The PRDM9 KRAB domain is required for meiosis and involved in protein interactions

International audiencePR domain-containing protein 9 (PRDM9) is a major regulator of the localization of meiotic recombination hotspots in the human and mouse genomes. This role involves its DNA-binding domain, which is composed of a tandem array of zinc fingers, and PRDM9-dependent trimethylation of histone H3 at lysine 4. PRDM9 is a member of the PRDM family of transcription regulators, but unlike other family members, it contains a Krüppel-associated box (KRAB)-related domain that is predicted to be a potential protein interaction domain. Here, we show that truncation of the KRAB domain of mouse PRDM9 leads to loss of PRDM9 function and altered meiotic prophase and gametogenesis. In addition, we identified proteins that interact with the KRAB domain of PRDM9 in yeast two-hybrid assay screens, particularly CXXC1, a member of the COMPASS complex. We also show that CXXC1 interacts with IHO1, an essential component of the meiotic double-strand break (DSB) machinery. As CXXC1 is orthologous to Saccharomyces cerevisiae Spp1 that links DSB sites to the DSB machinery on the chromosome axis, we propose that these molecular interactions involved in the regulation of meiotic DSB formation are conserved in mouse meiosis

Exploiting The CRISPR/CAS9 System to Study Alternative Splicing In Vivo: Application to Titin

The giant protein titin is the third most abundant protein in striated muscle. Mutations in its gene are responsible for diseases affecting the cardiac and/or the skeletal muscle. Titin has been reported to be expressed in multiple isoforms with considerable variability in the I-band, ensuring the modulation of the passive mechanical properties of the sarcomere. In the M-line, only the penultimate Mex5 exon coding for the specific is7 domain has been reported to be subjected to alternative splicing. Using the CRISPR-Cas9 editing technology, we generated a mouse model where we stably prevent the expression of alternative spliced variant(s) carrying the corresponding domain. Interestingly, the suppression of the domain induces a phenotype mostly in tissues usually expressing the isoform that has been suppressed, indicating that it fulfills (a) specific function(s) in these tissues allowing a perfect adaptation of the M-line to physiological demands of different muscles

<it>In vivo</it> gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens

<p>Abstract</p> <p>Background</p> <p>Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector.</p> <p>Results</p> <p>Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions <it>in vitro</it>. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells <it>in vivo</it>. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down.</p> <p>Conclusion</p> <p>This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results <it>in vivo</it>. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.</p

Efficient generation of mouse models with the CRISPR/Cas9 technology

The CRISPR-Cas9 system allows rapid generation of a large spectrum of genetically modified mouse models. Here we present efficient gene knock-out, knock-in and exon deletion projects undertaken at our transgenesis platform located in the near suburbs of Paris (SEAT-TAAM PHENOMIN). The delivery of different forms of spCas9 into the pronucleus of murine zygotes and the analysis of injected embryos at the blastocyst stage or beyond have allowed us to obtain mouse models from hybrid and inbred strains for studies in myology, immunology, reproduction biology, etc. with a success rate of about 10 % (KI)–70 % (KO) in the founder generation

Variable behavior of iPSCs derived from CML patients for response to TKI and hematopoietic differentiation.

Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents

Characterization of iPSC clones.

<p>(<b>A</b>) Representative immunofluorescence of pluripotency markers in human iPSC clones derived from CD34⁺ CB cells (CB-iPSC #11) and CD34⁺ from CML first patient (CML-iPSCs #1.22, #1.24 and #1.31) and from CML second patient (#2.1 and #2.2), staining with anti-OCT4, anti-SOX2, anti-KLF4, anti-NANOG, anti-SSEA-4 and anti-TRA1-60. MEFs surrounding human iPSCs served as a negative control for immunofluorescence (magnification x100 or x200). (<b>B</b>) Representative alcian blue staining of histological sections of teratoma derived from human CB-iPSC #11 and CML-iPSC #1.31 encompassing tissues with all three germ layers (magnification x25 and x200).</p

Transgene independence of CML-iPSCs survival in presence of TKI.

<p>(<b>A</b>) PCR for the integrated vectors OSK 1 and MshP53 in 11 subclones of CML-iPSC #1.31 pretreated with CRE adenovirus. Generation of transgene-free subclone CML-iPSC #1.31i: excision of the 2 vectors. (<b>B</b>) Immunohistochemistry of pluripotency markers: OCT4, SOX2, KLF4, NANOG, SSEA-4 and TRA1-60 in human transgene-free iPSC subclones (after excision) derived from CD34⁺ from CML patient (#1.22 exc and #1.31 exc) (<b>C</b>) Dose-effect of TKI exposure (with imatinib (left panel) or ponatinib (right panel)) for 6 days on human excised CML-iPSCs (# 1.22, #1.31) and CB-iPSC (#11) subclones survival. iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC clone without TKI. Mean ± SD of triplicate.</p

Effect of shRNA against BCR-ABL1 on CML-iPSC #1.31 clone proliferation.

<p>(<b>A</b>) Western blot analysis of BCR-ABL1 and ABL expression in CML-iPSC #1.31 with shRNA control (shC) and with shRNA against BCR-ABL1 (shBCR). (<b>B</b>) Left panel: Proliferation of CML-iPSC (#1.31) with shC or shBCR. iPSCs counts at day 6 expressed as percentages relative to same iPSC (CML-iPSC #1.31) with shC. Mean +/− SD, n = 3. Right panel: Dose-effect of imatinib exposure for 6 days on iPSCs (CML-iPSC #1.31, CML-iPSC #1.31 with shC or with sh BCR). iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC without TKI. Mean ± SD, n = 3.</p

BCR-ABL1 independent proliferation.

<p>(<b>A</b>) Dose-effect of imatinib exposure (0–5 µM) for 6 days on CML-iPSC clones #1.22 and #1.31. Colony frequency is evaluated by alkaline phosphatase staining conducted at day 6. (<b>B</b>) Dose-effect of imatinib exposure for 6 days on iPSCs survival. iPSCs counts were conducted at day 6 and are expressed as percentages relative to same iPSC . Mean +/− SD n = 3, *: p<0.05 versus clone #1.22 with the same exposure. (<b>C</b>) Dose-effect of ponatinib exposure for 6 days on CML-iPSC clones (#1.22 Ph-, #1.24 and #1. 31 Ph+) survival. iPSCs counts are conducted at day 6 and expressed as percentages relative to same iPSC without TKI. Mean +/- SD, n = 3. * p <0.05 vs iPSC #1.22 (internal control Ph-) at the same TKI exposure. (<b>D</b>) Western-blot analysis of ABL, phosphotyr (p-Tyr) pattern, CRKL and phosphoCRKL (p-CRKL) in CML-iPSCs in absence (−) or presence (+) of imatinib (20 µM) for 48 h.</p