Estrogen Receptor Chromatin Profiling by CUT&RUN.

Area of Science:

• Molecular Biology
• Neuroscience
• Endocrinology

Background:

• Gonadal steroid hormones regulate gene transcription via nuclear hormone receptors (HRs).
• Identifying HR genomic binding sites is crucial for understanding hormone signaling in health and disease.
• Traditional ChIP-seq lacks sensitivity for detecting transcription factor (TF) binding in limited cell numbers, such as specific neuronal subtypes.

Purpose of the Study:

• To present a detailed protocol for Cleavage Under Targets & Release Under Nuclease (CUT&RUN) to map genome-wide estrogen receptor alpha (ERα) binding in mouse brain tissue.
• To offer a sensitive method for detecting protein-DNA interactions in small cell populations.

Main Methods:

• Development and description of a stepwise CUT&RUN protocol.
• Application of the protocol to detect ERα genome-wide binding in mouse brain tissue.
• Analysis of TF binding sites using CUT&RUN with as few as 100-1000 cells.

Main Results:

• The CUT&RUN protocol enables sensitive detection of ERα genome-wide binding in mouse brain tissue.
• The method successfully identifies TF binding sites in limited cell numbers, overcoming ChIP-seq limitations.
• The protocol is adaptable for studying most TFs in brain tissue.

Conclusions:

• CUT&RUN is a highly sensitive technique for mapping HR and TF binding sites in the brain.
• This protocol facilitates the study of hormone signaling mechanisms in genetically defined neuronal subtypes.
• The described CUT&RUN method advances the understanding of gene regulation by steroid hormones in the central nervous system.