QNAS
QNAS
QnAs with Shiv Grewal Brian Doctrow, Science Writer
The chromosomes that make up eukaryotic genomes consist of a complex of DNA, RNA, and proteins called chromatin. Chromatin occurs in two forms: euchromatin, a loosely packaged form that is poised for transcription, and heterochromatin, a tightly packaged, transcriptionally repressed form. By determining which genes are expressed, heterochromatin influences cell differentiation, heritability of gene-expression patterns, and the ability of cells to adapt to their environment. Shiv Grewal, elected to the National Academy of Sciences in 2014, has dedicated his career to elucidating the mechanisms of heterochromatin assembly. Grewal’s most significant discoveries include the description of specific histone methylation patterns that distinguish heterochromatin from euchromatin, the genetic demonstration of the heritability of heterochromatin, and the uncovering of the role of RNA in heterochromatin assembly. PNAS recently spoke with Grewal, a Distinguished Investigator and Laboratory Chief at the National Cancer Institute, National Institutes of Health, about his research. PNAS: How did you first become interested in heterochromatin research?
Shiv Grewal. Image courtesy of Bill Branson and the National Cancer Institute.
Grewal: I was always fascinated by how repressive chromatin domains are assembled over large regions of the genome. I wanted to study this phenomenon in a genetically tractable system, and I knew that the genome of the fission yeast Schizosaccharomyces pombe contained a large recombinationally repressed domain at the mating-type (mat) locus. But the sequence of that region wasn’t known. So as a postdoctoral fellow, I took it upon myself to sequence the entire region, and I noticed that there was a repeat element in that region. A homology search revealed that the repeat element was
nearly identical to repeats that are associated with centromeres in S. pombe. That led me to think that maybe I had cloned the wrong region, but I very quickly realized that no, indeed, I had cloned the right one. To my surprise, when I deleted that repeat element I found that gene silencing and recombinational repression were affected across the entire domain. Subsequently, we determined that deletion of the repeat element affected the establishment of heterochromatin that coats the entire repressed domain. These results made it very clear that understanding how this repeat element triggers heterochromatin assembly was important. PNAS: In 2002, you published two papers in Science (1, 2), showing that small RNAs produced by the RNA interference (RNAi) pathway were required for heterochromatin formation in S. pombe. The role of small RNAs in regulating chromosome structure was named “Breakthrough of the Year” by Science in 2002. What was the significance of that discovery? Grewal: If you want to specify which chromatin domains are silenced and which are expressed, you need to target the appropriate enzymes that open or close chromatin. Before those publications, everyone believed that targeting enzymes to specific regions of the genome could only occur through DNA binding proteins. But that presents a significant logistical challenge; most heterochromatin regions contain transposons and their remnants, or repeat elements. How do cells quickly adapt to target heterochromatin to invasive repetitive DNA elements? My earlier finding that a repeat element triggered heterochromatin assembly across the mat locus set the stage for our later discovery that RNAi processes the repeat transcripts to target heterochromatin assembly. Our work brought RNA to the forefront as the specificity determinant, and not only introduced a new paradigm for targeting heterochromatin assembly activities, but also explained how RNA could be harnessed as a rapid and adaptive targeting mechanism. Importantly, the RNAi-based mechanism of heterochromatin targeting is conserved in C[aenorhabditis]. elegans, plants, and many other organisms. PNAS: Does RNA also target heterochromatin assembly to gene-rich regions?
This is a QnAs with a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 15548 in issue 51 of volume 112.
9388–9389 | PNAS | August 23, 2016 | vol. 113 | no. 34
www.pnas.org/cgi/doi/10.1073/pnas.1612149113
Grewal: Yes. It is important to note that there are two types of heterochromatin, and RNA is crucial to targeting both types. Constitutive heterochromatin is associated with centromeric repeats, telomeres, and the repressed mat locus that I mentioned earlier. At all three locations RNAi has a role in targeting or nucleation of heterochromatin. But then there are other heterochromatin domains distributed across the S. pombe genome that coat genes. These domains are akin to facultative heterochromatin that undergoes regulated assembly and disassembly in higher eukaryotes. We discovered a novel RNA-based mechanism involving the conserved nuclear RNA elimination machinery, called MTREC [Mtl1–Red1 core], which assembles facultative heterochromatin at gametogenic genes (normally silenced during vegetative growth) independent of RNAi. We also discovered that RNA elimination factors cooperate with RNAi to assemble heterochromatin at retrotransposons and developmentally regulated genes under specific growth conditions. From these discoveries emerged another new paradigm: that the cell is able to distinguish many types of RNA molecules and channel them to RNA processing machinery to target heterochromatin. PNAS: In your Inaugural Article (3) you explore the role of noncanonical transcription termination in heterochromatin formation. Why did you study transcription termination? Grewal: Transcription of target sequences is important for forming heterochromatin. This raises a fundamental question: What is the difference between transcriptional activity that generates a functional mRNA and transcriptional activity that triggers heterochromatin assembly? An important clue was that the RNA elimination factors that trigger heterochromatin assembly also associate with factors involved in 3′-end processing of RNA transcripts. We wondered whether these factors might be components of a specialized noncanonical termination machinery that promotes both RNA degradation and heterochromatin formation. We found that the conserved exoribonuclease Dhp1/Rat1/Xrn2, which is involved in 3′-end processing and transcription termination, acts together with elimination factors to degrade target transcripts and recruit heterochromatin assembly proteins. Our work suggests that cotranscriptional RNA processing factors not only determine the fate of target transcripts, but also specify sites of heterochromatin assembly. If normal termination occurs, then you get a functional mRNA. However, termination by a noncanonical
mechanism involving elimination factors is linked to RNA degradation and heterochromatin assembly. We believe these findings have important implications for understanding how certain genomic regions, including regulated genes and repeat elements, are specified as targets of heterochromatin assembly in higher eukaryotes. PNAS: You also found that heterochromatin domains can be inherited. What are the implications of this finding for genetics and development? Grewal: In work we published almost 15 years ago (1), we showed that heterochromatin can be epigenetically inherited in cis during both mitosis and meiosis. This work suggested that the unit of inheritance, what we call the gene, can include DNA plus its associated nucleoprotein complexes. Our subsequent work defined mechanisms by which histone modifiers both “write” and “read” methylated histones to promote epigenetic inheritance of heterochromatin. Others have recently confirmed these findings. The fact that the chromatin state can be stably propagated during cell division raises the important question of whether defects in epigenetic maintenance of chromatin structure could be responsible for phenotypic changes or certain human diseases. There is a lot of attention on epigenetics, and whether what you are exposed to in this generation might have implications for the next generation. I don’t know how much of that is true, but at least at the cellular level during development, the differentiated state of the cell is maintained every time that the cell divides. So the heritability of chromatin structure does play an important role in maintaining the hallmark gene-expression profile of a particular cell type. PNAS: What medical applications might your work have? Grewal: The inappropriate expression of genes that are normally expressed only in specific cell types, such as germ cells, can lead to cancer. But the mechanisms that keep those germ cell-specific genes repressed in somatic cells are not very well understood. We are driven by the possibility of applying what we have learned from S. pombe to similar mechanisms that are acting in mammalian cells. The well-conserved mechanisms that we study in S. pombe will help us find ways to exploit these pathways in human cells for therapeutic drug targeting to treat cancer and other developmental diseases.
1 Hall IM, et al. (2002) Establishment and maintenance of a heterochromatin domain. Science 297(5590):2232–2237. 2 Volpe TA, et al. (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297(5588): 1833–1837. 3 Chalamcharla VS, Folco HD, Dhakshnamoorthy J, Grewal SI (2016) A conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing. Proc Natl Acad Sci USA 112(51):15548–15555.
Doctrow
PNAS | August 23, 2016 | vol. 113 | no. 34 | 9389
QNAS
QnAs with Shiv Grewal Brian Doctrow, Science Writer
The chromosomes that make up eukaryotic genomes consist of a complex of DNA, RNA, and proteins called chromatin. Chromatin occurs in two forms: euchromatin, a loosely packaged form that is poised for transcription, and heterochromatin, a tightly packaged, transcriptionally repressed form. By determining which genes are expressed, heterochromatin influences cell differentiation, heritability of gene-expression patterns, and the ability of cells to adapt to their environment. Shiv Grewal, elected to the National Academy of Sciences in 2014, has dedicated his career to elucidating the mechanisms of heterochromatin assembly. Grewal’s most significant discoveries include the description of specific histone methylation patterns that distinguish heterochromatin from euchromatin, the genetic demonstration of the heritability of heterochromatin, and the uncovering of the role of RNA in heterochromatin assembly. PNAS recently spoke with Grewal, a Distinguished Investigator and Laboratory Chief at the National Cancer Institute, National Institutes of Health, about his research. PNAS: How did you first become interested in heterochromatin research?
Shiv Grewal. Image courtesy of Bill Branson and the National Cancer Institute.
Grewal: I was always fascinated by how repressive chromatin domains are assembled over large regions of the genome. I wanted to study this phenomenon in a genetically tractable system, and I knew that the genome of the fission yeast Schizosaccharomyces pombe contained a large recombinationally repressed domain at the mating-type (mat) locus. But the sequence of that region wasn’t known. So as a postdoctoral fellow, I took it upon myself to sequence the entire region, and I noticed that there was a repeat element in that region. A homology search revealed that the repeat element was
nearly identical to repeats that are associated with centromeres in S. pombe. That led me to think that maybe I had cloned the wrong region, but I very quickly realized that no, indeed, I had cloned the right one. To my surprise, when I deleted that repeat element I found that gene silencing and recombinational repression were affected across the entire domain. Subsequently, we determined that deletion of the repeat element affected the establishment of heterochromatin that coats the entire repressed domain. These results made it very clear that understanding how this repeat element triggers heterochromatin assembly was important. PNAS: In 2002, you published two papers in Science (1, 2), showing that small RNAs produced by the RNA interference (RNAi) pathway were required for heterochromatin formation in S. pombe. The role of small RNAs in regulating chromosome structure was named “Breakthrough of the Year” by Science in 2002. What was the significance of that discovery? Grewal: If you want to specify which chromatin domains are silenced and which are expressed, you need to target the appropriate enzymes that open or close chromatin. Before those publications, everyone believed that targeting enzymes to specific regions of the genome could only occur through DNA binding proteins. But that presents a significant logistical challenge; most heterochromatin regions contain transposons and their remnants, or repeat elements. How do cells quickly adapt to target heterochromatin to invasive repetitive DNA elements? My earlier finding that a repeat element triggered heterochromatin assembly across the mat locus set the stage for our later discovery that RNAi processes the repeat transcripts to target heterochromatin assembly. Our work brought RNA to the forefront as the specificity determinant, and not only introduced a new paradigm for targeting heterochromatin assembly activities, but also explained how RNA could be harnessed as a rapid and adaptive targeting mechanism. Importantly, the RNAi-based mechanism of heterochromatin targeting is conserved in C[aenorhabditis]. elegans, plants, and many other organisms. PNAS: Does RNA also target heterochromatin assembly to gene-rich regions?
This is a QnAs with a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 15548 in issue 51 of volume 112.
9388–9389 | PNAS | August 23, 2016 | vol. 113 | no. 34
www.pnas.org/cgi/doi/10.1073/pnas.1612149113
Grewal: Yes. It is important to note that there are two types of heterochromatin, and RNA is crucial to targeting both types. Constitutive heterochromatin is associated with centromeric repeats, telomeres, and the repressed mat locus that I mentioned earlier. At all three locations RNAi has a role in targeting or nucleation of heterochromatin. But then there are other heterochromatin domains distributed across the S. pombe genome that coat genes. These domains are akin to facultative heterochromatin that undergoes regulated assembly and disassembly in higher eukaryotes. We discovered a novel RNA-based mechanism involving the conserved nuclear RNA elimination machinery, called MTREC [Mtl1–Red1 core], which assembles facultative heterochromatin at gametogenic genes (normally silenced during vegetative growth) independent of RNAi. We also discovered that RNA elimination factors cooperate with RNAi to assemble heterochromatin at retrotransposons and developmentally regulated genes under specific growth conditions. From these discoveries emerged another new paradigm: that the cell is able to distinguish many types of RNA molecules and channel them to RNA processing machinery to target heterochromatin. PNAS: In your Inaugural Article (3) you explore the role of noncanonical transcription termination in heterochromatin formation. Why did you study transcription termination? Grewal: Transcription of target sequences is important for forming heterochromatin. This raises a fundamental question: What is the difference between transcriptional activity that generates a functional mRNA and transcriptional activity that triggers heterochromatin assembly? An important clue was that the RNA elimination factors that trigger heterochromatin assembly also associate with factors involved in 3′-end processing of RNA transcripts. We wondered whether these factors might be components of a specialized noncanonical termination machinery that promotes both RNA degradation and heterochromatin formation. We found that the conserved exoribonuclease Dhp1/Rat1/Xrn2, which is involved in 3′-end processing and transcription termination, acts together with elimination factors to degrade target transcripts and recruit heterochromatin assembly proteins. Our work suggests that cotranscriptional RNA processing factors not only determine the fate of target transcripts, but also specify sites of heterochromatin assembly. If normal termination occurs, then you get a functional mRNA. However, termination by a noncanonical
mechanism involving elimination factors is linked to RNA degradation and heterochromatin assembly. We believe these findings have important implications for understanding how certain genomic regions, including regulated genes and repeat elements, are specified as targets of heterochromatin assembly in higher eukaryotes. PNAS: You also found that heterochromatin domains can be inherited. What are the implications of this finding for genetics and development? Grewal: In work we published almost 15 years ago (1), we showed that heterochromatin can be epigenetically inherited in cis during both mitosis and meiosis. This work suggested that the unit of inheritance, what we call the gene, can include DNA plus its associated nucleoprotein complexes. Our subsequent work defined mechanisms by which histone modifiers both “write” and “read” methylated histones to promote epigenetic inheritance of heterochromatin. Others have recently confirmed these findings. The fact that the chromatin state can be stably propagated during cell division raises the important question of whether defects in epigenetic maintenance of chromatin structure could be responsible for phenotypic changes or certain human diseases. There is a lot of attention on epigenetics, and whether what you are exposed to in this generation might have implications for the next generation. I don’t know how much of that is true, but at least at the cellular level during development, the differentiated state of the cell is maintained every time that the cell divides. So the heritability of chromatin structure does play an important role in maintaining the hallmark gene-expression profile of a particular cell type. PNAS: What medical applications might your work have? Grewal: The inappropriate expression of genes that are normally expressed only in specific cell types, such as germ cells, can lead to cancer. But the mechanisms that keep those germ cell-specific genes repressed in somatic cells are not very well understood. We are driven by the possibility of applying what we have learned from S. pombe to similar mechanisms that are acting in mammalian cells. The well-conserved mechanisms that we study in S. pombe will help us find ways to exploit these pathways in human cells for therapeutic drug targeting to treat cancer and other developmental diseases.
1 Hall IM, et al. (2002) Establishment and maintenance of a heterochromatin domain. Science 297(5590):2232–2237. 2 Volpe TA, et al. (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297(5588): 1833–1837. 3 Chalamcharla VS, Folco HD, Dhakshnamoorthy J, Grewal SI (2016) A conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing. Proc Natl Acad Sci USA 112(51):15548–15555.
Doctrow
PNAS | August 23, 2016 | vol. 113 | no. 34 | 9389
