Sivakanthan Kasinathan

Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types.

ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo

Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.

High-resolution mapping defines the cooperative architecture of Polycomb response elements

Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb response elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of nonhistone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor ADF1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished by enhanced Polycomb recruitment both to PREs and to target promoters of repressed genes. These results suggest that the stability of multifactor complexes at promoters and regulatory elements is a crucial aspect of developmentally regulated gene expression.

A unique chromatin complex occupies young α-satellite arrays of human centromeres

Young α-satellite tandem dimers dominate functional human centromeres and precisely position pairs of 100-bp CENP-A nucleosomes. The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100–base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C–containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes.

Mapping Regulatory Factors by Immunoprecipitation from Native Chromatin

Occupied Regions of Genomes from Affinity‐purified Naturally Isolated Chromatin (ORGANIC) is a high‐resolution method that can be used to quantitatively map protein‐DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low‐salt solubilization to preserve protein‐DNA complexes, followed by immunoprecipitation and paired‐end sequencing for genome‐wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein‐DNA complexes, and high‐throughput sequencing to map sites of bound factors.

Non-B-Form DNA Is Enriched at Centromeres

Abstract Animal and plant centromeres are embedded in repetitive “satellite” DNA, but are thought to be epigenetically specified. To define genetic characteristics of centromeres, we surveyed satellite DNA from diverse eukaryotes and identified variation in <10‐bp dyad symmetries predicted to adopt non‐B‐form conformations. Organisms lacking centromeric dyad symmetries had binding sites for sequence‐specific DNA‐binding proteins with DNA‐bending activity. For example, human and mouse centromeres are depleted for dyad symmetries, but are enriched for non‐B‐form DNA and are associated with binding sites for the conserved DNA‐binding protein CENP‐B, which is required for artificial centromere function but is paradoxically nonessential. We also detected dyad symmetries and predicted non‐B‐form DNA structures at neocentromeres, which form at ectopic loci. We propose that centromeres form at non‐B‐form DNA because of dyad symmetries or are strengthened by sequence‐specific DNA binding proteins. This may resolve the CENP‐B paradox and provide a general basis for centromere specification.

Simple and Complex Centromeric Satellites in Drosophila Sibling Species

Centromeres are the chromosomal sites of assembly for kinetochores, the protein complexes that attach to spindle fibers and mediate separation of chromosomes to daughter cells in mitosis and meiosis. In most multicellular organisms, centromeres comprise a single specific family of tandem repeats—often 100–400 bp in length—found on every chromosome, typically in one location within heterochromatin. Drosophila melanogaster is unusual in that the heterochromatin contains many families of mostly short (5–12 bp) tandem repeats, none of which appear to be present at all centromeres, and none of which are found only at centromeres. Although centromere sequences from a minichromosome have been identified and candidate centromere sequences have been proposed, the DNA sequences at native Drosophila centromeres remain unknown. Here we use native chromatin immunoprecipitation to identify the centromeric sequences bound by the foundational kinetochore protein cenH3, known in vertebrates as CENP-A. In D. melanogaster, these sequences include a few families of 5- and 10-bp repeats; but in closely related D. simulans, the centromeres comprise more complex repeats. The results suggest that a recent expansion of short repeats has replaced more complex centromeric repeats in D. melanogaster.

Non-B-form DNA structures mark centromeres

Animal and plant centromeres are embedded in repetitive “satellite” DNA, but are thought to be epigenetically specified. To define genetic characteristics of centromeres, we surveyed satellite DNA from diverse eukaryotes and identified variation in <10-bp dyad symmetries predicted to adopt non-B-form conformations. Organisms lacking centromeric dyad symmetries had binding sites for sequence-specific DNA binding proteins with DNA bending activity. For example, human and mouse centromeres are depleted for dyad symmetries, but are enriched for non-B DNA and are associated with binding sites for the conserved DNA-binding protein CENP-B, which is required for artificial centromere function but is paradoxically non-essential. We also detected dyad symmetries and predicted non-B-form DNA structures at neocentromeres, which form at ectopic loci. We propose that centromeres form at non-B-form DNA because of dyad symmetries or are strengthened by sequence-specific DNA binding proteins. Our findings resolve the CENP-B paradox and provide a general basis for centromere specification.

Correspondence: Reply to ‘DNA shape is insufficient to explain binding’

Transcription factors (TFs) are DNA-binding proteins that regulate gene expression. Sequence-specific TFs recognize DNA via specific amino acid-base hydrogen bonds and contacts that read local DNA shape1. Studying base and shape readout modes of TFs in vivo has been challenging due to technical issues associated with current approaches for mapping TF-binding sites (TFBSs). We recently introduced Chromatin Endogenous Cleavage with sequencing (ChEC-seq), an in vivo mapping method based on fusing Micrococcal Nuclease (MNase) to a TF (ref. 2). Upon addition of calcium to permeabilized cells, tethered MNase cuts DNA adjacent to the bound TF and the released fragments are sequenced to provide a high-resolution genomewide TFBS map. We used ChEC-seq to map the budding yeast TFs Abf1, Reb1 and Rap1 and obtained data similar to highresolution ChIP-seq without the need for cross-linking, chromatin solubilization or antibodies. When cells were collected o1 min after calcium addition, most TFBSs contained a TF-specific sequence motif (‘fast’ sites). We also reported ‘slow’ sites with low motif scores that appeared after B10 min. We found that DNA shape features of high-scoring (mostly fast) and low-scoring (mostly slow) TFBSs corresponded closely, but differed from randomly chosen sites not overlapping highor low-scoring sites. In our study, DNA shape features of fast and slow sites were centred on the best match to the TF consensus motif; however, randomly chosen genomic intervals were not similarly centred on the best motif match. Rossi, Lai and Pugh now find that when random sites are motif-centred, the shape features correspond closely to slow site features3, which might suggest that DNA shape is insufficient to explain binding site selection by the TFs Abf1, Reb1 and Rap1. However, given that sequence and shape features covary4, it is problematic to rely on motif-dependent analyses to draw conclusions about whether a TF recognizes DNA shape5. To address this problem, we aligned DNA shape feature vectors for unique fast and slow ChEC-seq sites for each TF using a procedure that relied only on shape data and was not directly informed by sequence alignment. Given the possibility for overlap between nearby TFBSs, we identified unique sites that do not intersect with any other ChEC-seq sites within intervals ranging from 100 to 500 bp surrounding ChEC-seq peak maxima, with larger windows associated with increasing stringency. For Abf1 and Reb1, we found that average fast and slow site shape features were well correlated at a range of interval widths (Poo0.001; Fig. 1a–c). We also searched sites using a ‘shape profile’ defined using the average fast site features and found that score distributions for fast and slow sites only slightly differed (P40.03), but were very different from random and free MNase sites (Poo10 10) for Abf1 (Fig. 1b) and Reb1 (not shown). The major shape feature proximal to Abf1 motifs is a deformation to the helix indicative of motif-proximal poly(dA:dT) tracts (Fig. 1a), a sequence feature we observed at slow sites in our original study2. Consistent with the recognition of a preferred shape signature by Abf1 and Reb1 at fast and slow sites, random sites and free MNase sites were not well correlated with fast and slow sites (Fig. 1a–c). We do not observe shape features enriched for poly(dA:dT) tracts at free MNase sites (Fig. 1a,b), suggesting that the detection of this shared shape feature at fast and slow ChEC-seq sites is not simply due to the higher prevalence of these features within nucleosome-depleted regions. Shape features at Rap1 fast and slow sites were not well correlated (Po0.1; Fig. 1c,d). The robustness of the correlation between average fast and slow shape features for Abf1 and Reb1 across a range of interval widths (Fig. 1d) suggests that sampling of similar shapes by TFs may explain binding events, even within promoters where fast and slow sites co-occur. From these motif-independent analyses, we conclude that fast and slow binding sites for Abf1 and Reb1 have similar shape features. We next queried a TF-gene regulatory association database6, and asked whether TF-slow site associations had been previously observed in mapping or gene expression studies orthogonal to ChEC-seq. Consistent with our previous demonstration that slow sites were recovered as sites without the canonical motif in other studies2, the proportion of fast and slow sites documented or proposed to regulate proximal genes in previous studies (Fig. 1e) was similar across a range of interval widths. This suggests that slow sites with shape features similar to fast sites are likely true DOI: 10.1038/ncomms15644 OPEN

Corrigendum: ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.

Nature Communications 6, Article number: 8733 (2015); Published 22 October 2015; Updated 16 December 2015 An incorrect version of the Supplementary Data 1, in which data in columns E, F, G, H and K of the sheet ‘Reb1’ were sorted independently from the other columns, was inadvertently published withthis Article.