Mutations in two non‐canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects

SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. Whereas metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well‐studied BRAHMA and SPLAYED ATPases, as well as two closely related non‐canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here, we show that although strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems, and display few and aberrant cell divisions. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects, we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication‐coupled chromatin assembly are discussed.     

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition

1. Download : Download high-res image (165KB)
2. Download : Download full-size image

     

Essential role of ARID2 protein-containing SWI/SNF complex in tissue-specific gene expression

Unfolding of the gene expression program that converts precursor cells to their terminally differentiated counterparts is critically dependent on the nucleosome-remodeling activity of the mammalian SWI/SNF complex. The complex can be powered by either of two alternative ATPases, BRM or BRG1. BRG1 is critical for development and the activation of tissue specific genes and is found in two major stable configurations. The complex of BRG1-associated factors termed BAF is the originally characterized form of mammalian SWI/SNF. A more recently recognized configuration shares many of the same subunits but is termed PBAF in recognition of a unique subunit, the polybromo protein (PBRM1). Two other unique subunits, BRD7 and ARID2, are also diagnostic of PBAF. PBAF plays an essential role in development, apparent from the embryonic lethality of Pbmr1-null mice, but very little is known about the role of PBAF, or its signature subunits, in tissue-specific gene expression in individual differentiation programs. Osteoblast differentiation is an attractive model for tissue-specific gene expression because the process is highly regulated and remains tightly synchronized over a period of several weeks. This model was used here, with a stable shRNA-mediated depletion approach, to examine the role of the signature PBAF subunit, ARID2, during differentiation. This analysis identifies a critical role for ARID2-containing complexes in promoting osteoblast differentiation and supports a view that the PBAF subset of SWI/SNF contributes importantly to maintaining cellular identity and activating tissue-specific gene expression.     

CHB2, a member of the SWI3 gene family,is a global regulator in Arabidopsis

The SWI/SNF complex is an ATP-dependent chromatin remodeling complex that plays an important role in the regulation of eukaryotic gene expression. Very little is known about the function of SWI/SNF complex in plants compared with animals and yeast. SWI3 is one of the core components of the SWI/SNF chromatin remodeling complexes in yeast. We have identified a putative SWI3-like cDNA clone, CHB2 (AtSWI3B), from Arabidopsis thaliana by screening the expressed sequence tag database. CHB2 encodes a putative protein of 469 amino acids and shares 23% amino acid sequence identity and 64% similarity with the yeast SWI3. The Arabidopsis genome contains four SWI3-like genes, namely CHB1 (AtSWI3A), CHB2 (AtSWI3B), CHB3 (AtSWI3C) and CHB4 (AtSWI3D). The expression of CHB2, CHB3 and CHB4 mRNA was detected in all tissues analyzed by RT-PCR. The expression of CHB1 mRNA, however, could not be detected in the siliques, suggesting that there is differential expression among CHB genes in different Arabidopsis tissues. To investigate the role of CHB2 in plants, Arabidopsis plants were transformed with a gene construct comprising a CHB2 cDNA in the antisense orientation driven by the CaMV 35S promoter. Repression of CHB2 expression resulted in pleiotropic developmental abnormalities including abnormal seedling and leaf phenotypes, dwarfism, delayed flowering and no apical dominance, suggesting a global role for CHB2 in the regulation of gene expression. Our results indicate that CHB2 plays an essential role in plant growth and development.     

A cooperative activation loop among SWI/SNF, γ‐H2AX and H3 acetylation for DNA double‐strand break repair

Although recent studies highlight the importance of histone modifications and ATP‐dependent chromatin remodelling in DNA double‐strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser‐139 (S139ph) after DNA damage, binds to γ‐H2AX (the phosphorylated form of H2AX)‐containing nucleosomes in S139ph‐dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ‐H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ‐H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ‐H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ‐H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ‐H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ‐H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.     

The Swi/Snf chromatin remodeling complex is essential for hyphal development in Candida albicans

The ability of dimorphic transition between yeast and hyphal forms in Candida albicans is one of the vital determinants for its pathogenicity and virulence. We isolated C. albicans SWI1 as a suppressor of the invasive growth defect in a Saccharomyces cerevisiae mutant. Expression of C. albicans SWI1 in S. cerevisiae partially complemented the growth defect of a swi1 mutant in the utilization of glycerol. Swi1 is in a complex with Snf2 in C. albicans, and both proteins are localized in the nucleus independent of the growth form. Deleting SWI1 or SNF2 in C. albicans prevented true hyphal formation and resulted in constitutive pseudohypha-like growth in all media examined. Furthermore, swi1/swi1 mutant was defective in hypha-specific gene expression and avirulent in a mouse model of systemic infection. These data strongly suggest the conserved Swi/Snf complex in C. albicans is required for hyphal development and pathogenicity.     

IGFBP-2 stimulates calcium/calmodulin-dependent protein kinase kinase 2 activation leading to AMP-activated protein kinase induction which is required for osteoblast differentiation

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins-2 (IGFBP-2) function coordinately to stimulate osteoblast differentiation. Induction of AMP-activated protein kinase (AMPK) is required for differentiation and is stimulated by these two factors. These studies were undertaken to determine how these two peptides lead to activation of AMPK. Enzymatic inhibitors and small interfering RNA were utilized to attenuate calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) activity in osteoblasts, and both manipulations resulted in failure to activate AMPK, thereby resulting in inhibition of osteoblast differentiation. IGFBP-2 and IGF-I stimulated an increase in CaMKK2, and inhibition of IGFBP-2 binding its receptor resulted in failure to induce CaMKK2 and AMPK activation. Injection of a peptide that contained the IGFBP-2 receptor-binding domain into IGFBP-2^−/− mice activated CaMKK2 and injection of a CaMKK2 inhibitor into normal mice inhibited both CamKK2 and AMPK activation in osteoblasts. We conclude that induction of CaMKK2 by IGFBP-2 and IGF-I in osteoblasts is an important signaling event that occurs early in differentiation and is responsible for activation of AMPK, which is required for optimal osteoblast differentiation.     

Atlantic salmon (Salmo salar) muscle precursor cells differentiate into osteoblasts in vitro: Polyunsaturated fatty acids and hyperthermia influence gene expression and differentiation

The formation and mineralisation of bone are two critical processes in fast-growing Atlantic salmon (Salmo salar). The mechanisms of these processes, however, have not been described in detail. Thus, in vitro systems that allow the study of factors that influence bone formation in farmed Atlantic salmon are highly warranted. We describe here a method by which unspecialised primary cells from salmon white muscle can differentiate to osteoblasts in vitro. We have subsequently used the differentiated cells as a model system to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions, namely polyunsaturated fatty acids, PUFAs, and temperature. Muscle precursor cells changed their morphology from triangular or spindle-shaped cells to polygonal or cubical cells after 3 weeks in osteogenic medium. In addition, gene expression studies showed that marker genes for osteoblastogenesis; alp, col1a1, osteocalcin, bmp2 and bmp4 increased after 3 weeks of incubation in osteogenic media showing that these cells have differentiated to osteoblasts at this stage. Adding CLA or DHA to the osteoblast media resulted in a reduced PGE₂ production and increased expression of osteocalcin. Further, temperature studies showed that differentiating osteoblasts are highly sensitive to increased incubation temperature at early stages of differentiation. Our studies show that unspecialised precursor cells isolated from salmon muscle tissue can be caused to differentiate to osteoblasts in vitro. Furthermore, this model system appears to be suitable for the study of osteoblast biology in vitro.