- The Structure and Function of Chromatin
- Histone Modification: The What, How, and Why
- Chromatin Structure and Function
- Molecular Cell
- Chromatin modifications and their function pdf printer
- Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives
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The Structure and Function of Chromatin
The eukaryotic genome is packaged into a repeating subunit known as the nucleosome, which consists of bp of DNA wrapped nearly twice around an octamer of basic histone proteins. All eukaryotic DNA transactions, from transcription to DNA repair to recombination, occur in the context of the packaging of this nucleosomal packaging.
As the DNA wrapped around the nucleosome and the DNA located between nucleosomes differ in their accessibility and structural characteristics, the precise locations of nucleosomes along the genome have proven to be of great importance for understanding the function of the genome.
Our laboratory has long been interested in the structural biology of the genome — what are the rules underlying genomic packaging, and what are the functional implications of different packaging states? We have focused on the budding yeast S.
Histone Modification: The What, How, and Why
We have continued to investigate aspects of nucleosome positioning in budding yeast, using both traditional genetic approaches Weiner et al, Genome Res as well as comparative genomic studies Tsankov et al, PLoS Bio , Hughes et al, Mol Cell to illuminate the forces responsible for nucleosome positioning in vivo. The lessons learned from yeast nucleosome maps have largely proved general in a multitude of species subject to genome-wide nucleosome mapping, with a number of key concepts emerging Figure 1.
First, active and poised promoters are nucleosome-depleted, as are active enhancers.
Chromatin Structure and Function
Second, nucleosomes bordering regulatory elements are well-positioned. Third, highly transcribed genes tend to have lower nucleosome occupancy and less precise positioning, most likely due to action of RNA polymerase II and its associated factors.
Finally, the density of the nucleosome template average distance between two adjacent nucleosomes can vary, mostly due to trans factors. We continue to study nucleosome positioning to some extent in yeast, with an active interest in single molecule decomposition of ensemble measurements.
Epigenomic assays capture snapshots of complex cellular states. Unlike the DNA sequence, which is highly stable, chromatin can be highly dynamic, raising the question of the relevant time-scales that govern chromatin structures.
This question is related to, but distinct from, the question of heterogeneity within a cell population. This question applies throughout the range of structures found in vivo, from long-range chromosomal interactions to nucleosome chemical modifications. Understanding the function of the structures observed will ultimately require knowing the relevant time-scales for the structures in question. To characterize chromatin dynamics genome-wide, we have adapted genetically-encoded pule-chase methods to characterize replication-independent nucleosome turnover rates genome-wide Dion et al, Science These studies reveal replication-independent turnover at transcribed genes, with particularly rapid histone replacement occurring at regulatory regions promoters and enhancers.
Mutant studies from many labs including ours have enabled mechanistic insights into the cellular machinery responsible for histone replacement.
Over longer time scales, the question of histone dynamics is key to any mechanistic understanding of how chromatin states are copied from one generation to the next. To understand the mechanism by which chromatin states could be inherited, it is necessary to understand the unique challenges posed by histone protein dynamics during replication.
First, during genomic replication the passage of the replication fork disrupts histone-DNA contacts, and old histones must reassociate with daughter chromosomes at a location close to their original location on the mother chromosome. Otherwise, locus-specific epigenetic information would be randomly shuffled every generation.
Chromatin modifications and their function pdf printer
Second, old histones only account for one half of the histones on each new genome, and the remaining histones are newly synthesized during each S phase. This implies some information passage from old to new histones, as otherwise old chromatin states would rapidly be diluted by new histones.
To attempt to measure the extent of nucleosome movement during genomic replication, we made use of a genetic pulse-chase system developed by the van Leeuwen laboratory in which induction of Cre-Lox recombination results in a switch from one epitope-tagged histone H3 to a new epitope tag.
This system enabled us to switch off an ancestral H3 tag and subsequently follow the genomic disposition of the ancestral H3 for several cell divisions Radman-Livaja et al, PLoS Bio These results therefore constrain the maximum amount of information theoretically carried by chromatin between generations.
The beads on a string structure of the chromatin fiber is not composed of uniform beads, as the chemical makeup of individual nucleosomes can in principle be extremely variable from nucleosome to nucleosome, with important consequences for genome function.
Most dramatically, the histone proteins can be chemically modified, with a prodigious variety of post-translational modifications occurring acetylation, methylation, phosphorylation, ubiquitylation, and many more at multiple residues in all of the histones. These chemical modifications can alter the chemical and physical properties of the nucleosome, but also serve to modulate binding by proteins that recognize the modified state e.
Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives
This incredible diversity of histone modifications leads naturally to the question of what it all means — why do so many histone modifications occur in the cell? We have long been interested in the question of how combinatorial complexity contributes to chromatin regulation. How many distinct modification combinations occur Figure 2 , and can specific combinations of modifications be matched with specific outcomes such that the code can be deciphered? We have taken two approaches to these questions, based on 1 genome-wide mapping of histone modifications at mononucleosome resolution Liu et al, PLoS Bio , Weiner et al, Mol Cell , and 2 analysis of transcriptional changes in budding yeast carrying either mutant histones or deletions of histone-modifying enzymes Dion et al, PNAS , Weiner et al, PLoS Bio Focusing first on genomic mapping, large-scale efforts in histone state mapping in multiple model systems reveal a number of conserved aspects of chromatin structure.
Z variants, and H3K56 acetylation. Gene body nucleosomes are typically marked with H3K36me3 and H3K79me3, and are generally somewhat depleted of histone tail acetylations. Other histone marks distinguish repressed, poised, and active enhancers, with H3K27 methylation and acetylation marking repressed and active enhancers, respectively. Third, two major forms of repressive chromatin are associated with specific histone modifications.
Classical heterochromatin including telomeres and many repetitive sequences is marked with H3K9 methylation, while genes repressed by polycomb-group factors are marked with H3K27me3.
Finally, nucleosomes associated with centromeres often contain the H3-like CENP-A protein, and during M phase a broad pericentric domain of nucleosomes are marked with H3S10ph.
Overall, there is a striking correspondence of histone state with the function of genomic regions, and because of this mapping of histone modifications is an effective method to discover genes and regulatory elements. Turning to the functions of histone modifications, one systematic approach to understand the functions of combinatorial histone modifications has been whole genome analysis of gene expression changes observed in histone mutants.
Such studies typically find that histone mutants exhibit relatively little phenotypic complexity resulting from different combinations of histone mutants. This is seen both in studies focusing on specific histone point mutants and their combinations, as well as in studies focusing on deletion mutants of histone modifying enzymes.
For example, we systematically examined all 16 possible combinatorial mutations among the 4 lysines in the histone H4 tail, finding that mutation of three of the residues lysines 5, 8, and 12 had indistinguishable effects on gene expression, whereas lysine 16 was confirmed to have unique effects on gene expression Dion et al, PNAS Furthermore, gene expression defects in combinatorial mutants were little different from linear combinations of the component mutations — in other words, the effect of the H4K5,16R double mutant on gene expression could be predicted by adding the K5R and K16R datasets together.
In contrast to the modest effects of many histone modifying deletions observed at steady-state, single gene studies suggest that chromatin regulators have important roles in dynamic processes that are masked at steady-state. This has motivated us to study chromatin regulators in the context of transcriptional reprogramming, using diamide stress in yeast as a convenient paradigm for inducing massive transcriptional changes Weiner et al, PLoS Bio Grouping deletion mutants with similar gene expression defects identifies known complexes, and that joint analysis of histone mutants and deletion mutants associates many histone modifying enzymes with their target sites.
Together, these data provide a rich multi-modal view on the role of chromatin regulators in gene induction and repression dynamics, and suggest that understanding the myriad roles of chromatin structure in gene regulation on a genome-wide scale will require extending mutant analyses to kinetic studies. While the 1-dimensional structure of chromatin is increasingly well-understood, our understanding of the three-dimensional folding of the genome in the nucleus has traditionally lagged behind.
That said, over the past decade this gap has been dramatically narrowed thanks to the development by Dekker and colleagues of the 3C Chromosome Conformation Capture family of techniques.
In these methods, chromatin is subject to crosslinking in vivo to capture interactions between chromosomal loci. The genome is then fragmented, typically with restriction enzymes, and DNA ligation is used to capture interactions between chromosomal loci that were in contact with one another in vivo.
Genome-wide variants of 3C, such as Hi-C, have revealed a number of organizational features of eukaryotic genomes at increasingly fine resolutions, from the scale of full chromosomal territories, to multi-Mb active and inactive compartments, to hundred-kb contact domains TADs , to enhancer-promoter loops.
Our method provides insight into yeast genome folding at all length scales of interest.
At larger scales, the Rabl configuration of chromosomes is seen as clustering of centromeres, and interactions between the telomeres of chromosome arms of similar length. The concordance of multiple independent approaches to analysis of chromosome folding provides hope that we are approaching a true understanding of the structures adopted by chromosomes in the cell.
Chromatin Structure and Function Nucleosome positioning The eukaryotic genome is packaged into a repeating subunit known as the nucleosome, which consists of bp of DNA wrapped nearly twice around an octamer of basic histone proteins.
Histone dynamics Epigenomic assays capture snapshots of complex cellular states. Histone modifications The beads on a string structure of the chromatin fiber is not composed of uniform beads, as the chemical makeup of individual nucleosomes can in principle be extremely variable from nucleosome to nucleosome, with important consequences for genome function. Chromosome folding While the 1-dimensional structure of chromatin is increasingly well-understood, our understanding of the three-dimensional folding of the genome in the nucleus has traditionally lagged behind.