Full Length ArticleBiological functions of chromobox (CBX) proteins in stem cell self-renewal, lineage-commitment, cancer and development
Graphical abstract
Introduction
The reversible activation or suppression of gene transcription is mediated by chromatin remodeling which controls access of regulatory proteins to promoter and enhancer elements of genes. This remodeling is regulated by a large number of proteins and enzymes (>300) that recognize, add or remove chemical moieties to and from histone proteins (e.g., acetylation, methylation, phosphorylation) at multiple residues (e.g., lysines, arginines, serines/threonines) to form a ‘histone code’ of post-translational modifications (PTMs) [[1], [2], [3], [4]]. Beyond extensive knowledge about epigenetic mechanisms that has primarily been obtained in the fields of cancer, development, stem cells and model organisms, it has become increasingly apparent that fidelity of epigenetic control is also critical for normal skeletal development and bone formation [5]. The biological interpretation of these histone modifications is achieved by a broad class of histone code readers. For example, acetylated lysines interact with bromodomain (BRD) proteins, while methylated lysines interact with proteins that either have a chromobox (CBX) or chromodomain (CHD). Because there are many different types of methylated lysines in histones H3 and H4 that each impart different molecular responses, a large number of distinct lysine-specific methyl readers has evolved that recognize different amino acid positions (Fig. 1) [[6], [7], [8]].
This review focuses on the CBX class of methyl readers, because these epigenetic regulators have been rather unexplored in the context of osteogenesis, while they are likely to have important roles in bone formation. Notably, a key subset of CBX proteins recognizes H3K27me3 marks which are deposited by EZH2 and are highly suppressive for early stages of osteoblast differentiation [[9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]]. Other CBX proteins recognize H3K9me3 marks that are generated by the methyl transferases SUV39H1 and SUV39H2, which are both actively expressed in mesenchymal stromal cells and osteoblasts [20,21]. Because it is realistic to anticipate that CBX may have key roles in bone development, homeostasis and repair, we assessed the current state of knowledge obtained for CBX proteins in non-osseous tissues and cell types.
The currently known roles of CBX proteins in soft tissue development and homeostasis historically developed from studies on model organisms such as the fruit fly and certain fly mutants that would exhibit additional comb-like bristles (‘polycomb’) on their legs. At present, we know that Polycomb group (PcG) proteins are essential epigenetic regulators that modify the chromatin landscape in multiple cell types by interaction with PTMs in the N-terminal amino acid sequences of histone proteins (i.e., ‘histone tails’) [[1], [2], [3], [4], [5]]. Since their discovery in Drosophila as a set of genes responsible for controlling key stages of fly development [22], PcG proteins have been shown to be key transcriptional suppressors that play principal roles in many biological and cellular processes, including soft tissue development, stem cell pluripotency, senescence and cancer [23]. For example, prominent biological roles for CBX proteins have become apparent in the onset and progression of tumorigenesis in breast, colorectal and lung cancer, and suggest that they can act as both tumor suppressors and oncogenes [[24], [25], [26], [27], [28], [29]]. Even though PcG proteins are principal gene regulators that have been well-studied in human cancers and mouse genetics, these proteins have remained largely unexplored during bone tissue development.
At the molecular level, PcG proteins form two distinct, multi-protein complexes known as Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2) (Fig. 2). Both complexes support the efficient and developmental stage specific repression of genes to accommodate maintenance of stem cell fates, or adoption and retention of phenotype committed states. PRC1 complexes can be divided into canonical and non-canonical complexes according to the presence or absence of CBX proteins. The family of CBX proteins consists of at least eight members in both mouse and human genomes, each of which contains a chromobox (CBX) (Fig. 1). Each of these proteins has been shown to have a principal role in proper gene regulation and differentiation of non-skeletal cells [[30], [31], [32]]. At present, there are very few published studies that specifically focus on the role of CBX proteins in skeletal development and bone formation. One study has shown that CBX2 has a functional role in osteoblast differentiation [33], while our group has presented four preliminary studies on the expression and function of CBX proteins in mesenchymal stem cells or during osteoblast differentiation in culture [20,21,34,35].
The chromobox, which is a highly conserved protein module and a defining feature of CBX proteins, interprets the two principal gene suppressive epigenetic marks H3K9me3 and H3K27me3 (Fig. 1). The physical interactions of CBX proteins with these post-translational modifications in the N-terminal tail of H3 support gene repression [[36], [37], [38]]. H3K27me3 marks are recognized by one of five CBX isoforms (i.e., CBX2, CBX4, CBX6, CBX7 or CBX8) that reside in canonical PRC1 complexes. The CBX subunit is essential for binding of PRC1 complexes to heterochromatin. As such, CBX proteins are important interpreters and regulators of the epigenome. H3K9me3 marks are read by CBX1, CBX3 or CBX5 (alias HP1β, HP1γ and HP1α, respectively) as members of the heterochromatin protein 1 (HP1) family that support chromatin compaction.
Functional studies on how CBX proteins regulate gene expression during differentiation require loss-of-function (LOF) studies with CBX specific siRNAs. Such studies are performed with the assumption that RNA interference effectively diminishes both mRNA and protein levels for specific CBX proteins. However, this premise is clearly not true for the core histones proteins H2A, H2B, H3 and H4 which are highly abundant and are stably associated with chromatin [39,40]. Histone proteins have relatively long half-lives (>24 h) and can remain associated with chromosomes through multiple cell divisions, while they have relatively unstable mRNAs. Interestingly, CBX proteins that interact with PTMs of histones are also quite stable (half-life>24), even when their mRNA levels are diminished after 5 to 8 h. [41], In addition, other epigenetic regulators exhibit the same properties (i.e., stable protein and unstable mRNA). This stability of CBX proteins makes sense considering that these proteins support long-term suppression of specific chromosomal regions during successive cell divisions when cells must maintain their phenotype. One consequence of the existence of stable reservoirs of CBX proteins is that siRNAs for CBX proteins may primarily lower mRNA levels and inhibit ribosomal translation of new protein rather than reducing pre-existing pools of CBX proteins [41]. These limitations of CBX stability in LOF studies using siRNAs are not relevant to straight gene knockout studies where embryos develop without a specific CBX protein, but could perhaps be relevant to inducible and/or conditional gene knockout models for CBX proteins.
Section snippets
PRC2 components in mammals
The structure and composition of PRC2 complexes in relation to CBX proteins has been well-studied in several model organisms. In Drosophila, the PRC2 core complex consists of Enhancer of Zeste [E(z)], Suppressor of Zeste [Su(z)], and Extra Sex Combs (Esc) (Fig. 2). The mammalian PRC2 core complex is comprised of Enhancer of Zeste 1 or 2 (EZH1 or EZH2, respectively), Embryonic Ectoderm Development (EED), and Suppressor of Zeste 12 (SUZ12), as well as Rbap46 (RBBP7) or Rbap48 (RBBP4)(Fig. 2). The
CBX proteins in stem cell identity and differentiation
Epigenetic gene repression by CBX proteins [69,[70], [71], [72]] is critical for the maintenance and self-renewal capabilities of pluripotent and multipotent stem cells [46,73,74]. The CBX proteins of canonical PRC1 complexes (CBX2/4/6/7/8) have dynamic functions by balancing self-renewal and differentiation of pluripotent embryonic stem cells (ESCs) during differentiation. CBX7 is primarily responsible for maintenance of pluripotency in ESCs and hematopoietic stem cells (HSCs), CBX7 is
Conclusion
The studies discussed in this review present the general concept that epigenetic regulation by each of the eight CBX proteins in mouse and human plays a crucial role in soft tissue development and cancer. Current studies suggest that CBX proteins are critical for balancing self-renewal and differentiation in both pluripotent and multipotent stem cells. However, there are considerable molecular complexities, because different CBX isoforms are differentially expressed in distinct cell types,
Funding
This work was supported by funding from the National Institutes of Health (R01 AR069049 to AvW). We also thank William and Karen Eby for their generous philanthropic support. We also acknowledge intramural support from the Department of Orthopedic Surgery (to AAB).
CRediT authorship contribution statement
AJVW, LB, AAB, ANL, AD, RT, CRP and ZW have participated in experimental studies on CBX proteins in our research group and have contributed ideas at different stages of the project.
AJVW, LB, AAB, CRP and ZW collectively conceived the overall scope of this review and produced the initial drafts.
AJVW, LB, AAB, ANL, AD, RT, CRP and ZW provided final edits to improve the accuracy of the presentation.
Ackowledgments
We would like to thank all past and present members of our laboratory, as well as institutional and extramural colleagues for stimulating discussions and generously sharing ideas. We are particularly grateful to our former students Pengfei Zan, Merel Mol and Esther Liu, as well as our long term collaborators Gary Stein, Janet Stein, Jonathan Gordon, Martin Montecino, Mario Galindo, Peter Kloen, Marianna Kruithof-de Julio, Simon Cool, David Deyle, Eric Lewallen, Matthew Abdel, David Lewallen and
References (189)
- et al.
Epigenetics: a landscape takes shape
Cell
(2007) - et al.
Role of H3K9me3 heterochromatin in cell identity establishment and maintenance
Curr Opin Genet Dev
(2019) Structural biology of the chromodomain: form and function
Gene
(2012)- et al.
Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development
J Biol Chem
(2018) - et al.
Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice
Journal of Biological Chemistry
(2018) - et al.
Enhancer of zeste homolog 2 inhibition stimulates bone formation and mitigates bone loss caused by ovariectomy in skeletally mature mice
Journal of Biological Chemistry
(2016) - et al.
Epigenetic control of skeletal development by the histone methyltransferase Ezh2
Journal of Biological Chemistry
(2015) - et al.
Inhibition of the epigenetic suppressor EZH2 primes osteogenic differentiation mediated by BMP2
J Biol Chem
(2020) - et al.
Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing
Gene
(2017) - et al.
PRC1 complex diversity: where is it taking us?
Trends Cell Biol
(2014)