Sharmistha Kundu

MNase titration reveals differences between nucleosome occupancy and chromatin accessibility.

Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation.

Combined modulation of polycomb and trithorax genes rejuvenates β cell replication

Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclin-dependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the age-dependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass.

Dominant Role for Signal Transduction in the Transcriptional Memory of Yeast GAL Genes

ABSTRACT Several recent studies have shown that the transcriptional induction of yeast GAL genes occurs with faster kinetics if the gene has been previously expressed. Depending on the experimental regimen, this transcriptional “memory” phenomenon can persist for 1 to 2 cell divisions in the absence of an inducer (short-term memory) or for >6 cell divisions (long-term memory). Long-term memory requires the GAL1 gene, suggesting that memory involves the cytoplasmic inheritance of high levels of Gal1 that are expressed in the initial round of expression. In contrast, short-term memory requires the SWI/SNF chromatin-remodeling enzyme, and thus, it may involve the inheritance of distinct chromatin states. Here we have reevaluated the roles of SWI/SNF, the histone variant H2A.Z, and components of the nuclear pore in both the short-term and long-term memory of GAL genes. Our results suggest that the propagation of novel chromatin structures does not contribute to the transcriptional memory of GAL genes, but rather, memory of the previous transcription state is controlled primarily by the inheritance of the Gal3p and Gal1p signaling factors.

Role of chromatin states in transcriptional memory.

Establishment of cellular memory and its faithful propagation is critical for successful development of multicellular organisms. As pluripotent cells differentiate, choices in cell fate are inherited and maintained by their progeny throughout the lifetime of the organism. A major factor in this process is the epigenetic inheritance of specific transcriptional states or transcriptional memory. In this review, we discuss chromatin transitions and mechanisms by which they are inherited by subsequent generations. We also discuss illuminating cases of cellular memory in budding yeast and evaluate whether transcriptional memory in yeast is nuclear or cytoplasmically inherited.

Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation

Master regulatory genes require stable silencing by the polycomb group (PcG) to prevent misexpression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in ESCs and neural progenitors using 5C and super-resolution microscopy. The genomic loci of repressed PcG targets formed discrete, small (20-140 Kb) domains of tight interaction that corresponded to locations bound by canonical polycomb repressive complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1 and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in size and boundary characteristics. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher-order structure.

Mutation of a nucleosome compaction region disrupts Polycomb-mediated axial patterning

Polycomb group gene silencing Histone proteins wrap around DNA to form nucleosomes that package metazoan DNA into the nucleus. Chromatin compaction is also believed to be critical for the repression of homeotic genes by the Polycomb repressive complex 1 (PRC1) during development. Lau et al. used mutagenesis and expression analyses to examine this gene silencing mechanism. Altering the PRC1 compaction region affected transcriptional repression and patterning of the mouse body axis. Thus, chromatin compaction may drive the stable and heritable silencing of genes involved in body patterning. Science, this issue p. 1081 Nucleosome compaction by PRC1 is likely necessary for maintaining gene repression during development. Nucleosomes play important structural and regulatory roles by tightly wrapping the DNA that constitutes the metazoan genome. The Polycomb group (PcG) proteins modulate nucleosomes to maintain repression of key developmental genes, including Hox genes whose temporal and spatial expression is tightly regulated to guide patterning of the anterior-posterior body axis. CBX2, a component of the mammalian Polycomb repressive complex 1 (PRC1), contains a compaction region that has the biochemically defined activity of bridging adjacent nucleosomes. Here, we demonstrate that a functional compaction region is necessary for proper body patterning, because mutating this region leads to homeotic transformations similar to those observed with PcG loss-of-function mutations. We propose that CBX2-driven nucleosome compaction is a key mechanism by which PcG proteins maintain gene silencing during mouse development.

Enhancer regions show high histone H3.3 turnover that changes during differentiation

The organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation. DOI: http://dx.doi.org/10.7554/eLife.15316.001

Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction

Activation of transcription requires alteration of chromatin by complexes that increase the accessibility of nucleosomal DNA. Removing nucleosomes from regulatory sequences has been proposed to play a significant role in activation. We tested whether changes in nucleosome occupancy occurred on the set of genes that is activated by the unfolded protein response (UPR). We observed no decrease in occupancy on most promoters, gene bodies, and enhancers. Instead, there was an increase in the accessibility of nucleosomes, as measured by micrococcal nuclease (MNase) digestion and ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing), that did not result from removal of the nucleosome. Thus, changes in nucleosome accessibility predominate over changes in nucleosome occupancy during rapid transcriptional induction during the UPR.

Multitasking by Polycomb response elements

Development requires the expression of master regulatory genes necessary to specify a cell lineage. Equally significant is the stable and heritable silencing of master regulators that would specify alternative lineages. This regulated gene silencing is carried out by Polycomb group (PcG) proteins, which must be correctly recruited only to the subset of their target loci that requires lineage-specific silencing. A recent study by Erceg and colleagues (pp. 590-602) expands on a key aspect of that targeting: The same DNA elements that recruit PcG complexes to a repressed locus also encode transcriptional enhancers that function in different lineages where that locus must be expressed. Thus, PcG targeting elements overlap with enhancers.