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Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology
Courtney M Mazur1, Parthena E Kotsalidis1, Majd George1
1Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Abstract:
Osteocytes play critical roles in bone, making them attractive targets for therapeutics aimed at improving bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here, we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin β3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61high cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule-binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost, and impaired dendrite morphology in vitro. Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify the role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.
Insights
Researchers identified CD61 as a marker for osteocyte maturation. A genome-wide screen revealed CLIP2 is crucial for osteocyte function, impacting dendrite development and gene expression.
Area of Science:
- Bone Biology
- Cellular and Molecular Medicine
- Genomics
Background:
- Osteocytes are crucial for bone health, but the genes regulating their maturation and function remain largely unknown.
- Understanding these genes is vital for developing therapeutics to enhance bone mass and strength.
Purpose of the Study:
- To identify novel genes involved in osteocyte differentiation and dendrite development.
- To establish CD61 as a reliable marker for osteocyte maturation.
Main Methods:
- Genome-wide CRISPR-interference (CRISPRi) screening was performed in an osteocyte-like cell line (Ocy454).
- A flow cytometry assay was developed to quantify surface CD61 protein levels.
- CLIP2 gene function was assessed following inhibition.
Main Results:
- CD61 (integrin β3) was identified as a marker of osteocyte maturation, with increased surface expression correlating with osteocyte marker gene levels.
- CLIP2 was identified as a gene essential for CD61 expression.
- CLIP2 inhibition decreased CD61 expression, reduced osteocyte-specific gene expression (Dmp1, Sost), and impaired osteocyte dendrite morphology.
Conclusions:
- Surface CD61 is a valuable marker for assessing osteocyte maturity.
- The microtubule cytoskeleton, influenced by CLIP2, plays a significant role in osteocyte differentiation, morphology, and function.
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