RESUMO
Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.
Assuntos
Timina DNA Glicosilase , Proteína Supressora de Tumor p53 , Animais , Camundongos , Ciclo Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
t(9;11)-Induced leukemia is present both in children and adults, and depending on age, can cause predominantly acute lymphoblastic (ALL) or acute myeloid leukemia (AML), respectively. In general, in infants, it is characterized by poor (ALL) or intermediate (AML) prognosis, whereas in adults, it is classified as being of intermediate-high risk [15,24,31]. Its hallmark is the chromosomal translocation between chromosomes 9 and 11, leading to the formation of the MLL-AF9 fusion gene. The expressed chimeric protein was shown to be crucial for leukemia progression. MLL-AF9 recruits - among other factors - the super elongation complex (SEC), leading to aberrant activation of target genes [4,5,9,17,24]. The Polycomb group of proteins plays crucial roles in many processes, such as embryogenesis, differentiation, and maintaining cell homeostasis, and recently reports linking it to MLL-AF9 have emerged. This review will focus on its role in t(9;11)-related leukemia, highlighting the possible therapeutic-targeting strategies.
Assuntos
Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Adulto , Criança , Humanos , Lactente , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Proteínas do Grupo Polycomb/genética , Translocação Genética/genéticaRESUMO
Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.
Assuntos
Leucemia Promielocítica Aguda , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Humanos , Fator 4 Semelhante a Kruppel , Leucemia Promielocítica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição/metabolismo , Tretinoína/farmacologiaRESUMO
In this work we demonstrate that the previously described reaction of sequence specific Ni(ii)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys2His2 zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(ii) ions. It results in loss of the Zn(ii) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(ii)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(ii)-susceptible sequence between the edited zinc finger and an affinity tag allows for removal of the latter sequence by Ni(ii) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(ii) ions. Mass spectra revealed that a Ni(ii) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(ii)-dependent protein hydrolysis is influenced by the Ni(ii) concentration, pH and temperature of the reaction provides a platform for novel regulated DNA effector design.
