of variable number of tandem repeat (VNTR) type are in relation with the presence of tandemly repeated nucleotide sequences in MUC4 gene.

Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs.

A lambda gt11 cDNA library constructed from human tracheo-bronchial mucosa was screened with a polyclonal antiserum raised to chemically deglycosylated pronase glycopeptides from human bronchial mucins. Out of 20 positives clones, one partial cDNA clone was isolated and allowed to map a novel human tracheo-bronchial mucin gene. It contains 48 nucleotide tandem repeats quite perfectly identical which encodes a protein containing about 50% of hydroxy amino-acids. This clone hybridized to polydisperse messages produced by human tracheo-bronchial and human colonic mucosae. The gene (proposed name MUC 4) from which cDNA is derived maps to chromosome 3.