The molecular basis of heterochromatin assembly and epigenetic inheritance

Abstract

Heterochromatin plays a fundamental role in gene regulation, genome integrity, and silencing of repetitive DNA elements. Histone modifications are essential for the establishment of heterochromatin domains, which is initiated by the recruitment of histone-modifying enzymes to nucleation sites. This leads to the deposition of histone H3 lysine-9 methylation (H3K9me), which provides the foundation for building high-concentration territories of heterochromatin proteins and the spread of heterochromatin across extended domains. Moreover, heterochromatin can be epigenetically inherited during cell division in a self-templating manner. This involves a "read-write" mechanism where pre-existing modified histones, such as tri-methylated H3K9 (H3K9me3), support chromatin association of the histone methyltransferase to promote further deposition of H3K9me. Recent studies suggest that a critical density of H3K9me3 and its associated factors is necessary for the propagation of heterochromatin domains across multiple generations. In this review, I discuss the key experiments that have highlighted the importance of modified histones for epigenetic inheritance.

Keywords: epigenetic; heterochromatin; histone methylation; inheritance; read-write; silencing.

Figure 1.. HP1 binds to methylated nucleosomes and multimerizes to recruit effector proteins for heterochromatin assembly.

(Left) S. pombe Swi6/HP1 is concentrated at specific foci within the nucleus. Blue DAPI staining shows the nucleus of an S. pombe cell, while the red foci show high concentrations of Swi6/HP1 protein bound to heterochromatic loci. (Center) Methylation of histone H3 lysine 9 (H3K9me) by Clr4/Suv39h provides the foundation for recruiting and concentrating heterochromatin proteins such as Swi6/HP1. Swi6/HP1 molecules stoichiometrically bind to H3K9 methylated nucleosomes via their amino-terminal chromodomain and form dimers (curved dotted lines) through their carboxy-terminal chromoshadow domain. In addition, HP1 proteins are believed to engage in non-stoichiometric multivalent interactions (straight dotted lines) to form high-concentration spatial nuclear territories, which provides a multifunctional scaffold to amplify the chromatin association of various effector proteins. Specifically, the formation of high-concentration HP1 territories helps to attract and retain otherwise diffusible proteins required for a multitude of heterochromatin functions, including factors involved in histone deacetylation (HDACs), histone methylation (HMTs), histone chaperones (FACT), RNA interference (RNAi), RNA processing and peripheral tethering (RIXC), and chromosome architecture (SMC), among many others.

Figure 2.. RNA- and DNA-based mechanisms guide the nucleation of heterochromatin domains.

(A) DNA binding proteins (DBP) bind to specific DNA sequences and through adaptor proteins recruit histone-modifying activities, such as the histone methyltransferase Clr4/Suv39h and HDACs, to assemble heterochromatin. (B) In the RNAi-dependent pathway, the RNA-induced transcriptional silencing (RITS) complex containing Ago1, Tas3 and Chp1, is a key component of a self-reinforcing loop mechanism that couples the generation of siRNAs to heterochromatin assembly. Long non-coding RNAs (lncRNAs) generated by RNAPII are processed by RNAi machinery to generate siRNAs. The siRNAs guide RITS to nascent transcripts where it helps target the Clr4 complex (named ClrC) via the Stc1 adaptor protein to methylate H3K9. H3K9me in turn promotes stable chromatin association of RITS via the Chp1 chromodomain protein. RITS acts together with the Rdp1-containing complex (RDRC) and Dicer (Dcr1) to process nascent transcripts into siRNAs. siRNA biogenesis is facilitated by Ers1 that connects RDRC with Swi6/HP1. The siRNAs further promote H3K9me by Clr4 to efficiently assemble heterochromatin and silence target loci. (C) In the RNAi-independent pathway, nuclear RNA elimination factors and RNAPII termination factors target the Clr4 complex to nucleate heterochromatin. The YTH family RNA-binding protein Mmi1 binds to transcripts containing determinant of selective removal (DSR) elements and recruits the cleavage and polyadenylation (CPF) complex to trigger RNAPII termination at non-canonical termination sites via a mechanism that also requires the 5’→ 3’ exoribonuclease Xrn2 (named Dhp1 in S. pombe). On the other hand, Mmi1 associates with the Enhancer of Rudimentary Homolog Erh1 (ERH in mammals). Mmi1-Erh1 recruits MTREC (PAXT in mammals), which promotes RNA degradation through the 3’ → 5’ exoribonuclease activity of Rrp6/exosome. MTREC and termination factors act together to recruit the Clr4 complex to methylate H3K9 and nucleate heterochromatin. In this mechanism, premature termination at non-canonical sites is coupled to RNA degradation and heterochromatin assembly.

Figure 3.. Nucleation and spreading of heterochromatin.

The formation of the archetypical heterochromatin domain at the silent mat region in S. pombe involves two distinct nucleation mechanisms that independently recruit/retain heterochromatin factors at specific sites. In addition to RNAi machinery that nucleates heterochromatin at the centromere homologous cenH element, the DNA binding proteins (DBPs), including ATF/CREB family proteins, collaborate with Swi6/HP1 and other factors to engage Clr4/Suv39h at a nearby site. Methylation of H3K9 by Clr4/Suv39h not only provides the foundation for forming high concentration HP1 territories but it also serves as an epigenetic template for loading additional Clr4/Suv39h. The ability of Clr4/Suv39h to bind H3K9me3 and catalyze the further deposition of H3K9me allows the concentration gradient of heterochromatin factors to expand and spread across an extended domain surrounded by inverted repeat (IR-R and IR-L) boundary elements (middle). In this read-write mechanism, the chromatin association of Clr4/Suv39h is further strengthened by Swi6/HP1, which also attracts other factors that safeguard the H3K9me3 epigenetic template. Among these, Clr3 HDAC suppresses histone turnover to maintain H3K9me3, and FACT is believed to aid the retention of parental methylated histones during replication. Moreover, the Rix1-containing RNA processing complex RIXC, which localizes to IR boundary elements containing B-boxes (transcription factor TFIIIC binding site), tethers the heterochromatin domain to the nuclear periphery. Tethering restricts the heterochromatin domain to a specific nuclear volume, presumably enabling heterochromatin factors to be efficiently concentrated. However, considering that RIXC binds Swi6/HP1 with high affinity, RIXC bound to boundary elements might help directly concentrate Swi6/HP1 to facilitate heterochromatin assembly.

Figure 4.. Heterochromatin is epigenetically inherited in cis in a self-templating manner.

In a foundational experiment, genetically identical haploid cells differing only in the level of heterochromatin at the silent mat region were crossed to make a diploid. Remarkably, this experiment showed that the differential H3K9 methylation patterns are maintained in the shared nuclear environment of the diploid cell. During replication, heterochromatin provides a template for its own reassembly, so not only is DNA replicated but the chromatin also replicates in a self-templating manner, leading to the propagation of differential chromatin states for multiple generations. Importantly, these distinct chromatin states are epigenetically propagated in mitotically dividing cells and are transmitted through gametogenesis into gametes. See Hall et al. (2002) for details.

Figure 5.. Fundamental principles underlying self-propagation of heterochromatin domains.

Two principles govern the stable propagation of heterochromatin: (1) Epigenetic self-templated inheritance of heterochromatin domains requires a high density of H3K9me3 to support an effective local concentration of Clr4/Suv39h on chromatin. (2) The ability of the Clr4/Suv39h methyltransferase to both bind to and deposit H3K9me, referred to as the read-write mechanism, is critical for heterochromatin propagation. HDACs recruited by HP1 proteins and other factors suppress histone turnover and maintain a high density of H3K9me3. During DNA replication, just as parental DNA strands act as a template, parental H3K9me3 transferred to sister chromatids and HP1 provide an epigenetic template for the recruitment of Clr4, which in turn modifies new histones to propagate heterochromatin through its read-write activity.

Figure 6.. Robust chromatin association of HDAC activity is required to preserve the H3K9me3 epigenetic template.

(A) Heterochromatin differs dramatically from euchromatin in the rate of histone turnover. Stably propagated heterochromatin domains show preferential enrichment of HDACs recruited by HP1 and DNA-binding proteins. As a result, the dynamic balance between HDACs and HATs is shifted in favor of HDACs. Deacetylation by HDACs preserves methylated nucleosomes in part by suppressing turnover of histones, presumably by preventing the engagement of bromodomain-containing ATP-dependent chromatin remodeling complexes, which have a higher affinity for nucleosomes with acetylated H3 tails. On the other hand, euchromatic regions enriched for HATs show high histone acetylation and turnover, particularly at regulatory elements (shown in red) such as gene promoters targeted by remodeling enzymes. (B) Propagation of a heterochromatin domain established by artificial recruitment of Clr4. TetR-Clr4 binds to tet operators (tetO) at an ectopic site via a fusion of the Clr4 catalytic domain to the TetR DNA binding domain. Methylation of H3K9 by TetR-Clr4 recruits HP1 and associated HDACs as well as the endogenous Clr4 complex that spreads via the read-write mechanism. Upon the release of TetR-Clr4, epigenetic inheritance of heterochromatin can only occur if the high density of H3K9me3 is maintained by HDACs. (Top) The low level of HDACs at an ectopic heterochromatin domain located in an otherwise euchromatic environment is insufficient to maintain the high density of H3K9me3 and Clr4 required for heterochromatin propagation. (Bottom) However, restoring robust chromatin association of an HDAC (Clr3 in S. pombe) by fusing it to two chromodomains (HDAC-CDx2), which preserves H3K9me3, is sufficient to prime the system to support self-propagation of heterochromatin through the read-write mechanism. Heterochromatin propagation at the ectopic site can also be recapitulated by loss of the major HATs (not shown), further reinforcing the idea that it is the dynamic balance between opposing activities of HDACs and HATs at a given locus that determines heritability of the silenced chromatin domain.

Figure 7.. Overlapping mechanisms maintain a critical density of methylated histones that serve as the carriers of epigenetic information.

Normally, multiple pathways mediate the recruitment of histone-modifying enzymes, such as Clr4/Suv39h and HDACs, which collaborate to maintain a high local concentration of H3K9me3 to support read-write heterochromatin propagation. If RNA- or DNA-based mechanisms, or both, are impaired, the levels of H3K9me3 and its interacting proteins maintained mainly by the read-write mechanism vary depending on the chromosomal context and other conditions affecting nucleosome stability, thus resulting in a metastable state and defective heterochromatin propagation. Defects in DNA-mediated and RNA-mediated mechanisms can be bypassed by enhancing H3K9me-mediated recruitment via alternative mechanisms. For example, when H3K9me3 is stabilized (indicated by dark blue nucleosomes) by enhanced chromatin association of HDACs (e.g. by fusion of Clr3 HDAC to chromodomains), which acts in part by suppressing histone turnover, H3K9me3 modified nucleosomes alone are sufficient to support self-propagation of the heterochromatin domain for multiple cell divisions.