The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin
Abstract
Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein Origin Recognition Complex-Associated (ORCA/LRWD1) preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affects replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.
Article and author information
Author details
Reviewing Editor
- Michael R Botchan, University of California, Berkeley, United States
Version history
- Received: January 15, 2015
- Accepted: April 27, 2015
- Accepted Manuscript published: April 29, 2015 (version 1)
- Version of Record published: May 26, 2015 (version 2)
Copyright
© 2015, Giri et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,051
- views
-
- 771
- downloads
-
- 35
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
Elastic cartilage constitutes a major component of the external ear, which functions to guide sound to the middle and inner ears. Defects in auricle development cause congenital microtia, which affects hearing and appearance in patients. Mutations in several genes have been implicated in microtia development, yet, the pathogenesis of this disorder remains incompletely understood. Here, we show that Prrx1 genetically marks auricular chondrocytes in adult mice. Interestingly, BMP-Smad1/5/9 signaling in chondrocytes is increasingly activated from the proximal to distal segments of the ear, which is associated with a decrease in chondrocyte regenerative activity. Ablation of Bmpr1a in auricular chondrocytes led to chondrocyte atrophy and microtia development at the distal part. Transcriptome analysis revealed that Bmpr1a deficiency caused a switch from the chondrogenic program to the osteogenic program, accompanied by enhanced protein kinase A activation, likely through increased expression of Adcy5/8. Inhibition of PKA blocked chondrocyte-to-osteoblast transformation and microtia development. Moreover, analysis of single-cell RNA-seq of human microtia samples uncovered enriched gene expression in the PKA pathway and chondrocyte-to-osteoblast transformation process. These findings suggest that auricle cartilage is actively maintained by BMP signaling, which maintains chondrocyte identity by suppressing osteogenic differentiation.
-
- Cancer Biology
- Cell Biology
Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.