Identifying distinct heterochromatin regions using combinatorial epigenetic probes in live cells

Highlights

• Live cell probes were developed and validated for detecting heterochromatin regions enriched in ^meCpG, H3K9me3, or H3K27me3.

• The probes enabled live-cell tracking of single cell chromatin marked with dual epigenetic modifications.

• Co-localization of two epigenetic modifications was also demonstrated via FRET imaging.

Abstract

The 3D spatial organization of the genome controls gene expression and cell functionality. Heterochromatin (HC), which is the densely compacted and largely silenced part of the chromatin, is the driver for the formation and maintenance of nuclear organization in the mammalian nucleus. It is functionally divided into highly compact constitutive heterochromatin (cHC) and transcriptionally poised facultative heterochromatin (fHC). Long regarded as a static structure, the highly dynamic nature of the heterochromatin is being slowly understood and studied. These changes in HC occur on various temporal scales during the cell cycle and differentiation processes. Most methods that capture information about the heterochromatin are static techniques that cannot provide a readout of how the HC organization evolves with time. The delineation of specific areas such as fHC are also rendered difficult due to its diffusive nature and lack of specific features. Another degree of complexity in characterizing changes in heterochromatin occurs due to the heterogeneity in the HC organization of individual cells, necessitating single cell studies. Overall, there is a need for live cell compatible tools that can stably track the heterochromatin as it undergoes re-organization. In this work, we present an approach to track cHC and fHC based on the epigenetic hallmarks associated with them. Unlike conventional immunostaining approaches, we use small recombinant protein probes that allow us to dynamically monitor the HC by binding to modifications specific to the cHC and fHC, such as H3K9me3, DNA methylation and H3K27me3. We demonstrate the use of the probes to follow the changes in HC induced by drug perturbations at the single cell level. We also use the probe sets combinatorically to simultaneously track chromatin regions enriched in two selected epigenetic modifications using a FRET based approach that enabled us tracking distinctive chromatin features in situ.

Introduction

The mammalian nucleus is functionally divided into several regions comprising primarily of the chromatin, the nucleolus, and transcription factories [1]. Gene rich regions tend to be loosely compacted (euchromatin or EC) and are commonly found near the center of the nucleus. Densely compacted, largely gene poor regions [[2], [3]] (heterochromatin or HC) are most abundant near the nuclear periphery [4]. Accumulating evidence suggests that the 3D spatial arrangement of chromatin largely encodes cell functionality [[5], [6]], dictates inter- and intra- chromosomal interactions [7], partially programs transcription profile [8], and thus ultimately determines the phenotype [9]. In healthy cells, euchromatin and heterochromatin regions typically have well-defined boundaries which are essential for distinctive gene activity and maintaining integrity during cellular processes [10].

Heterochromatin formation is the driving force behind the observed 3D architecture of the genome and forms a basis for understanding the spatial organization within the nucleus. The formation of heterochromatin occurs very early in development and is crucial for differentiation [11], as well as the establishment and maintenance of nuclear organization in mammalian cells [[12], [13]]. Repetitive DNA regions and developmentally regulated genes are the major components of HC within the genome [2]. Functionally, heterochromatin may be divided into two sub-classes [2,14]: i. constitutive heterochromatin (cHC) which is highly stable, heavily compacted, and gene poor, and, ii. facultative heterochromatin (fHC) which tends to be less compacted, developmentally regulated, and transcriptionally poised. The functional distinction between cHC and fHC is important because though both are transcriptionally silent, fHC retains the potential to interconvert between cHC and EC [15].

Both the cHC and fHC modulate chromatin to maintain nuclear stability and prevent the access of transcriptional machinery to repetitive DNA elements, curtailing their transcription and recombination. The boundaries and spatial organization of both the cHC and fHC must be maintained. Disruptions in the cHC and fHC can result in genomic instability [[16], [17]], transcription of transposable elements and silenced repeats [18], and loss of genomic organization. For instance, heterochromatin de-condensation is commonly seen in cancer cells, such as in breast and ovarian cancer where the inactive X chromosome is de-compacted leading to the expression of X linked genes [19]. The dramatic loss of heterochromatin at the nuclear periphery, leading to poor staining in malignant cancer cells, has long been used as a maker by pathologists to identify specific cancers [20]. The destabilization of HC and subsequent epigenomic instability is, however, an understudied topic area in cancer biology warranting careful studies. The distinction between cHC and fHC is difficult to accomplish by conventional cytological stains [14]. Delineating these two areas is thus of key importance in processes where the heterochromatin is disrupted.

HC is typically identified in single cells via the simple stains that bind to DNA. These include 4′6-diamidino-2-phenylindole (DAPI) for fixed cells and Hoechst 33342 for live cells, which primarily bind to AT-rich heterochromatin regions, thus differentiating it from the euchromatin regions of lower DNA density. Other approaches to identify heterochromatin are banding and FISH [[21], [22]], which rely on the tightly compacted nature of heterochromatin DNA, or the use of specific DNA sequences, respectively, to identify heterochromatin regions. The distinction between fHC and cHC, however, has been difficult to accomplish without the use of antibodies specific to epigenetic marks found at these regions [23]. For instance, the cHC is primarily characterized by a high density of H3K9me3 and DNA methylation [2] while the fHC is enriched in H3K27me3 [14]. However, antibody-based methods require a fixation step which is not conducive to live cell applications.

In this work, we present an approach to monitor fHC and cHC regions based on epigenetic readouts. The probes directly inform the spatial distribution and abundance of epigenetic modifications and thus offer a convenient tool to reveal the contributions of HC to various biological processes.