RESUMO
Interactions between proteins and DNA are crucial for all biological systems. Many studies have shown the dependence of protein-DNA interactions on the surrounding salt concentration. How these interactions are maintained in the hypersaline environments that halophiles inhabit remains puzzling. Towards solving this enigma, we identified the DNA motif recognized by the Halobactrium salinarum ROS-dependent transcription factor (hsRosR), determined the structure of several hsRosR-DNA complexes and investigated the DNA-binding process under extreme high-salt conditions. The picture that emerges from this work contributes to our understanding of the principles underlying the interplay between electrostatic interactions and salt-mediated protein-DNA interactions in an ionic environment characterized by molar salt concentrations.
Assuntos
Proteínas Arqueais/química , DNA Arqueal/química , Halobacterium salinarum/genética , Cloreto de Potássio/química , Tolerância ao Sal/genética , Fatores de Transcrição/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Arqueal/genética , DNA Arqueal/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Halobacterium salinarum/metabolismo , Haloferax/genética , Haloferax/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Cloreto de Potássio/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Estresse Fisiológico , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations.
Assuntos
Deformidades Congênitas dos Membros/genética , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/fisiologia , Animais , Região Branquial/metabolismo , Elementos Facilitadores Genéticos/genética , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox , Histona Desacetilases/genética , Proteínas de Homeodomínio/genética , Botões de Extremidades/metabolismo , Deformidades Congênitas dos Membros/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Proteínas Repressoras/genética , Fator de Transcrição AP-2 , Fatores de Transcrição/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Protein-DNA interactions are highly dependent on salt concentration. To gain insight into how such interactions are maintained in the highly saline cytoplasm of halophilic archaea, we determined the 3-D structure of VNG0258H/RosR, the first haloarchaeal DNA-binding protein from the extreme halophilic archaeon Halobactrium salinarum. It is a dimeric winged-helix-turn-helix (wHTH) protein with unique features due to adaptation to the halophilic environment. As ions are major players in DNA binding processes, particularly in halophilic environments, we investigated the solution structure of the ionic envelope and located anions in the first shell around the protein in the crystal using anomalous scattering. Anions that were found to be tightly bound to residues in the positively charged DNA-binding site would probably be released upon DNA binding and will thus make significant contribution to the driving force of the binding process. Unexpectedly, ions were also found in a buried internal cavity connected to the external medium by a tunnel. Our structure lays a solid groundwork for future structural, computational and biochemical studies on complexes of the protein with cognate DNA sequences, with implications to protein-DNA interactions in hyper-saline environments.
