MCM5 mutation leads to silence of Stat1-bcl2 and accelerates the apoptosis of immature T lymphocytes with DNA damage

doi:10.21203/rs.3.rs-1993621/v1

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MCM5 mutation leads to silence of Stat1-bcl2 and accelerates the apoptosis of immature T lymphocytes with DNA damage

https://doi.org/10.21203/rs.3.rs-1993621/v1

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During T lymphocyte development, immature T lymphocyte undergoes V(D)J rearrangement of T-cell receptor (TCR) genes to further develop to functional T cells. Upon this situation, additional DNA damage coming from the mutation of DNA replication genes sensitively makes immature T cells undergo apoptosis. While the mechanisms how immature T lymphocytes undergo fast cell death upon genomic instability have not been resolved. In our current study, we showed that in zebrafish mcm5 mutants, the immature T cells specifically and cell-autonomously undergo rapid apoptosis during T lymphocytes maturation. In mechanism, mcm5 loss of function results in DNA damage and up-regulation of tp53 signaling, sequentially gives rise to immature T lymphocytes apoptosis. Meanwhile upon mcm5 mutant, the absence of MCM5 decreases MCM5-Stat1 complex and disrupts phosphorylation of Stat1, which sequentially inhibits bcl2a transcription and accelerates the apoptosis of immature T lymphocytes. As the evidence, after blocking DNA synthesizing using chemical DNA synthesis inhibitors, MCM5 facilitates the up-regulation of bcl2a transcription to avoid rapid death of immature T lymphocytes. Our study first provides a mechanism as to how immature T cells with DNA damage coming from endogenous DNA replication stress are rapidly cleared off during early development, as well explains the extra biological role of MCM5 beside the role in DNA duplication regulation.

Zebrafish

T lymphocytes

DNA damage

Bcl2

tp53

absence of enhancing Stat1-bcl2 axis facilitates immature T cells apoptosis in mcm5 mutants

T-cell acute lymphoblastic leukemia (T-ALL) is the most common leukemia ^1,2. Genomic instability promotes T-ALL initiation, metastasis and further deterioration ^3,4. During this process, genome instabilities such as deletion of p16INK4A ⁵, mutant of Mini-chromosome maintenance proteins 4/2 (MCM4/2) ^6–8, impaired Nonhomologous end joining (NHEJ) ² and DNA damage response (DDR) ⁹, defection of RNA splicing ^10,11, all of them collaborate with MYC to elevate cell proliferation, tumor initiation and expand early transformed T cells. Even leukaemic self-renewal and malignant haematopoiesis are constrained by promoting myeloid differentiation in myeloid stem cells ¹², the efficient clearance of immature lymphocytes with defect genome is regarded as a common way to avoid lymphoblastic leukemia ^13–15, this principle is supported by the fact that impeded cell apoptosis in p53 mutants or overexpression of anti-apoptotic signaling fuels lymphoblastic leukemia ^16–20.

During the development of T lymphocytes, it was reported that immature lymphocytes suffering serious and unrepaired DNA damage caused by gene mutation appear sensitive to undergo cell death ^8,21,22, and the remaining lymphocytes with unrepaired DNA damage finally develop to thymomas in the later development stage since DNA damage accumulation ^8,23,24. These evidences suggested that, the immature T cells with serious genomic instability tends be efficiently cleared off to avoid leukemia. On the other hand, DNA damage occurs from both the endogenous gene mutation and environmental stress ^25,26. When DNA damage is caused by mild and transient environment replication stress, the anti-apoptosis signaling would be activated to prevent cell from rapid death, which is beneficial for life continues and evolution ^24,27. Thus, it is important for the organism to evolve mechanisms to distinguish endogenous DNA damage stress from transient environmental DNA replication stress. Using such a mechanism, the organism could be able to clear the lymphocytes with heritable DNA damage came from endogenous mutation to avoid leukemia, and maintain life continue and enhance evolution when facing the mild environmental replication stress. While the mechanism as to how immature T cells with genome instability coming from gene mutation is efficiently wiped off and how the immature T cells under mild environmental replication stress is kept from rapid apoptosis is far from being resolved.

MCM5 is a member of the crucial component of DNA replication licensing system and MCM5 loss leads to DNA duplication defects ²⁸. Previous work demonstrated that the number of MCM5 is excess over that of replication origins ²⁹ and the excess MCM5 plays a critical role in regulation of gene transcription during embryonic development ³⁰. Here, we identified that, mcm5 loss disturbed DNA duplication of immature T lymphocyte and sequentially led to up-regulation of p53 signaling, which resulting in immature T lymphocyte apoptosis. Meanwhile, the absence of MCM5 disrupts the phosphorylation of Stat1 and the subsequent bcl2a transcription, accelerating the apoptosis of immature T lymphocyte with genomic instability. While under transient environmental DNA replication stress, MCM5 facilitates bcl2 transcription to prevent immature T lymphocyte from rapid death. Our result first provided a mechanism as to how immature T cells with DNA damage coming from endogenous replication stress are rapidly cleared off during early development.

Fish and Fish Maintenance

Wild type (AB), transgenic line Tg(c-myb:EGFP)³¹, Tg(coro1a:EGFP)³², Tg(gata1:desRed) ³³, Tg(lyz:desRed)³², Δ113p53^M/M ³⁴, mcm5^+/- ³⁵, mcm3^+/-, Tg(HSP70l:MCM5-t2a-mCherry) and Tg(Rag2:desRed) ³⁶ fishes were maintained in standard conditions at about 28.5℃. The developmental stages were characterized as previously described ³⁷.

MCM3 mutant construction

One sgRNA was designed according to the exon4 of mcm3 genomic DNA and the sgRNA was synthesized in vitro as the manual descripted (HiScribe™ T7 High Yield RNA Synthesis Kit，NEB，NO.E2040S). The detailed process was followed by previous reports ³⁵. An additional “A” was inserted into the genomic DNA and that resulted in a pre-stop codon at the NO. 178 amino acid in the CDS region of mcm3.

Cas9-sgRNA Ribonucleoprotein Complexes (RNPs) preparation and injection

To generate the mosic mutantin the F0 embryos, we optimized the CRISPR-cas9 gene editing process according to the previous reports ^38,39. In brief, the four/or three forward primers (targeting bcl2a/or GFP) and the reverse primer were designed on the web site (https://www.crisprscan.org/?page=gene), then the sgRNAs were synthesized in vitro using HiScribe™ T7 High Yield RNA Synthesis Kit (NEB，E2040S). Cas9 protein (EnGen^®Spy Cas9 NLS, NEB, M0646T）was purchased from NEB company. To examine the mutant efficiency of bcl2a, the injected embryos was collected and prepared the genomic DNA, and the semi-quantitative RT-PCR was used to evaluate as previous report ³⁷.

Chemical treatment

As previous report the zebrafish embryos were treated with 25nM Camptothecin(Beyotime, SC0141) and 2.5μM Roscovitine(Sigma, R7772) to disturb DNA replication (57). In brief, the chemical was diluted with egg water as the concentration descripted above, the embryos were incubated with the chemical from 3dpf to the stage required.

MO and mRNA injection

Morpholino oligos (MO) for mcm5⁴⁰, p53 ⁴¹, stat1a ⁴², and control were obtained from Gene Tools. mcm5 mRNA, Δ113p53mRNA and bcl2a mRNA were synthesized in vitro using mMESSAGE Kit (Ambion, AM1340). The concentration of MO was as following: mcm5 MO, 300uM; p53 MO, 200 μM; stat1a MO, 500 μM; control MO, 500 μM. The concentration for mRNA injection was as following: mcm5 mRNA, 30ng/μl; bcl2a mRNA, 30ng/μl; Δ113p53mRNA, 30ng/μl. All the MOs and mRNAs were injected at 1-4 cell stage.

RT-qPCR

RT- qPCR was performed using the Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies) and the CFX96 Real-Time System (BIO-RAD) according to manufactures instruction. Transcription of beta-actin was used for normalization. Primers are listed in table S 1. All the experiments were repeated at least for 3 times.

Whole-Mount in situ hybridization

In situ hybridization was performed as described previous study ³⁷. The previous probes mcm5, rag2, rag1, foxn1, ccl25a, ikaros, scl, hbbe3, gata1, lmo2, pu.1 c-myb, il7r, GH were used as previous reports ^36,37,43. The CDs of mcm3, bcl2a and the specific region of Δ113p53 were amplified using PCR and cloned into the vector pcs2⁺, then linearized the plasmids and synthesized the individual antisense probe. To synthesize p53 specific probe, the CDs of p53was amplified using PCR (Sp6 promoter was added to the start end of the Reverse Primer), part of the PCR product was used for sequencing to evaluate whether the right PCR product was got, the remaining PCR product was used as the template to synthesize antisense probes³⁷.

Sudan black staining and O-dianisidine staining

The Sudan black staining is referenced to previous report ⁴⁴. O-dianisidine staining (Aladdin119-90-4 ) was carried out as previous report ⁴⁵. The staining embryos could be store at 4℃ to avoid light for photography.

Western Blot and cell transfection

To examine the protein level of Tp53(Genetex, GTX128135), γH2A (Genetex, GTX127340), BCL2a (abcam, ab182858), Stat1 (Santa cruz, SC-464) and p-Stat1 (Santa cruz, SC-8394) in embryos with different treatment, about 50–100 embryos were collected for protein extraction. Western blotting was performed as described previously⁴⁶. 293T cells were cultured to a density of 80%-90% in 6-well plates. Transfection reagent (Invitrogen,L3000001) and siRNA (Purchased from RIBO Bio) or plasmid were mixed in EP tubes and placed at room temperature for 10-15 minutes before being added into 6-well plates. The transfection effect was detected by Western blot 72h after transfection.

Co-Immunoprecipitation (Co-IP)

Co-immunoprecipitation was performed as described previously ⁴⁷. In brief, as the Co-immunoprecipitation experiment, add 4μg antibody (Anti-Mouse-Flag, Sigma, F1804) to the remaining samples and incubate overnight at 4℃. Then prepare new tube to rinse 70μ L protein G (Invitrogen,10003D) with 1XPBS, centrifuge at 2000rpm for 5 min, discard the supernatant and get the rinsed protein G. Next the protein G was added into the samples and incubate at 4℃ overnight. Then centrifuge it for 5 min at 4℃ (2000rpm) and discard the supernatant, wash the precipitate 4-5 times with 1mL NP-40 buffer for 15 min each time. Finally add 2X Loading buffer into the precipitate, boil the mix at 100℃ for 7 minutes, centrifuge at 2000rpm for 5 minutes, collect the supernatant and store at -20℃ for Western Blot detection. The primary antiobody Anti-rabbit-HA (abcam, AB236632) and anti-GFP (Cloud Clone, PAD025Ge07) was used in this research.

Immunostaining

The embryos were fixed overnight with PEM at 4℃, washed with PBS (5min 3x) and blocked with PBTN (4% BSA, 0.02%NaN_{3 ,}in PT) for 2 hours at 4℃. Then the primary antibody H3p (GTX128116), γH2A (Genetex, GTX127340) were diluted with PBTN in accordance with 1:100, incubated on shaker at 4℃ for overnight. Then wash the embryos with PT (0.3% Triton-X-100, in 1X PBS) at least 20min for 8 times. The secondary antibody (GeneTex 26800) was diluted with PBTN in accordance with 1:500 and added. Incubate the embryos overnight at 4℃ (Keep in dark). Finally, the embryos were washed with PT for more than 8 times (30min every time) and preceded for imaging.

EDU experiment

The EDU staining was processed as previous reports ⁴⁸. BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, C0078S) was used for this experiment. After EdU staining reaction the embryos were washed with PT for 3 times, flowing Immunostaining for GFP.

Pro-apoptotic cell staining

Embryos were dechorionated and fixed in 4% paraformaldehyde overnight at 4℃, then washed with PBST 3 times (10 minutes each time) and stored in 100% methanol overnight. After that the embryos were washed 3 times with PBST for 3 times, then In Situ Cell Death Detection Fluorescein kit (Roche11684795910) was applied to examine cell apoptosis according to the manufacturer’s instructions.

Statistical analysis

The data were analyzed with Novoexpress, ImageJ, statistical software in Graphpad Prism 8 for Windows (GraphPad Software). Student’s t tests (two-tailed, unequalvariance) were performed to determine the statistical significance of the differences. Quantitative data were presented as means S.D. Experiments were performed at least three times for each experiment. NS, not significant, “*” p < 0.05, “**” p < 0.01 and “***” p < 0.001.

mcm5 specifically regulates T lymphocytes maturation during definitive hematopoiesis

Mcm5 is a crucial component of DNA duplication initiators and loss of Mcm5 function results in DNA replication defects and genomic instability ²⁸. In zebrafish, mcm5 is highly expressed in proliferating cells including the hematopoietic stem cells (HSCs) and cells in thymus (⁴⁰ and Fig. S1). To study the process of T cell development under genomic instability caused by endogenous DNA replication stress, a mcm5 nonsense mutation was generated (³⁵ and Fig. S2). The data showed that the primitive hematopoiesis was not disturbed in mcm5^−/− embryos (Fig. S3 A-F). This result was confirmed by the data in mcm5 morphants, where the transcription of maternal mcm5 mRNA was blocked ⁴⁰(Fig. S3 A, C). At the secondary wave of hematopoiesis, even though the erythrogenesis was not affected greatly (Fig.S4 A, B), the expression of rag1, il7r, ikaros, rag2 was disappeared on 5dpf (Fig. 1A, B). Further the data showed that the expression of rag2 was intact on 3.5dpf in mcm5 mutant embryos (Fig. 1C, E), and it was greatly downregulated on 4dpf (Fig. 1D, E). To confirm the specific role of mcm5 in T cell maturation, mcm5 mRNA was used to rescue the T cell phenotype in mcm5 mutants. The data showed that the T cell developmental defect was rescued by injection of mcm5 mRNA (Fig. 1F, G).

Since defects in thymus epithelium, HSCs specification and migration will result in T lymphocyte developmental defects ⁴³, then we examined whether these events are disturbed in mcm5^−/− embryos. The expression of ccl25a and foxn1 indicated that no significant defect for thymus epithelium development was observed in mcm5^−/− embryos (Fig. S5 A-C), the HSC specification (Fig.S6 A, C) and migration (Fig. S6 B, D) was also normal in mcm5^−/− embryos. These data implied that mcm5 cell autonomously regulates the maturation of T lymphocyte during definitive hematopoiesis.

Immature T cells undergo increased cell-apoptosis in mcm5^−/− embryos

In the transgenic line Tg(c-myb:GFP) only HSCs but not immature T lymphocytes are labeled with GFP from 4dpf (Fig. 2A, B and Fig S7 A), whereas in Tg(coro1a:GFP) embryos the GFP labeled cells were most overlapped with immature T lymphocytes in the thymus epithelium area (Fig. 2A, C and Fig.S7 B). Next we used Tg(coro1a:GFP): mcm5^−/− embryos to follow the detailed process of T lymphocyte development upon mcm5 loss of function. Indeed, the number of coro1a:GFP labeled cells was not different between siblings and mcm5^−/− embryos on 3.5dpf (Fig. 2D and Fig. S8 B, C, G, H), but it was reduced greatly in mcm5^−/− embryos on 4dpf (Fig. 2D and Fig. S8 E, J). This phenotype can be rescued by injecting mcm5 mRNA (Fig. 2D, E). In addition, from 3.2dpf to 4dpf, more coro1a:GFP labeled cells appeared to undergo apoptosis in mcm5^−/− embryos (Fig. S8 G-I, white arrow), and Tunel, H3p antibody staining and Edu staining experiments further confirmed that the immature T lymphocytes displayed proliferation defects and an increased pro-apoptotic phenotype (Fig. 2F, G and Fig. S9). To further determine if mcm5 was only involved in T lymphocyte maturation while not in early events during T lymphocyte development, the Tg(Hsp70l:mcm5-desRed) transgenic line was generated and mcm5 was induced to express on 3dpf by heat shock in mcm5^−/− embryos (Fig. S10 A, B). After this treatment, the immature T cells were restored in mcm5^−/− embryos (Fig. 2D, E and Fig. S10 C). These results further demonstrated that the immature T cells, but not the early progenitors, undergo apoptosis in mcm5^−/− embryos from 3.5dpf and thereafter.

Tp53 signaling mediates the increased T cell apoptosis upon mcm5 loss of function

To find out the mechanism as to how immature T lymphocyte undergoes apoptotic cell death in mcm5^−/− embryos, we did RNA-seq analysis comparing siblings and mcm5^−/− embryos (Fig.S11). The results indicated that the genes being related to cell cycle, apoptosis and Tp53 signaling were altered in mcm5^−/− embryos (Fig. 3A and Figure S11). This is consistent with the results of in situ, RT-qPCR and Western Blot: even though the level of tp53 transcripts was not changed (Fig. 3B, C), the protein levels of Tp53 and the transcription of mdm2 and tp21 were upregulated (Fig. 3B, C). Further, the data showed that inhibition of Tp53 blocked apoptosis of immature lymphocytes (Fig. 3D) and partially restored the number of T lymphocyte (Fig. 3E, F). These results demonstrated that up-regulation of Tp53 signaling mediates T cell apoptosis upon MCM5 loss of function.

Immature T cells in mcm5 mutants are more sensitive to DNA damage

Previous study reported that disruption of DNA replication results in T cell apoptosis ⁸. Our data showed that the expression of γ H₂A was increased in immature lymphocytes in mcm5 mutants (Fig. 4. B, C), suggesting the possibility that DNA replication defect in mcm5 mutants is the apoptosis inducer. To test this hypothesis, camptothecin and roscovitine were used to transiently block DNA synthesis separately ²⁷, and then we examined whether transient disruption of DNA duplication lead to T lymphocyte cell cycle arrest and the subsequent apoptosis (Fig. 4A). Both chemical treatments resulted in decreased number of T lymphocytes on 4dpf (Fig. 4E, F), accompanied with more serious DNA duplication defects and Tunel staining than that in mcm5^−/− embryos (Fig. 4B-D). In addition, p53 downstream genes, p21 and mdm2 were up-regulated (Fig. S12). These results suggested that DNA damage induced upregulation of Tp53 signaling, then mediated immature T cell apoptosis. Interestingly, even though the pro-apoptotic signaling in chemical treated embryos was more severe than that in mcm5^−/− embryos, the number of immature T cells on 4dpf was much more than that in mcm5^−/− embryos (Fig. 4E, F). An immediate question was why serious DNA damage in chemical treatment resulted in fewer numbers of immature T cell death.

Silence of bcl2a in mcm5 mutants accelerates the death of pro-apoptotic immature T cells

It is well known that when cell facing DNA duplication and the other developmental stress, the pro-apoptotic and anti-apoptotic pathways are simultaneously stimulated to orchestrate cell survival or death ^27,49,50. To find out why the serious DNA damage in embryos treated with chemical resulted in fewer numbers of immature T cell death, we examined whether the anti-apoptotic signaling Bcl2 and Δ113p53 were different between the embryos treated with chemical and embryos with mcm5 mutant. Our RNA-seq data had shown that tp53 was greatly upregulated (Fig. 3A and Fig. S11), but the results of in situ and RT-qPCR showed that the expression of tp53 was not different between siblings and mcm5 mutants (Fig. 3B, C). These results raised the possibility that Δ113p53 isoform might be upregulated in mcm5^−/− embryos. Indeed, our data showed that in mcm5^−/− embryos, the expression of Δ113p53 was upregulated (Fig. 5A). Further data showed that injection of Δ113p53 mRNA rescued the T cell defect in mcm5 mutant embryos on 4dpf (Fig. 5B, E), and in Δ113p53^−/−:mcm5^−/− double mutant embryos the T cell defect was more serious than that in mcm5^−/− embryos (Fig. 5B, Fig. S13). Even Δ113p53 was upregulated in mcm5 mutants, but it’s expression was also increased in embryos treated with camptothecin (Fig. 5A), these data demonstrated that the upregulation of Δ113p53 was not the underlying reason why the embryos treated with chemical displayed more numbers of immature T cells. Sequentially, we examined the expression of bcl2a in all these embryos. The data showed that bcl2a was downregulated in mcm5 mutants (Fig. 5C), whereas in chemical treated embryos the expression of bcl2a was upregulated (Fig. 5C), and Bcl2 protein was also upregulated (Fig. 5D). These results implied that the downregulation of bcl2a enhanced the T cell apoptosis in mcm5 mutants. To evaluate this hypothesis, bcl2a mRNA was injected in mcm5^−/− embryos. The data showed that T cell numbers were partially restored on 4dpf in mcm5^−/− embryos injected with bcl2a mRNA (Fig. 5E, F). In addition, when the function of bcl2a was knocked-out transiently by injecting sgRNR-Cas9 complex into one-cell embryos (Fig. S14 B), the number of T lymphocytes was greatly decreased compared with controls (Fig.S14 C, c2-c6). All these results above suggested that the expression of bcl2a cannot be activated efficiently and this accelerates the death of pro-apoptotic immature T cells in mcm5 mutants.

Downregulation of stat1signaling mediates silence of bcl2 cascade in mcm5 mutants

Previous literature has reported that activation of Stat1 enhances the expression of bcl2, and sequentially promotes leukemia ¹⁷, and in zebrafish stat1a morphants the neutrophils are increased ⁴². In mcm5 mutants, we found that the neutrophils were also increased (Fig. S15 A, B) and the expression of stat1 downstream genes were downregulated (Fig. S15 C). These facts implied the possibility that Stat1 signaling is downregulated in mcm5 mutants. To test this hypothesis, the protein of Stat1 and p-Stat1 was examined. The level of p-Stat1 was reduced while Stat1 was not decreased in mcm5 mutants (Fig. 6A), and this role was conserved in human cells (Fig. S16). To establish whether activated Stat1 mediates Bcl2 signaling to protect immature T cells from apoptosis under DNA replication stress, we injected stat1a MO into the embryos to block stat1a translation, sequentially treated the embryos with camptothecin on 3dpf, then examined T lymphocyte development and the expression of bcl2a. We observed that inhibition of stat1a resulted in a severe decrease of immature T cells compared to controls on 4dpf (Fig. 6B), and blocked the upregulation of bcl2a transcription in embryos treated with camptothecin (Fig. 6C). In addition, after inhibiting stat1a, injection of bcl2a mRNA partially restored T lymphocyte development in the embryos treated with camptothecin (Fig. 6D). These results demonstrated that the enhanced Stat1-Bcl2 signaling protects immature T cells from terminal apoptosis under transient environmental stress, while the absence of up-regulating Stat1-bcl2 axis accelerates the apoptosis of immature T cells in mcm5 mutants.

To uncover how MCM5 facilitates Stat1a to promote bcl2a transcription, the interaction of Stat1a and MCM5 was examined using Co-immunoprecipitation (Co-IP). The experiments showed that zebrafish MCM5 and Stat1a bind to each other (Fig. 6E), indicating the possibility that MCM5 directly binds to Stat1a and facilitates Stat1a phosphorylation to enhance bcl2a expression. Since previous study reported that MCM3 works as a partner for MCM5 to activate the expression of target genes in vitro ⁵¹, next we examined whether MCM3 loss of function is similar to mcm5 mutants. Indeed, in mcm3 mutants (Fig. S 17 A-E), rag1 expression and Coro-GFP labeled cell was decreased (Fig. 6F and Fig. S 18 A), accompanying that more immature T cells displayed increased pro-apoptotic signal and DNA damage than that in controls (Fig. S18 B-D), the transcription of bcl2a is downregulated whereas Δ113p53 is elevated (Fig. 6G and Fig. S18 E). In addition, the number of immature T cell was less than that in embryos treated with camptothecin, and injection of bcl2a mRNA partially rescued the T lymphocyte developmental defect (Fig. S18 A). These data further supported that MCM5/Stat1 complex is required to enhance the expression of bcl2a to prevent immature T cell from rapid death under DNA replication stress.

Previous literatures showed that in zebrafish, part of HSCs migrate into thymus to develop as T lymphocytes and be matured from 7dpf. So thymus, as the T cell developmental niches, is critical for T cell maturation during this process ^43,52. Our data identified that in mcm5^−/− embryos, the immature T lymphocytes were sensitive to DNA damage during T cell maturation (Fig. 1A-D, Fig. 2D, E and Fig. S8), and this stage-specific role of mcm5 was via a T lymphocyte cell-autonomous way while not be related to thymus development (Fig. S5). In mechanism, our data showed that mcm5 loss of function decreased the level of phosphorylation of Stat1 and the expression of bcl2a, the downstream gene of Stat1 signaling (Fig. 6A and Fig. 5C, D ¹⁷), which partially explained why T cell maturation is sensitive to DNA damage in mcm5 mutants ²⁷.

During hematopoiesis T lymphocytes display more sensitive to developmental stress including DNA replication stress ^21,22. One possible mechanism is relative to T cell specification and maturation, since in this stage the developing T cells undergo V(D)J rearrangement of T-cell receptor (TCR) genes and more additional specific genes are transcribed ^53,54. While in zebrafish top3a^−/− embryos the developing lymphocytes displayed specific and sensitive impairment, but repressing the function of rag1 didn’t rescue the severe phenotype²¹. This data implied the complicated mechanism underlying T lymphocyte development exists, which is far from being resolved. Although the mechanism why the developing T lymphocytes are sensitive to DNA damage is not resolved, the biological significance of this phenomenon is obvious because, DNA damage accumulation contributes carcinogenesis as animals get older ^8,23. So to avoid leukemia, the immature T lymphocyte with heritable DNA damage tends to undergo death efficiently ²¹. But in paradox, to maintain life the organism/cells, including the immature T lymphocytes, must evolve a mechanism to protect them from rapid death under transient replication stress. Indeed, our data demonstrated that the anti-apoptotic factor Δ113p53 was highly expressed upon endogenous DNA stress and transient exogenous DNA replication stress (Fig. 5A), but only upon endogenous heritable DNA damage in mcm5 mutants, silence of stat1-bcl2 cascade accelerate the rapid T cell death.

Mouse Pole3 was discovered to play an essential role in T cell differentiation, and this role was non-replicative functions of the ubiquitous POLE complex ⁵⁵, implying the additional role of DNA replication factors exists besides the role in DNA replication regulation⁵⁶. Our research identified MCM5 plays a critical anti-apoptosis role through Stat1-bcl2a axis (Fig. 6H), it first gives a mechanism as to how immature lymphocytes with endogenous heritable DNA damage are efficiently cleared off via apoptotic cell death: orchestrating upregulation of Tp53 cascade and silencing of Bcl2 enhancement. This work highlights the means to find oncotarget genes whose loss/gain of function could simultaneously result in upregulation of apoptotic signaling and silencing of anti-apoptotic signaling.

Acknowledgments:

We would like to thank Dr. Li Li, Dr. Yiyue Zhang, Dr. Jun Chen, Dr. Honghui Huang to support the plasmids and different fish lines, Dr. Karuna Sampath for editing the manuscript and critical comments, Dr. Huaqing Sun, Dr. Xiaohong Liu and Dr. Tao Zhang for reading and nice commence to the manuscript; we also would like to thank the members working in our fish facility for their help taking care of all the fish lines in this study.

Funding:

This work was supported by the National Natural Science Foundation of China (No. 32070805), the Science and Technology Department of Sichuan Province (2021ZYD0074, 2021JDRC0171) and Disciplinary Construction Innovation Team Foundation of Chengdu Medical College: CMC-XK-2102.

Author contributions:

Conceptualization: SZH；Methodology: SZH, ML；

Investigation: SZH, ML, JMX, KZ, YD, YYL, YDL,

Visualization: ML, SZH, JMX, YF, BYS, SRL；

Funding acquisition: SZH, ML；

Project administration: ML, SZH；

Supervision: SZH, ML；

Writing – original draft: SZH, XDL, YF, YD；

Writing – review & editing: SZH, YF, ML, YYL;

Competing interests:

Authors declare that they have no competing interests

Data and materials availability:

All data are available in the main text or the supplementary materials.

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MCM5 mutation leads to silence of Stat1-bcl2 and accelerates the apoptosis of immature T lymphocytes with DNA damage

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