A Critical Role of Nuclear m6A Reader YTHDC1 in Leukemogenesis by Regulating MCM Complex-Mediated DNA Replication

YTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in Acute Myeloid Leukemia (AML) and that it is required for proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We find that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem/progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs, but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides the compelling evidence to show an oncogenic role and a distinct mechanism of YTHDC1 in AML.

Systemic Analysis of the DNA Replication Regulator MCM Complex in Ovarian Cancer and Its Prognostic Value

Microliposome maintenance (MCM) 2, MCM3, MCM4, MCM5, MCM6, and MCM7 are DNA replication regulators and are involved in the progression of multiple cancer types, but their role in ovarian cancer is still unclear. The purpose of this study is to clarify the biological function and prognostic value of the MCM complex in ovarian cancer (OS) progression. We analyzed DNA alterations, mRNA and protein levels, protein structure, PPI network, functional enrichment, and prognostic value in OC based on the Oncomine, cBioPortal, TCGA, CPTAC, PDB, GeneMANIA, DAVID, KEGG, and GSCALite databases. The results indicated that the protein levels of these DNA replication regulators were increased significantly. Moreover, survival analysis showed a prognostic signature based on the MCM complex, which performed moderately well in terms of OS prognostic prediction. Additionally, protein structure, functional enrichment, and PPI network analyses indicated that the MCM complex synergistically promoted OC progression by accelerating DNA replication and the cell cycle. In conclusion, our study suggested that the MCM complex might be a potential target and prognostic marker for OC patients.

Homozygous mutation in MCM7 causes autosomal recessive primary microcephaly and intellectual disability

BackgroundMinichromosomal maintenance (MCM) complex components 2, 4, 5 and 6 have been linked to human disease with phenotypes including microcephaly and intellectual disability. The MCM complex has DNA helicase activity and is thereby important for the initiation and elongation of the replication fork and highly expressed in proliferating neural stem cells.MethodsWhole-exome sequencing was applied to identify the genetic cause underlying the neurodevelopmental disease of the index family. The expression pattern of Mcm7 was characterised by performing quantitative real-time PCR, in situ hybridisation and immunostaining. To prove the disease-causative nature of identified MCM7, a proof-of-principle experiment was performed.ResultsWe reported that the homozygous missense variant c.793G>A/p.A265T (g.7:99695841C>T, NM_005916.4) in MCM7 was associated with autosomal recessive primary microcephaly (MCPH), severe intellectual disability and behavioural abnormalities in a consanguineous pedigree with three affected individuals. We found concordance between the spatiotemporal expression pattern of Mcm7 in mice and a proliferative state: Mcm7 expression was higher in early mouse developmental stages and in proliferative zones of the brain. Accordingly, Mcm7/MCM7 levels were detectable particularly in undifferentiated mouse embryonal stem cells and human induced pluripotent stem cells compared with differentiated neurons. We further demonstrate that the downregulation of Mcm7 in mouse neuroblastoma cells reduces cell viability and proliferation, and, as a proof-of-concept, that this is counterbalanced by the overexpression of wild-type but not mutant MCM7.ConclusionWe report mutations of MCM7 as a novel cause of autosomal recessive MCPH and intellectual disability and highlight the crucial function of MCM7 in nervous system development.

Shared and distinct roles of Esc2 and Mms21 in suppressing genome rearrangements and regulating intracellular sumoylation

Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements (dGCRs). How Mms21 targets its substrates in the cell is insufficiently understood. Here, we demonstrate that Esc2, a protein with SUMO-like domains (SLDs), recruits the Ubc9 SUMO conjugating enzyme to specifically facilitate Mms21-dependent sumoylation and suppress dGCRs. The D430R mutation in Esc2 impairs its binding to Ubc9 and causes a synergistic growth defect and accumulation of dGCRs with mutations that delete the Siz1 and Siz2 E3 ligases. By contrast, esc2-D430R does not appreciably affect sensitivity to DNA damage or the dGCRs caused by the catalytically inactive mms21-CH. Moreover, proteome-wide analysis of intracellular sumoylation demonstrates that esc2-D430R specifically down-regulates sumoylation levels of Mms21-preferred targets, including the nucleolar proteins, components of the SMC complexes and the MCM complex that acts as the catalytic core of the replicative DNA helicase. These effects closely resemble those caused by mms21-CH, and are relatively unaffected by deleting Siz1 and Siz2. Thus, by recruiting Ubc9, Esc2 facilitates Mms21-dependent sumoylation to suppress the accumulation of dGCRs independent of Siz1 and Siz2.

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress

Telomeres are intrinsically difficult-to-replicate regions of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a separation-of-function TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that specifically inhibited the TRF2-ORC interaction in human cells without substantially inhibiting TRF2 interactions with its other binding partners. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions in human cells. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

Role of Amygdalin in Blocking DNA Replication in Breast Cancer In Vitro

Background: Amygdalin has anticancer benefits because of its active component, hydrocyanic acid. However, the underlying molecular mechanism is unclear. Objective: This study aimed to investigate the molecular mechanism by which amygdalin exerts antiproliferative effects in the human Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line. Methods: MCF-7 cells were exposed to amygdalin at a particular IC50 value for 24 and 48 hours and compared to nontreated cells. An Affymetrix whole-transcript expression array was used to analyze the expression of 32 genes related to DNA replication. Results: Among the 32 genes, amygdalin downregulated the expression of 16 genes and 19 genes by >1.5-fold at 24 and 48 hours, respectively. At 24 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, PRIM1, and PRIM2; DNA polymerase δ complex: POLD3; DNA polymerase complex: POLE4, minichromosome maintenance protein (MCM) complex (helicase): MCM2, MCM3, MCM4, MCM6, and MCM7; clamp and clamp loader: PCNA; nuclease: FEN1; and DNA ligase: LIG1. At 48 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, and PRIM1; DNA polymerase δ complex: POLD3; DNA polymerase complex: POLE and POLE2; MCM complex (helicase): MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7; clamp and clamp loader: PCNA, RFC2, and RFC3; RNase H: RNASEH2A; nucleases: DNA2 and FEN1; and DNA ligase: LIG1. Conclusion: Amygdalin treatment caused downregulation of several genes that play critical roles in DNA replication in the MCF-7 cell line. Thus, it might be useful as an anticancer agent.