Accepted Manuscript Chromatin Remodeling: From Transcription to Cancer Moshe Yaniv PII:
S2210-7762(14)00061-1
DOI:
10.1016/j.cancergen.2014.03.006
Reference:
CGEN 271
To appear in:
Cancer Genetics
Received Date: 10 March 2014 Accepted Date: 13 March 2014
Please cite this article as: Yaniv M, Chromatin Remodeling: From Transcription to Cancer, Cancer Genetics (2014), doi: 10.1016/j.cancergen.2014.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chromatin Remodeling: From Transcription to Cancer
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Moshe Yaniv
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Department of Developmental Biology and Stem Cells
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[email protected] tel: 33 1 4568 8512 fax: 33 1 4568 8979
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Institut Pasteur 28 rue du Docteur Roux 75724 Paris cedex 15, France
Running title: epigenetic and cancer
Key words: tumor suppressor, development, differentiation, cell cycle
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Summary
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In this short review article I have tried to trace the path that led my laboratory from the early studies of the structure of papovaviruses minichromosomes and transcription control to the investigation of chromatin remodeling complexes of the SWI/SNF family.
I discuss briefly the genetic and biochemical studies that lead to the discovery of the
SWI/SNF complex in yeast and drosophila and summarize some of the studies on the
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developmental role of the murin complex. The discovery of the tumor suppressor function of the SNF5/Ini1/SMARCA1b gene in human and the identification of frequent mutations in
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other subunits of this complex in different human tumors opened a fascinating field of research on this epigenetic regulator with the hope to better understand tumor development and hopefully to develop novel treatments.
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Introduction
The Genetic material of our cells, the DNA, is packaged in the nucleus in a structure called chromatin that remained ill defined biochemically for a long time. Breakthroughs in the
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seventies of last century both biochemically and by electron microscopy, followed later by X ray crystallography unraveled the structure of the nucleosome, the basic unit of the chromatin. An octamer composed of two molecules of each one of the four core histones,
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H3,H4, H2A and H2B is surrounded by one and three quarters turns of double stranded DNA comprising 147 base pairs ( 1). The nucleosomes are aligned like beads on a string along the long DNA chain of chromosomes. This chain of nucleosomes is further packaged in higher order structures that are not fully established yet. One possible structure is a 30 nanometers solenoid or an helix composed by packaging of six nucleosomes per turn. The linker histone H1 contributes to the formation of these higher order structures. The chromatin has to be further compacted to fit our genome of more than two meters in a
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nucleus with a diameter of roughly 10 microns. Furthermore the degree of compaction can very between active chromatin, the euchromatin and the inactive heterochromatin.
The nucleosome is a very stable structure with strong electrostatic interactions between
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the negatively charged DNA and the basic histones. This interaction can be disrupted only by NaCl concentrations above one molar and are thus very stable in the nucleus. The
packaging of the DNA double helix around the histones core in the nucleosome restricts the access of specific DNA sequences to recognition by proteins involved in DNA repair or
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recognition of transcription control elements. The same difficulty is faced by RNA or DNA polymerases that have to separate locally both strands of DNA. Progression of these
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enzymes along the nucleosome fibers is hampered by the strong contacts between histones and DNA.
Early on when we studied the structure of the SV40 and Polyomavirus minichromosomes containing roughly 5200 base pairs of circular DNA packed in 24 nucleosomes. We investigated if the histones can slide easily along the DNA chain or in other words whether
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the nucleosome is a static or dynamic structure? Testing for the accessibility of restriction sites in the viral genome to restriction enzymes like EcoR1, we found that an important fraction of minichromosomes are protected from digestion after prolonged incubation strongly arguing that the core histones do not move rapidly along the DNA chain (2 ). On the
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other hand we and others could show that the template for DNA replication and transcription is the viral chromatin(3). It became clear that we miss an activity or activities
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to facilitate DNA dependent transactions like replication, transcription or repair.
The discovery of the SWI/SNF chromatin remodeling complex Hints that biological systems that can regulate nucleosome mobilization came from two distinct areas of research. Genetic studies in the yeast sacharomeyes cereviciae on one hand and drosophila melanogaster on the other discovered genetic complexes that control the expression of a group of genes. In yeast these were mutations that block sucrose
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fermentation, the SNF genes (4). Mutations in what became apparent as the same group of genes also blocked mating type switch, the SWI genes (5). These mutations were suppressed by mutations that reduced the number of histone genes indicating a link with chromatin structure and its capacity to block transcription(6) .
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For drosophila these were mutations in the trithorax group of genes that were selected as suppressors of polycomb homeotic mutations, the polycomb proteins were thought to function at the level of chromatin. (7). Cloning of the yeast SWI2/SNF2 gene revealed that it encoded for a protein with motifs related to RNA or DNA helicases with ATP binding motif
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and DNA dependent ATPase activity ( 8). A highly homologous protein was encoded by the
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drosophila Brm gene (7 ).
To check if such genes are conserved in higher eukaryotes both the Crabtree laboratory as well as our group set up to search for the possible existence of human homologues of the yeast or drosophila helicase. Crabtree cloned a cDNA encoding BRG1, Brahma related gene 1, also called SMARCA4 ( 9) and we cloned a highly homologous protein, that we called human BRM or human Brahama, also called SMARCA2 (10). SMARC stands for SWI/SNF
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related, actin containing, regulators of chromatin. A second mammalian gene related to another member of the genetic Snf complex , an homologue of the yeast Snf5 gene was cloned as a HIV integrase interacting protein, and called INI1 ( 11 ). The same cDNA was isolated by Claude Sardet in a two hybrid screen and further characterized by us as the
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human homologue of the yeast Snf5 gene, also called SMARCB1 (12).
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For BRM it was shown that re-expression of the helicase in human cell lines lacking one or both of the helicases strongly increased transcriptional activation by the glucocorticoid hormone receptor (GR) (10). BRG1 could rescue SWI2/SNF2 mutants in yeast (9). These studies were reminiscent of the genetic data in yeast and drosophila showing that the SWI/SNF complex is involved in transcription activation.
In parallel to these studies several groups isolated biochemically protein complexes which facilitated ordered nucleosome assembly in vitro and nucleosome mobilization or disruption in the presence of ATP ( 13-16). All together there are at least four families of 4
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chromatin remodeling complexes in yeast, droshophila and mammalian cells defined by the nature of the helicase related protein: SWI/SNF, ISWI, INO80/SWR1and CHD. Each class contains several distinct complex subtypes with distinct biochemical activity and biological
can be found in a recent review by Hargreaves and Crabtree ( 17).
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functions . Detailed description of these complexes is beyond the scope of this article and
As for the mammalian SWI/SNF complex, it is composed of 10-12 distinct subunits with composition partially different between different cell types (18) and a mass close to 2 mega
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daltons. The biological activity of the complex can be assayed with chromatin reconstituted in vitro. It can mobilize nucleosomes and facilitate the transcription of chromatinized
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templates. Similarly by mobilizing (moving ) the histones relative to the DNA sequences it exposes restriction sites to cleavage by restriction endonucleases (19). How this is done is not yet fully understood. Electron microscopy images of a purified yeast RSC complex, highly homologous to the genuine SWI/SNF complex of the same organism, demonstrates that the complex is much bigger that the nucleosome itself, more than one Md versus 200 Kd and that it has a cavity that can accommodate the nucleosome ( 20). It is believed that
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ATP hydrolysis is accompanied by transient disruption of DNA-histone contacts along 1020 base pairs of the nucleosome, pulling out of this DNA segment and its translocation or movement along the histone octamer core to finally result in the sliding of the DNA relative
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to the histone core (21 -23)
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The functions of the SWI/SNF complex in development and differentiation
Having isolated several of the genes encoding subunits of the SWI/SNF complex in the mouse, it was tempting to study in more detail their role in the development of higher organisms. Inactivation of the Brm helicase gave rise to viable mice that showed over growth and failure to arrest primary fibroblasts on confluency in culture( 24 ). We observed
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increased compensatory expression of BRG1 in the absence of BRM. On the contrary, the BRG1 helicase was shown to be essential for early embryonic growth (25 ). SNF5/Ini1 is another subunit of what was defined as the core complex and is encoded by a single gene in mouse or man. Its inactivation resulted in very early embryonic lethality ( 26-28), identical
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to the phenotype observed with BRG1-/- embryos. In both cases development was arrested around the implantation time. Recovery of mutant blastocystes and their culture in vitro revealed that the inner cell mass cells do not multiply in the absence of one or the other of the core complex subunits and that they undergo apoptosis(25,26) . The very early lethality
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of BRG1-/- mice was in agreement with the observations that BRG1 is expressed maternally and is the only SWI/SNF helicase present in very early development( 29). SNF5/Ini1 is also
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expressed maternally and its de novo transcription initiates very early , at the 2-4 cell stage of development (26 ). The fact that the phenotype of the BRG1 and SNF5/Ini1 knock out mice was identical strongly argues that both proteins function in the same biochemical complex, at least in early development. This difference in the behavior of the two alternative helicase subunits of the complex suggest that BRG1 is the basic helicase of the embryonic complex and that BRM takes over partially in a number of cell types during
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differentiation or growth arrest.
To investigate the function of the SWI/SNF complex at later stages of development we and others used conditional inactivation of different genes encoding subunits of the
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complex in different cell types and at different stages of development. We have used a floxed SNF5 allele and crossed these mice with a strain expressing the CRE recombinase in
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the hepatoblasts of the developing liver starting at day 10.5- 11 of development. The mutant mice developed up to birth but died shortly after birth from acute liver failure. They showed a major defect in maintaining normal plasma glucose concentrations caused by failure to accumulate glycogen and to synthesize glucose. A decreased expression of several of the corresponding biosynthetic enzymes was observed. In addition the mutant hepatocytes exhibited defects in cell-cell adhesion due to decreased synthesis of E-Cadherin and of proteins involved in gap, tight and adherens junctions. These observations suggested that the SWI/SNF complex is important for the terminal differentiation of epithelial cells. In addition we observed an increased proliferation of parachymal cells with changes in the 6
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expression of a number of cell cycle markers including p53 targets genes like p21, indicating a defect in growth arrest and apoptosis (30 ).
Global transcriptome analysis of liver specific genes up regulated during normal
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development showed a decrease in the expression of two thirds but not all of these genes. These studies clearly demonstrated that the SWI/SNF complex is essential for the terminal differentiation of the hepatocytes (we did not check other cell types), for the formation of
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polarized epithelia and for normal glucose homeostasis (30).
Evidence that the SWI/SNF complex is involved in growth control and differentiation came
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also from studies by other groups. Re-introduction of SNF5 into the Mon cell line isolated from human malignant rhabdoid tumors resulted in growth arrest and adipocyte differentiation further demonstrating the lack of fully functional SWI/SNF complex is required for specific linage differentiation (31). Subunits of the SWI/SNF complex were shown to be essential for survival and differentiation of myoblasts and other cell types (32). The complex plays a major role in the pluripotency and differentiation of ES cells (
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33, 34) . Finally specific subunits of the complex were shown to regulate neuronal differentiation ( 35).
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The SWI/SNF complex, cell transformation and cancer
Following the expression of BRM and BRG1 in different cell lines Christian Muchardt
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realized that certain human tumor cell lines lack either BRM or both BRM and BRG1(10). In addition, BRG1 and BRM were shown to interact and cooperate with pRb in repressing E2F target genes and in inducing growth arrest ( 36- 39 ). Furthermore we observed that Ras transformation of NIH 3T3 mouse fibroblasts results in strongly reduced BRM expression and that re-expression of BRM in these cells reversed their transformed phenotype( 40). This suppression activity depended on an intact helicase activity since a point mutation in the ATP binding site abolished growth suppression in soft agar. Colonies that grew in agar
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these experiments over-expressed the mutant helicase protein suggesting that it behaved as a dominant negative effector and facilitated cell transformation. These data clearly demonstrated that the SWI/SNF complex is involved in growth control and behaves as a negative regulator of growth. This hypothesis was further supported by
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observations that the level of the BRM protein strongly increased upon confluency of
epithelial cells, it was very low in mouse embryonal carcinoma cells or ES cells and went up upon differentiation( 40).
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While the data accumulated from the studies on the SWI/SNF complex suggested that it may be involved in growth control and cancer no full evidence existed until the surprising
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discovery that SNF5/Ini1 is inactivated bi-allelically in the large majority of malignant rhabdoid tumors of very young children (41,42 ). This was the first example for a tumor suppressor function of a subunit of a chromatin remodeling complex.
Following this observation, we and others investigated mice heterozygous for the Snf5 gene or with the conditional floxed allele of the Snf5 gene. We observed that these mice
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developed with age rhabdoid like tumors in their nervous system or in the periphery. In these tumors the second wild type allele of the Snf5 gene was lost further substantiating the tumor suppressor nature of this gene (26-28). While the histopathology of the murin tumors was identical to that of the human samples a major difference was observed
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between the two organisms. While in humans the penetrance of the disease in carriers of a mutant gene was close to 100% , in the case of the mice tumor appeared much slower (
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taking account of the life expectancy of humans and mice) and with much lower penetrance
Studies of mouse primary fibroblasts isolated from heterozygous mice suggested that inactivation of the second allele of Snf5 in culture by expressing a Cre recombinase from an infecting recombinant Adenovirus induces the p53 pathway and cell death. Based on these data we and the laboratory of Roberts crossed the Snf5 heterozygous mice with a p53-/- mouse. These mice developed rhabdoid tumors much faster than the original Snf5+/- mice with 100% penetrance. This model is certainly one of the fastest evolving 8
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tumors in a mouse model. These tumors were rhabdoid in nature and not of the typical lymphomas or sarcoma’s observed in p53-/- mice( 43 -45). The difference observed between humans and mice concerning the role of p53 is not fully understood. One explanation may be related to the rate of loss of the second allele. It may be fast in humans,
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slow in the mouse and accelerated in the absence of p53. Another possibility may involve a difference between human and mouse precursor cells of the tumors: the growth arrest and pro-apoptotic functions of p53 will be activated in the mouse but not human cells lacking
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SNF5/Ini1.
Further screening of tumor cell lines for the expression of BRM and BRG1 revealed that a
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number of tumor cell lines fail to express these proteins with frequencies varying between tumor types. In many cases the lack of expression was associated to an epigenetic CpG methylation of the BRG1 gene(46-49). Following these pioneering studies on SNF5/Ini1 the explosion of exonic and whole genome sequencing of tumor derived cell lines or primary tumor samples a major surprise became evident. A considerable proportion of tumors, roughly 20%, carry mutations in different subunits of the SWI/SNF complex (50,51). This
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chromatin remodeling complex is becoming a major tumor suppressor function in human cells. As described in different contributions in this volume a number of targets for the SWI/SNF complex were identified in recent years. Many of these are involved in cell cycle control like the CDK inhibitors ink4a-ARF-ink4b locus where the SNF5 dependent activity
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is required for polycomb eviction(52) p27 (24,), cyclin D1( 53 ) and several E2F target genes among others. In addition as mentioned above we believe that genes involved in
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epithelial differentiation and cell polarity are also targets for this complex. Their decreased expression will cause a block in terminal differentiation and favor cell transformation. What is less clear is why different tumors accumulate mutations in different subunits of the complex. Several of the subunits are encoded by more than one gene and may have functions in specific cell types that may be sensitive to their inactivation. Others like SNF5/Ini1 may have an essential survival function in many cell types and only a limited number of tumor cell types can survive in its absence.
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In addition to gene regulation, the SWI/SNF complex or specific sub-complexes may be involved in other processes in the cell that may be also involved in tumor formation. In the case of Saccharomyces cerevisiae it was clearly shown that the RSC complex and to a lesse extent the SWI/SNF complex are involved in chromosome segregation and centrosome
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function (54-58 ). Accumulating evidence in murin or human cells also demonstrate that the SWI/SNF complexes are involved in DNA damage control and daughter chromosome segregation ( 44,59, 60) . Such defects can also facilitate cell transformation , however they
mutations observed in rhabdoid tumor cells.
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Epigenetic and Cancer
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are somewhat incompatible with the low rate of chromosome rearrangements or point
Can we define the SWI/SNF complex as an epigenetic tumor suppressor complex? This brief summary of some of the properties and biological functions of this complex clearly indicates that this complex positively or negatively regulates a considerable number of genes at the transcriptional level in different cell types ranging from early embryonic cells
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to fully differentiated cell types. Among these genes, many involve signaling pathways that play an important role in development and cancer like the sonic hedgehog ( 61) or the Wntbeta catenin pathways ( 62 ), LIF /Stat3 signaling in ES cells by facilitating poly comb
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repression( 63) the AKT signaling pathway( 64) etc. In addition it regulates an important class of genes that control cell cycle progression including the transcriptional program of p53( 44,. 65) as well as many differentiation programs. In its absence, cell differentiation is
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blocked while cell cycle progression becomes insensitive to external cues. The activity of the SWI/SNF complex is dependent on its recruitments to specific genes by gene specific transcription factors or by histone modifications like acetylation that can interact with bromodomains present in different subunits of the complex. In addition several of the subunits has non specific DNA binding motifs. The changes in gene expression profiles caused by absence or the defects in specific subunits is transmitted during cell divisions and expansion of tumor cells. In this respect we can clearly talk about an epigenetic regulator. However it’s effect on gene expression and growth is limited to
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somatic cells and does not involve germ line transmission to the off-springs. The SWI/SNF complex can be viewed as the prototype of an epigenetic regulator of gene expression that is involved in tumor suppression. The broad implication of different subunits of the complex in different types of human cancer suggest that partial to global loss of SWI/SNF
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activity may result in a multitude of deregulated cellular programs and the final out-come , cell survival , cell death or malignant transformation will differ from one cell type to
another. It will be important to find ways to restore its activity in tumor cells that will
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DOI:
10.1016/j.cancergen.2014.03.006
Reference:
CGEN 271
To appear in:
Cancer Genetics
Received Date: 10 March 2014 Accepted Date: 13 March 2014
Please cite this article as: Yaniv M, Chromatin Remodeling: From Transcription to Cancer, Cancer Genetics (2014), doi: 10.1016/j.cancergen.2014.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chromatin Remodeling: From Transcription to Cancer
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Moshe Yaniv
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Department of Developmental Biology and Stem Cells
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[email protected] tel: 33 1 4568 8512 fax: 33 1 4568 8979
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Institut Pasteur 28 rue du Docteur Roux 75724 Paris cedex 15, France
Running title: epigenetic and cancer
Key words: tumor suppressor, development, differentiation, cell cycle
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Summary
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In this short review article I have tried to trace the path that led my laboratory from the early studies of the structure of papovaviruses minichromosomes and transcription control to the investigation of chromatin remodeling complexes of the SWI/SNF family.
I discuss briefly the genetic and biochemical studies that lead to the discovery of the
SWI/SNF complex in yeast and drosophila and summarize some of the studies on the
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developmental role of the murin complex. The discovery of the tumor suppressor function of the SNF5/Ini1/SMARCA1b gene in human and the identification of frequent mutations in
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other subunits of this complex in different human tumors opened a fascinating field of research on this epigenetic regulator with the hope to better understand tumor development and hopefully to develop novel treatments.
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Introduction
The Genetic material of our cells, the DNA, is packaged in the nucleus in a structure called chromatin that remained ill defined biochemically for a long time. Breakthroughs in the
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seventies of last century both biochemically and by electron microscopy, followed later by X ray crystallography unraveled the structure of the nucleosome, the basic unit of the chromatin. An octamer composed of two molecules of each one of the four core histones,
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H3,H4, H2A and H2B is surrounded by one and three quarters turns of double stranded DNA comprising 147 base pairs ( 1). The nucleosomes are aligned like beads on a string along the long DNA chain of chromosomes. This chain of nucleosomes is further packaged in higher order structures that are not fully established yet. One possible structure is a 30 nanometers solenoid or an helix composed by packaging of six nucleosomes per turn. The linker histone H1 contributes to the formation of these higher order structures. The chromatin has to be further compacted to fit our genome of more than two meters in a
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nucleus with a diameter of roughly 10 microns. Furthermore the degree of compaction can very between active chromatin, the euchromatin and the inactive heterochromatin.
The nucleosome is a very stable structure with strong electrostatic interactions between
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the negatively charged DNA and the basic histones. This interaction can be disrupted only by NaCl concentrations above one molar and are thus very stable in the nucleus. The
packaging of the DNA double helix around the histones core in the nucleosome restricts the access of specific DNA sequences to recognition by proteins involved in DNA repair or
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recognition of transcription control elements. The same difficulty is faced by RNA or DNA polymerases that have to separate locally both strands of DNA. Progression of these
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enzymes along the nucleosome fibers is hampered by the strong contacts between histones and DNA.
Early on when we studied the structure of the SV40 and Polyomavirus minichromosomes containing roughly 5200 base pairs of circular DNA packed in 24 nucleosomes. We investigated if the histones can slide easily along the DNA chain or in other words whether
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the nucleosome is a static or dynamic structure? Testing for the accessibility of restriction sites in the viral genome to restriction enzymes like EcoR1, we found that an important fraction of minichromosomes are protected from digestion after prolonged incubation strongly arguing that the core histones do not move rapidly along the DNA chain (2 ). On the
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other hand we and others could show that the template for DNA replication and transcription is the viral chromatin(3). It became clear that we miss an activity or activities
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to facilitate DNA dependent transactions like replication, transcription or repair.
The discovery of the SWI/SNF chromatin remodeling complex Hints that biological systems that can regulate nucleosome mobilization came from two distinct areas of research. Genetic studies in the yeast sacharomeyes cereviciae on one hand and drosophila melanogaster on the other discovered genetic complexes that control the expression of a group of genes. In yeast these were mutations that block sucrose
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fermentation, the SNF genes (4). Mutations in what became apparent as the same group of genes also blocked mating type switch, the SWI genes (5). These mutations were suppressed by mutations that reduced the number of histone genes indicating a link with chromatin structure and its capacity to block transcription(6) .
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For drosophila these were mutations in the trithorax group of genes that were selected as suppressors of polycomb homeotic mutations, the polycomb proteins were thought to function at the level of chromatin. (7). Cloning of the yeast SWI2/SNF2 gene revealed that it encoded for a protein with motifs related to RNA or DNA helicases with ATP binding motif
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and DNA dependent ATPase activity ( 8). A highly homologous protein was encoded by the
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drosophila Brm gene (7 ).
To check if such genes are conserved in higher eukaryotes both the Crabtree laboratory as well as our group set up to search for the possible existence of human homologues of the yeast or drosophila helicase. Crabtree cloned a cDNA encoding BRG1, Brahma related gene 1, also called SMARCA4 ( 9) and we cloned a highly homologous protein, that we called human BRM or human Brahama, also called SMARCA2 (10). SMARC stands for SWI/SNF
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related, actin containing, regulators of chromatin. A second mammalian gene related to another member of the genetic Snf complex , an homologue of the yeast Snf5 gene was cloned as a HIV integrase interacting protein, and called INI1 ( 11 ). The same cDNA was isolated by Claude Sardet in a two hybrid screen and further characterized by us as the
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human homologue of the yeast Snf5 gene, also called SMARCB1 (12).
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For BRM it was shown that re-expression of the helicase in human cell lines lacking one or both of the helicases strongly increased transcriptional activation by the glucocorticoid hormone receptor (GR) (10). BRG1 could rescue SWI2/SNF2 mutants in yeast (9). These studies were reminiscent of the genetic data in yeast and drosophila showing that the SWI/SNF complex is involved in transcription activation.
In parallel to these studies several groups isolated biochemically protein complexes which facilitated ordered nucleosome assembly in vitro and nucleosome mobilization or disruption in the presence of ATP ( 13-16). All together there are at least four families of 4
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chromatin remodeling complexes in yeast, droshophila and mammalian cells defined by the nature of the helicase related protein: SWI/SNF, ISWI, INO80/SWR1and CHD. Each class contains several distinct complex subtypes with distinct biochemical activity and biological
can be found in a recent review by Hargreaves and Crabtree ( 17).
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functions . Detailed description of these complexes is beyond the scope of this article and
As for the mammalian SWI/SNF complex, it is composed of 10-12 distinct subunits with composition partially different between different cell types (18) and a mass close to 2 mega
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daltons. The biological activity of the complex can be assayed with chromatin reconstituted in vitro. It can mobilize nucleosomes and facilitate the transcription of chromatinized
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templates. Similarly by mobilizing (moving ) the histones relative to the DNA sequences it exposes restriction sites to cleavage by restriction endonucleases (19). How this is done is not yet fully understood. Electron microscopy images of a purified yeast RSC complex, highly homologous to the genuine SWI/SNF complex of the same organism, demonstrates that the complex is much bigger that the nucleosome itself, more than one Md versus 200 Kd and that it has a cavity that can accommodate the nucleosome ( 20). It is believed that
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ATP hydrolysis is accompanied by transient disruption of DNA-histone contacts along 1020 base pairs of the nucleosome, pulling out of this DNA segment and its translocation or movement along the histone octamer core to finally result in the sliding of the DNA relative
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to the histone core (21 -23)
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The functions of the SWI/SNF complex in development and differentiation
Having isolated several of the genes encoding subunits of the SWI/SNF complex in the mouse, it was tempting to study in more detail their role in the development of higher organisms. Inactivation of the Brm helicase gave rise to viable mice that showed over growth and failure to arrest primary fibroblasts on confluency in culture( 24 ). We observed
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increased compensatory expression of BRG1 in the absence of BRM. On the contrary, the BRG1 helicase was shown to be essential for early embryonic growth (25 ). SNF5/Ini1 is another subunit of what was defined as the core complex and is encoded by a single gene in mouse or man. Its inactivation resulted in very early embryonic lethality ( 26-28), identical
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to the phenotype observed with BRG1-/- embryos. In both cases development was arrested around the implantation time. Recovery of mutant blastocystes and their culture in vitro revealed that the inner cell mass cells do not multiply in the absence of one or the other of the core complex subunits and that they undergo apoptosis(25,26) . The very early lethality
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of BRG1-/- mice was in agreement with the observations that BRG1 is expressed maternally and is the only SWI/SNF helicase present in very early development( 29). SNF5/Ini1 is also
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expressed maternally and its de novo transcription initiates very early , at the 2-4 cell stage of development (26 ). The fact that the phenotype of the BRG1 and SNF5/Ini1 knock out mice was identical strongly argues that both proteins function in the same biochemical complex, at least in early development. This difference in the behavior of the two alternative helicase subunits of the complex suggest that BRG1 is the basic helicase of the embryonic complex and that BRM takes over partially in a number of cell types during
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differentiation or growth arrest.
To investigate the function of the SWI/SNF complex at later stages of development we and others used conditional inactivation of different genes encoding subunits of the
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complex in different cell types and at different stages of development. We have used a floxed SNF5 allele and crossed these mice with a strain expressing the CRE recombinase in
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the hepatoblasts of the developing liver starting at day 10.5- 11 of development. The mutant mice developed up to birth but died shortly after birth from acute liver failure. They showed a major defect in maintaining normal plasma glucose concentrations caused by failure to accumulate glycogen and to synthesize glucose. A decreased expression of several of the corresponding biosynthetic enzymes was observed. In addition the mutant hepatocytes exhibited defects in cell-cell adhesion due to decreased synthesis of E-Cadherin and of proteins involved in gap, tight and adherens junctions. These observations suggested that the SWI/SNF complex is important for the terminal differentiation of epithelial cells. In addition we observed an increased proliferation of parachymal cells with changes in the 6
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expression of a number of cell cycle markers including p53 targets genes like p21, indicating a defect in growth arrest and apoptosis (30 ).
Global transcriptome analysis of liver specific genes up regulated during normal
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development showed a decrease in the expression of two thirds but not all of these genes. These studies clearly demonstrated that the SWI/SNF complex is essential for the terminal differentiation of the hepatocytes (we did not check other cell types), for the formation of
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polarized epithelia and for normal glucose homeostasis (30).
Evidence that the SWI/SNF complex is involved in growth control and differentiation came
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also from studies by other groups. Re-introduction of SNF5 into the Mon cell line isolated from human malignant rhabdoid tumors resulted in growth arrest and adipocyte differentiation further demonstrating the lack of fully functional SWI/SNF complex is required for specific linage differentiation (31). Subunits of the SWI/SNF complex were shown to be essential for survival and differentiation of myoblasts and other cell types (32). The complex plays a major role in the pluripotency and differentiation of ES cells (
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33, 34) . Finally specific subunits of the complex were shown to regulate neuronal differentiation ( 35).
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The SWI/SNF complex, cell transformation and cancer
Following the expression of BRM and BRG1 in different cell lines Christian Muchardt
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realized that certain human tumor cell lines lack either BRM or both BRM and BRG1(10). In addition, BRG1 and BRM were shown to interact and cooperate with pRb in repressing E2F target genes and in inducing growth arrest ( 36- 39 ). Furthermore we observed that Ras transformation of NIH 3T3 mouse fibroblasts results in strongly reduced BRM expression and that re-expression of BRM in these cells reversed their transformed phenotype( 40). This suppression activity depended on an intact helicase activity since a point mutation in the ATP binding site abolished growth suppression in soft agar. Colonies that grew in agar
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these experiments over-expressed the mutant helicase protein suggesting that it behaved as a dominant negative effector and facilitated cell transformation. These data clearly demonstrated that the SWI/SNF complex is involved in growth control and behaves as a negative regulator of growth. This hypothesis was further supported by
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observations that the level of the BRM protein strongly increased upon confluency of
epithelial cells, it was very low in mouse embryonal carcinoma cells or ES cells and went up upon differentiation( 40).
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While the data accumulated from the studies on the SWI/SNF complex suggested that it may be involved in growth control and cancer no full evidence existed until the surprising
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discovery that SNF5/Ini1 is inactivated bi-allelically in the large majority of malignant rhabdoid tumors of very young children (41,42 ). This was the first example for a tumor suppressor function of a subunit of a chromatin remodeling complex.
Following this observation, we and others investigated mice heterozygous for the Snf5 gene or with the conditional floxed allele of the Snf5 gene. We observed that these mice
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developed with age rhabdoid like tumors in their nervous system or in the periphery. In these tumors the second wild type allele of the Snf5 gene was lost further substantiating the tumor suppressor nature of this gene (26-28). While the histopathology of the murin tumors was identical to that of the human samples a major difference was observed
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between the two organisms. While in humans the penetrance of the disease in carriers of a mutant gene was close to 100% , in the case of the mice tumor appeared much slower (
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taking account of the life expectancy of humans and mice) and with much lower penetrance
Studies of mouse primary fibroblasts isolated from heterozygous mice suggested that inactivation of the second allele of Snf5 in culture by expressing a Cre recombinase from an infecting recombinant Adenovirus induces the p53 pathway and cell death. Based on these data we and the laboratory of Roberts crossed the Snf5 heterozygous mice with a p53-/- mouse. These mice developed rhabdoid tumors much faster than the original Snf5+/- mice with 100% penetrance. This model is certainly one of the fastest evolving 8
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tumors in a mouse model. These tumors were rhabdoid in nature and not of the typical lymphomas or sarcoma’s observed in p53-/- mice( 43 -45). The difference observed between humans and mice concerning the role of p53 is not fully understood. One explanation may be related to the rate of loss of the second allele. It may be fast in humans,
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slow in the mouse and accelerated in the absence of p53. Another possibility may involve a difference between human and mouse precursor cells of the tumors: the growth arrest and pro-apoptotic functions of p53 will be activated in the mouse but not human cells lacking
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SNF5/Ini1.
Further screening of tumor cell lines for the expression of BRM and BRG1 revealed that a
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number of tumor cell lines fail to express these proteins with frequencies varying between tumor types. In many cases the lack of expression was associated to an epigenetic CpG methylation of the BRG1 gene(46-49). Following these pioneering studies on SNF5/Ini1 the explosion of exonic and whole genome sequencing of tumor derived cell lines or primary tumor samples a major surprise became evident. A considerable proportion of tumors, roughly 20%, carry mutations in different subunits of the SWI/SNF complex (50,51). This
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chromatin remodeling complex is becoming a major tumor suppressor function in human cells. As described in different contributions in this volume a number of targets for the SWI/SNF complex were identified in recent years. Many of these are involved in cell cycle control like the CDK inhibitors ink4a-ARF-ink4b locus where the SNF5 dependent activity
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is required for polycomb eviction(52) p27 (24,), cyclin D1( 53 ) and several E2F target genes among others. In addition as mentioned above we believe that genes involved in
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epithelial differentiation and cell polarity are also targets for this complex. Their decreased expression will cause a block in terminal differentiation and favor cell transformation. What is less clear is why different tumors accumulate mutations in different subunits of the complex. Several of the subunits are encoded by more than one gene and may have functions in specific cell types that may be sensitive to their inactivation. Others like SNF5/Ini1 may have an essential survival function in many cell types and only a limited number of tumor cell types can survive in its absence.
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In addition to gene regulation, the SWI/SNF complex or specific sub-complexes may be involved in other processes in the cell that may be also involved in tumor formation. In the case of Saccharomyces cerevisiae it was clearly shown that the RSC complex and to a lesse extent the SWI/SNF complex are involved in chromosome segregation and centrosome
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function (54-58 ). Accumulating evidence in murin or human cells also demonstrate that the SWI/SNF complexes are involved in DNA damage control and daughter chromosome segregation ( 44,59, 60) . Such defects can also facilitate cell transformation , however they
mutations observed in rhabdoid tumor cells.
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Epigenetic and Cancer
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are somewhat incompatible with the low rate of chromosome rearrangements or point
Can we define the SWI/SNF complex as an epigenetic tumor suppressor complex? This brief summary of some of the properties and biological functions of this complex clearly indicates that this complex positively or negatively regulates a considerable number of genes at the transcriptional level in different cell types ranging from early embryonic cells
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to fully differentiated cell types. Among these genes, many involve signaling pathways that play an important role in development and cancer like the sonic hedgehog ( 61) or the Wntbeta catenin pathways ( 62 ), LIF /Stat3 signaling in ES cells by facilitating poly comb
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repression( 63) the AKT signaling pathway( 64) etc. In addition it regulates an important class of genes that control cell cycle progression including the transcriptional program of p53( 44,. 65) as well as many differentiation programs. In its absence, cell differentiation is
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blocked while cell cycle progression becomes insensitive to external cues. The activity of the SWI/SNF complex is dependent on its recruitments to specific genes by gene specific transcription factors or by histone modifications like acetylation that can interact with bromodomains present in different subunits of the complex. In addition several of the subunits has non specific DNA binding motifs. The changes in gene expression profiles caused by absence or the defects in specific subunits is transmitted during cell divisions and expansion of tumor cells. In this respect we can clearly talk about an epigenetic regulator. However it’s effect on gene expression and growth is limited to
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somatic cells and does not involve germ line transmission to the off-springs. The SWI/SNF complex can be viewed as the prototype of an epigenetic regulator of gene expression that is involved in tumor suppression. The broad implication of different subunits of the complex in different types of human cancer suggest that partial to global loss of SWI/SNF
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activity may result in a multitude of deregulated cellular programs and the final out-come , cell survival , cell death or malignant transformation will differ from one cell type to
another. It will be important to find ways to restore its activity in tumor cells that will
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