RESUMO
Insect reproductive capacity can affect effective pest control and infertility studies and has become an important focus in recent molecular genetic research. Nucleosome assembly protein (Nap) is highly conserved across multiple species and is involved in forming the sperm nucleus in many species. We used clustered regularly interspaced palindromic repeats/Cas9 technology to knockout BmNap in Bombyx mori and observed that the mutations caused female infertility, whereas male fertility was not affected. BmNap mutants grew and mated normally; however, female mutants laid smaller eggs that could not be fertilised and did not hatch. In addition, female sterility produced by the mutation could be inherited stably via male mutants; therefore, Nap could be used as a potential target for lepidopteran pest control through population regulation. In the current study, we elucidated a new function of BmNap, increased the understanding of the oogenesis regulation network in Lepidoptera and promoted the development of insect sterility technologies.
RESUMO
Proliferating cell nuclear antigen (PCNA), the homotrimeric eukaryotic sliding clamp protein, recruits and coordinates the activities of a multitude of proteins that function on DNA at the replication fork. Chromatin assembly factor 1 (CAF-1), one such protein, is a histone chaperone that deposits histone proteins onto DNA immediately following replication. The interaction between CAF-1 and PCNA is essential for proper nucleosome assembly at silenced genomic regions. Most proteins that bind PCNA contain a PCNA-interacting peptide (PIP) motif, a conserved motif containing only eight amino acids. Precisely how PCNA is able to discriminate between binding partners at the replication fork using only these small motifs remains unclear. Yeast CAF-1 contains a PIP motif on its largest subunit, Cac1. We solved the crystal structure of the PIP motif of CAF-1 bound to PCNA using a new strategy to produce stoichiometric quantities of one PIP motif bound to each monomer of PCNA. The PIP motif of CAF-1 binds to the hydrophobic pocket on the front face of PCNA in a similar manner to most known PIP-PCNA interactions. However, several amino acids immediately flanking either side of the PIP motif bind the IDCL or C-terminus of PCNA, as observed for only a couple other known PIP-PCNA interactions. Furthermore, mutational analysis suggests positively charged amino acids in these flanking regions are responsible for the low micromolar affinity of CAF-1 for PCNA, whereas the presence of a negative charge upstream of the PIP prevents a more robust interaction with PCNA. These results provide additional evidence that positive charges within PIP-flanking regions of PCNA-interacting proteins are crucial for specificity and affinity of their recruitment to PCNA at the replication fork.
Assuntos
Fator 1 de Modelagem da Cromatina , Modelos Moleculares , Antígeno Nuclear de Célula em Proliferação , Ligação Proteica , Saccharomyces cerevisiae , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/química , Fator 1 de Modelagem da Cromatina/genética , Cristalografia por Raios X , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Motivos de Aminoácidos , Sítios de Ligação , Sequência de Aminoácidos , Conformação ProteicaRESUMO
Nucleosome assembly proteins (NAPs) have been identified as histone chaperons. Testis-Specific Protein, Y-Encoded-Like (TSPYL) is a newly arisen NAP family in mammals. TSPYL2 can be transcriptionally induced by DNA damage and TGFß causing proliferation arrest. TSPYL1, another TSPYL family member, has been poorly characterized and is the only TSPYL family member known to be causal of a lethal recessive disease in humans. This study shows that TSPYL1 and TSPYL2 play an opposite role in TGFß signaling. TSPYL1 partners with the transcription factor FOXA1 and histone methyltransferase EZH2, and at the same time represses TGFBR1 and epithelial-mesenchymal transition (EMT). Depletion of TSPYL1 increases TGFBR1 expression, upregulates TGFß signaling, and elevates the protein stability of TSPYL2. Intriguingly, TSPYL2 forms part of the SMAD2/3/4 signal transduction complex upon stimulation by TGFß to execute the transcriptional responses. Depletion of TSPYL2 rescues the EMT phenotype of TSPYL1 knockdown in A549 lung carcinoma cells. The data demonstrates the prime role of TSPYL2 in causing the dramatic defects in TSPYL1 deficiency. An intricate counter-balancing role of TSPYL1 and TSPYL2 in regulating TGFß signaling is also unraveled.
Assuntos
Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Transição Epitelial-Mesenquimal/genética , Linhagem Celular TumoralRESUMO
Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.
Assuntos
Histonas , Schizosaccharomyces , Histonas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Saccharomyces cerevisiae/genética , DNA/metabolismo , Nucleossomos/metabolismoRESUMO
The long-term effectiveness of targeted cancer therapies is limited by the development of resistance. Although epigenetic reprogramming has been implicated in resistance, the mechanisms remain elusive. Herein, we demonstrate that increased chromatin accessibility is involved in adaptive BRAF inhibitor (BRAFi)-resistance in melanoma cells. We observed loss of chromatin assembly factor 1 (CAF-1) and its related histone H3 lysine 9 trimethylation (H3K9me3) with adaptive BRAFi resistance. We further showed that depletion of CAF-1 provides chromatin plasticity for effective reprogramming by AP1 components to promote BRAFi resistance. Our data suggest that therapeutic approaches to restore H3K9me3 levels may compensate for the loss of CAF-1 and, in turn, suppress resistance to BRAF inhibitors.