- Email: [email protected]
Finding the Middlemen in Genome Organization
Developmental Cell
Previews Finding the Middlemen in Genome Organization Xianrong Wong1 and Karen L. Reddy1,* 1Department of Biological Chemistry, Center for Epigenetics, Johns Hopkins University, 855 N. Wolfe Street, Baltimore, MD 21205, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2015.12.007
Chromatin domains associated with the nuclear lamina are generally heterochromatic and transcriptionally repressed. How they are recruited to and maintained at the nuclear periphery remains unclear. A recent study by Gonzalez-Sandoval et al. (2015) in Cell identifies a chromatin-binding protein that links repressive chromatin with the inner nuclear membrane. The spatial organization of chromatin is thought to influence genome activity, and many studies have highlighted, in particular, the importance of the nuclear periphery, encompassing the nuclear envelope and the underlying nuclear lamina, in developmental gene regulation (reviewed in Luperchio et al., 2014). The nuclear lamina is composed of type V intermediate filament proteins (lamins) that lend structural integrity to the nucleus and anchor inner nuclear membrane (INM) proteins to the nuclear envelope. Whereas in C. elegans there is only one lamin gene, in mammalian cells there are several. The INM/lamina has been implicated in multiple processes, including regulation of signaling, response to mechanical stress, and, most importantly, gene expression and genome organization, especially scaffolding of heterochromatin. During development, cell-typespecific transcription factors and a changing epigenetic landscape work together to promote differentiation of specific lineages and to restrict alternate lineages. The role that lamina-proximal domains of chromatin, the so-called lamin-associated domains (LADs), play in this framework is just beginning to be understood. Of particular interest is the relationship between lamina association and the epigenome. Recent studies have implicated both lamin A/C and the INM protein LBR in scaffolding of heterochromatin at the nuclear periphery (Solovei et al., 2013). Cells devoid of both LBR and lamin A/C exhibit an inverted chromatin configuration in which heterochromatin occupies the nuclear interior and euchromatin occupies the periphery. These data imply that lamin A/C and LBR proteins are important for scaffolding heterochromatin to the INM/lamina or perinuclear zone, but
not for formation or maintenance of heterochromatin (based solely on cytological observations, not genome-wide chromatin analyses). However, whether LBR and lamin A interact directly with core nucleosomal proteins remains unclear (Roux et al., 2012), raising the possibility that other lamina-chromatin linker proteins must exist (Figure 1). One candidate is PRR14 (Proline Rich 14), which has been implicated in tethering heterochromatin at the nuclear periphery (Poleshko et al., 2013). However, disruption of this protein affects both chromatin status and morphology, making its specific role unclear. In a recent issue of Cell, work by Gonzalez-Sandoval et al. identified a putative linker protein in C. elegans that contains a chromodomain capable of recognizing methylated H3K9 (GonzalezSandoval et al., 2015). Several studies have highlighted the role that heterochromatin plays in directing genomic regions to the nuclear lamina (Harr et al., 2015; Kind et al., 2013; Towbin et al., 2012). Specifically, LADs exhibit characteristic heterochromatic modifications: histone H3 lysine 9 di- and trimethylation (H3K9me 2/3) along the entirety of LADs, and histone H3 lysine 27 trimethylation (H3k27me3) at LAD borders (Guelen et al., 2008; Harr et al., 2015). And these modifications, especially H3K9me2/3, are important for directing perinuclear association in various cellular and developmental systems (Bian et al., 2013; Harr et al., 2015; Kind et al., 2013; Towbin et al., 2012). Intriguingly, in murine fibroblasts, both H3k27me3 and H3k9me2/3 are necessary for the peripheral association of specific LAD border regions enriched in cell-type-specific and developmentally important genes (Harr et al., 2015). However, it remains unclear exactly how H3K9me2/3 and/or
670 Developmental Cell 35, December 21, 2015 ª2015 Elsevier Inc.
H3K27me3 modifications are ‘‘read’’ and then directed to and anchored at the nuclear lamina. So, the question remains: How are heterochromatic domains recruited to the inner nuclear membrane or lamina? The recent study by Gonzalez-Sandoval and colleagues identifies a potential linker in C. elegans (Gonzalez-Sandoval et al., 2015). The authors used a clever RNAi screening strategy to identify proteins important for keeping a lacO array, containing H3K9me3 and H3K27me3, at the nuclear lamina in embryos. Strikingly, in both a wild-type and a ‘‘sensitized’’ background, designed to eliminate possible redundant mechanisms for anchoring at the lamina (lacking the H3K9me2/3 binders HPL-1 and LIN-61), loss of only one protein product, CEC-4, dislodges the array. CEC-4 encodes a previously uncharacterized chromodomain protein that localizes to the nuclear periphery and binds methylated H3K9 (mono-, di-, and tri-), raising the possibility that CEC-4 modulates the step-wise association of differentially methylated H3K9 methylated chromatin with the periphery (Towbin et al., 2012). Importantly, loss of CEC-4 does not cause loss of heterochromatin on the delocalized array and does not disrupt overall gene expression. Furthermore, using chromatin immunoprecipitation (ChIP) of LEM-2, an INM protein, the authors demonstrate that disruption of perinuclear anchoring by loss of CEC-4 extended to endogenous chromosomal sub-domains, although the chromatin state of these regions was not monitored. The authors thus uncouple perinuclear anchoring from establishment/maintenance of heterochromatin, reminiscent of the studies with LBR and lamin A/C. These data strongly suggest that CEC-4 is the linker
Developmental Cell
Previews coupled with well-conserved epigenetic and developmental programs, allowed identification of a heterochromatin anchoring protein. It will be exciting to see similar studies applied to mammalian systems, both to understand how anchoring of heterochromatin contributes to the development and maintenance of differentiated tissues and to elucidate how LAD domains behave during mitosis and how they reorganize after each cell division.
ACKNOWLEDGMENTS K.L.R. is partially supported by RO1GM106024-03.
Figure 1. The Lamina Chromatin Interface Current model of lamina-associated genic regions. Lamin-associated domains (LADs), which are heterochromatic regions at the nuclear periphery (red in the figure), are enriched in H3K9me2/3 modifications. The borders of LADs (transition from red to green nucleosomes) are enriched in H3K27me3 (in mammals). Potential linkers/anchoring proteins (yellow cloud), such as PRR14 in mammalian cells and CEC4 in C. elegans, interact with both the underlying chromatin and the lamina/inner nuclear membrane.
for LADs in C. elegans embryos (in larvae, loss of CEC-4 did not disrupt perinuclear localization). Thus, if heterochromatin state is maintained (on the array and at a gross level) in the absence of CEC-4 (and LBR or LMNA/C in mammalian cells), is this anchoring of heterochromatin to the periphery important at all? Indeed, loss of CEC-4 did not alter normal development or brood size, but the authors speculated that alternative anchoring pathways present in L1 larvae might compensate for loss of the linker protein. To circumvent this possibility, the authors promoted muscle development by ectopically expressing a master regulator of muscle development (MyoD or HLH-1). In wildtype embryos, 100% of the cells of the resulting embryos become muscle in this ectopic system, and normal development fails. Strikingly, in CEC-4 mutants, 25% of the embryos go on to complete embryogenesis, suggesting that loss of CEC-4 prevents the animal from ‘‘locking in’’ the induced differentiation program. This is a very provocative finding that demon-
strates directly, for the first time, that genome association with the nuclear periphery is functionally important for developmental progression. These data agree with much of the previous cell and molecular work suggesting that one potential role of perinuclear anchoring is to repress alternate lineage genes. Importantly, in C. elegans, a cell’s developmental fate is normally determined by its lineage, unlike the situation in mammals, where cells are more reliant on external cues and cellcell communication. Perhaps the placement of genes within LADs is more vital in situations in which cells have to choose among several different alternative fates, whether that is part of the normal developmental program (e.g., in mammals) or a result of perturbed development (as in this study). Gonzalez-Sandoval and colleagues (2015) employed powerful genetic and screening strategies in C. elegans to study a fascinating and important question in genome organization. The relatively simple circuitry at the lamina of worms (which have only one lamin isotype)
REFERENCES Bian, Q., Khanna, N., Alvikas, J., and Belmont, A.S. (2013). J. Cell Biol. 203, 767–783. Gonzalez-Sandoval, A., Towbin, B.D., Kalck, V., Cabianca, D.S., Gaidatzis, D., Hauer, M.H., Geng, L., Wang, L., Yang, T., Wang, X., et al. (2015). Cell 163, 1333–1347. Guelen, L., Pagie, L., Brasset, E., Meuleman, W., Faza, M.B., Talhout, W., Eussen, B.H., de Klein, A., Wessels, L., de Laat, W., and van Steensel, B. (2008). Nature 453, 948–951. Harr, J.C., Luperchio, T.R., Wong, X., Cohen, E., Wheelan, S.J., and Reddy, K.L. (2015). J. Cell Biol. 208, 33–52. Kind, J., Pagie, L., Ortabozkoyun, H., Boyle, S., de Vries, S.S., Janssen, H., Amendola, M., Nolen, L.D., Bickmore, W.A., and van Steensel, B. (2013). Cell 153, 178–192. Luperchio, T.R., Wong, X., and Reddy, K.L. (2014). Curr. Opin. Genet. Dev. 25, 50–61. Poleshko, A., Mansfield, K.M., Burlingame, C.C., Andrake, M.D., Shah, N.R., and Katz, R.A. (2013). Cell Rep. 5, 292–301. Roux, K.J., Kim, D.I., Raida, M., and Burke, B. (2012). J. Cell Biol. 196, 801–810. Solovei, I., Wang, A.S., Thanisch, K., Schmidt, C.S., Krebs, S., Zwerger, M., Cohen, T.V., Devys, D., Foisner, R., Peichl, L., et al. (2013). Cell 152, 584–598. Towbin, B.D., Gonza´lez-Aguilera, C., Sack, R., Gaidatzis, D., Kalck, V., Meister, P., Askjaer, P., and Gasser, S.M. (2012). Cell 150, 934–947, http://dx.doi.org/10.1016/j.cell.2012.06.051.
Developmental Cell 35, December 21, 2015 ª2015 Elsevier Inc. 671
Previews Finding the Middlemen in Genome Organization Xianrong Wong1 and Karen L. Reddy1,* 1Department of Biological Chemistry, Center for Epigenetics, Johns Hopkins University, 855 N. Wolfe Street, Baltimore, MD 21205, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2015.12.007
Chromatin domains associated with the nuclear lamina are generally heterochromatic and transcriptionally repressed. How they are recruited to and maintained at the nuclear periphery remains unclear. A recent study by Gonzalez-Sandoval et al. (2015) in Cell identifies a chromatin-binding protein that links repressive chromatin with the inner nuclear membrane. The spatial organization of chromatin is thought to influence genome activity, and many studies have highlighted, in particular, the importance of the nuclear periphery, encompassing the nuclear envelope and the underlying nuclear lamina, in developmental gene regulation (reviewed in Luperchio et al., 2014). The nuclear lamina is composed of type V intermediate filament proteins (lamins) that lend structural integrity to the nucleus and anchor inner nuclear membrane (INM) proteins to the nuclear envelope. Whereas in C. elegans there is only one lamin gene, in mammalian cells there are several. The INM/lamina has been implicated in multiple processes, including regulation of signaling, response to mechanical stress, and, most importantly, gene expression and genome organization, especially scaffolding of heterochromatin. During development, cell-typespecific transcription factors and a changing epigenetic landscape work together to promote differentiation of specific lineages and to restrict alternate lineages. The role that lamina-proximal domains of chromatin, the so-called lamin-associated domains (LADs), play in this framework is just beginning to be understood. Of particular interest is the relationship between lamina association and the epigenome. Recent studies have implicated both lamin A/C and the INM protein LBR in scaffolding of heterochromatin at the nuclear periphery (Solovei et al., 2013). Cells devoid of both LBR and lamin A/C exhibit an inverted chromatin configuration in which heterochromatin occupies the nuclear interior and euchromatin occupies the periphery. These data imply that lamin A/C and LBR proteins are important for scaffolding heterochromatin to the INM/lamina or perinuclear zone, but
not for formation or maintenance of heterochromatin (based solely on cytological observations, not genome-wide chromatin analyses). However, whether LBR and lamin A interact directly with core nucleosomal proteins remains unclear (Roux et al., 2012), raising the possibility that other lamina-chromatin linker proteins must exist (Figure 1). One candidate is PRR14 (Proline Rich 14), which has been implicated in tethering heterochromatin at the nuclear periphery (Poleshko et al., 2013). However, disruption of this protein affects both chromatin status and morphology, making its specific role unclear. In a recent issue of Cell, work by Gonzalez-Sandoval et al. identified a putative linker protein in C. elegans that contains a chromodomain capable of recognizing methylated H3K9 (GonzalezSandoval et al., 2015). Several studies have highlighted the role that heterochromatin plays in directing genomic regions to the nuclear lamina (Harr et al., 2015; Kind et al., 2013; Towbin et al., 2012). Specifically, LADs exhibit characteristic heterochromatic modifications: histone H3 lysine 9 di- and trimethylation (H3K9me 2/3) along the entirety of LADs, and histone H3 lysine 27 trimethylation (H3k27me3) at LAD borders (Guelen et al., 2008; Harr et al., 2015). And these modifications, especially H3K9me2/3, are important for directing perinuclear association in various cellular and developmental systems (Bian et al., 2013; Harr et al., 2015; Kind et al., 2013; Towbin et al., 2012). Intriguingly, in murine fibroblasts, both H3k27me3 and H3k9me2/3 are necessary for the peripheral association of specific LAD border regions enriched in cell-type-specific and developmentally important genes (Harr et al., 2015). However, it remains unclear exactly how H3K9me2/3 and/or
670 Developmental Cell 35, December 21, 2015 ª2015 Elsevier Inc.
H3K27me3 modifications are ‘‘read’’ and then directed to and anchored at the nuclear lamina. So, the question remains: How are heterochromatic domains recruited to the inner nuclear membrane or lamina? The recent study by Gonzalez-Sandoval and colleagues identifies a potential linker in C. elegans (Gonzalez-Sandoval et al., 2015). The authors used a clever RNAi screening strategy to identify proteins important for keeping a lacO array, containing H3K9me3 and H3K27me3, at the nuclear lamina in embryos. Strikingly, in both a wild-type and a ‘‘sensitized’’ background, designed to eliminate possible redundant mechanisms for anchoring at the lamina (lacking the H3K9me2/3 binders HPL-1 and LIN-61), loss of only one protein product, CEC-4, dislodges the array. CEC-4 encodes a previously uncharacterized chromodomain protein that localizes to the nuclear periphery and binds methylated H3K9 (mono-, di-, and tri-), raising the possibility that CEC-4 modulates the step-wise association of differentially methylated H3K9 methylated chromatin with the periphery (Towbin et al., 2012). Importantly, loss of CEC-4 does not cause loss of heterochromatin on the delocalized array and does not disrupt overall gene expression. Furthermore, using chromatin immunoprecipitation (ChIP) of LEM-2, an INM protein, the authors demonstrate that disruption of perinuclear anchoring by loss of CEC-4 extended to endogenous chromosomal sub-domains, although the chromatin state of these regions was not monitored. The authors thus uncouple perinuclear anchoring from establishment/maintenance of heterochromatin, reminiscent of the studies with LBR and lamin A/C. These data strongly suggest that CEC-4 is the linker
Developmental Cell
Previews coupled with well-conserved epigenetic and developmental programs, allowed identification of a heterochromatin anchoring protein. It will be exciting to see similar studies applied to mammalian systems, both to understand how anchoring of heterochromatin contributes to the development and maintenance of differentiated tissues and to elucidate how LAD domains behave during mitosis and how they reorganize after each cell division.
ACKNOWLEDGMENTS K.L.R. is partially supported by RO1GM106024-03.
Figure 1. The Lamina Chromatin Interface Current model of lamina-associated genic regions. Lamin-associated domains (LADs), which are heterochromatic regions at the nuclear periphery (red in the figure), are enriched in H3K9me2/3 modifications. The borders of LADs (transition from red to green nucleosomes) are enriched in H3K27me3 (in mammals). Potential linkers/anchoring proteins (yellow cloud), such as PRR14 in mammalian cells and CEC4 in C. elegans, interact with both the underlying chromatin and the lamina/inner nuclear membrane.
for LADs in C. elegans embryos (in larvae, loss of CEC-4 did not disrupt perinuclear localization). Thus, if heterochromatin state is maintained (on the array and at a gross level) in the absence of CEC-4 (and LBR or LMNA/C in mammalian cells), is this anchoring of heterochromatin to the periphery important at all? Indeed, loss of CEC-4 did not alter normal development or brood size, but the authors speculated that alternative anchoring pathways present in L1 larvae might compensate for loss of the linker protein. To circumvent this possibility, the authors promoted muscle development by ectopically expressing a master regulator of muscle development (MyoD or HLH-1). In wildtype embryos, 100% of the cells of the resulting embryos become muscle in this ectopic system, and normal development fails. Strikingly, in CEC-4 mutants, 25% of the embryos go on to complete embryogenesis, suggesting that loss of CEC-4 prevents the animal from ‘‘locking in’’ the induced differentiation program. This is a very provocative finding that demon-
strates directly, for the first time, that genome association with the nuclear periphery is functionally important for developmental progression. These data agree with much of the previous cell and molecular work suggesting that one potential role of perinuclear anchoring is to repress alternate lineage genes. Importantly, in C. elegans, a cell’s developmental fate is normally determined by its lineage, unlike the situation in mammals, where cells are more reliant on external cues and cellcell communication. Perhaps the placement of genes within LADs is more vital in situations in which cells have to choose among several different alternative fates, whether that is part of the normal developmental program (e.g., in mammals) or a result of perturbed development (as in this study). Gonzalez-Sandoval and colleagues (2015) employed powerful genetic and screening strategies in C. elegans to study a fascinating and important question in genome organization. The relatively simple circuitry at the lamina of worms (which have only one lamin isotype)
REFERENCES Bian, Q., Khanna, N., Alvikas, J., and Belmont, A.S. (2013). J. Cell Biol. 203, 767–783. Gonzalez-Sandoval, A., Towbin, B.D., Kalck, V., Cabianca, D.S., Gaidatzis, D., Hauer, M.H., Geng, L., Wang, L., Yang, T., Wang, X., et al. (2015). Cell 163, 1333–1347. Guelen, L., Pagie, L., Brasset, E., Meuleman, W., Faza, M.B., Talhout, W., Eussen, B.H., de Klein, A., Wessels, L., de Laat, W., and van Steensel, B. (2008). Nature 453, 948–951. Harr, J.C., Luperchio, T.R., Wong, X., Cohen, E., Wheelan, S.J., and Reddy, K.L. (2015). J. Cell Biol. 208, 33–52. Kind, J., Pagie, L., Ortabozkoyun, H., Boyle, S., de Vries, S.S., Janssen, H., Amendola, M., Nolen, L.D., Bickmore, W.A., and van Steensel, B. (2013). Cell 153, 178–192. Luperchio, T.R., Wong, X., and Reddy, K.L. (2014). Curr. Opin. Genet. Dev. 25, 50–61. Poleshko, A., Mansfield, K.M., Burlingame, C.C., Andrake, M.D., Shah, N.R., and Katz, R.A. (2013). Cell Rep. 5, 292–301. Roux, K.J., Kim, D.I., Raida, M., and Burke, B. (2012). J. Cell Biol. 196, 801–810. Solovei, I., Wang, A.S., Thanisch, K., Schmidt, C.S., Krebs, S., Zwerger, M., Cohen, T.V., Devys, D., Foisner, R., Peichl, L., et al. (2013). Cell 152, 584–598. Towbin, B.D., Gonza´lez-Aguilera, C., Sack, R., Gaidatzis, D., Kalck, V., Meister, P., Askjaer, P., and Gasser, S.M. (2012). Cell 150, 934–947, http://dx.doi.org/10.1016/j.cell.2012.06.051.
Developmental Cell 35, December 21, 2015 ª2015 Elsevier Inc. 671