HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries.
J Vis Exp
; (145)2019 03 31.
Article
em En
| MEDLINE
| ID: mdl-30985763
ABSTRACT
CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Genes Homeobox
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
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Fator de Ligação a CCCTC
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
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Screening_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article