Bipartite binding interface recruiting HP1 to chromosomal passenger complex at inner centromeres.

Maintenance of ploidy depends on the mitotic kinase Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC) whose proficient activity is supported by HP1 enriched at inner centromeres. HP1 is known to associate with INCENP of the CPC in a manner that depends on the PVI motif conserved across HP1 interactors. Here, we found that the interaction of INCENP with HP1 requires not only the PVI motif but also its C-terminally juxtaposed domain. Remarkably, these domains conditionally fold the ß-strand (PVI motif) and the α-helix from a disordered sequence upon HP1 binding and render INCENP with high affinity to HP1. This bipartite binding domain termed SSH domain (Structure composed of Strand and Helix) is necessary and sufficient to attain a predominant interaction of HP1 with INCENP. These results identify a unique HP1-binding module in INCENP that ensures enrichment of HP1 at inner centromeres, Aurora B activity, and thereby mitotic fidelity.

Interplay between charge distribution and DNA in shaping HP1 paralog phase separation and localization.

The heterochromatin protein 1 (HP1) family is a crucial component of heterochromatin with diverse functions in gene regulation, cell cycle control, and cell differentiation. In humans, there are three paralogs, HP1α, HP1ß, and HP1γ, which exhibit remarkable similarities in their domain architecture and sequence properties. Nevertheless, these paralogs display distinct behaviors in liquid-liquid phase separation (LLPS), a process linked to heterochromatin formation. Here, we employ a coarse-grained simulation framework to uncover the sequence features responsible for the observed differences in LLPS. We highlight the significance of the net charge and charge patterning along the sequence in governing paralog LLPS propensities. We also show that both highly conserved folded and less-conserved disordered domains contribute to the observed differences. Furthermore, we explore the potential co-localization of different HP1 paralogs in multicomponent assemblies and the impact of DNA on this process. Importantly, our study reveals that DNA can significantly reshape the stability of a minimal condensate formed by HP1 paralogs due to competitive interactions of HP1α with HP1ß and HP1γ versus DNA. In conclusion, our work highlights the physicochemical nature of interactions that govern the distinct phase-separation behaviors of HP1 paralogs and provides a molecular framework for understanding their role in chromatin organization.

Functions of HP1 in preventing chromosomal instability.

Chromosomal instability (CIN), caused by errors in the segregation of chromosomes during mitosis, is a hallmark of many types of cancer. The fidelity of chromosome segregation is governed by a sophisticated cellular signaling network, one crucial orchestrator of which is Heterochromatin protein 1 (HP1). HP1 dynamically localizes to distinct sites at various stages of mitosis, where it regulates key mitotic events ranging from chromosome-microtubule attachment to sister chromatid cohesion to cytokinesis. Our evolving comprehension of HP1's multifaceted role has positioned it as a central protein in the orchestration of mitotic processes.

Mode of SUV420H2 heterochromatin localization through multiple HP1 binding motifs in the heterochromatic targeting module.

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

Single Genomic Loci Labeling and Manipulation Using SIMBA System.

The SIMBA (Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies) system is an innovative CRISPR-based imaging technique that leverages dCas9-SunTag and FRB-mCherry-HP1α, with scFv-FKBP acting as a bridge. This powerful system enables simultaneous visualization and manipulation of genomic loci. The dCas9-SunTag fusion protein allows for precise targeting of specific genomic sites, and the FRB-mCherry-HP1α fusion protein facilitates the condensation of chromatin at the targeted loci. The scFv-FKBP bridge protein links dCas9-SunTag and FRB-mCherry-HP1α, ensuring efficient and specific recruitment of HP1α to the desired genomic loci. This integrated approach allows us to visualize and manipulate genomic regions of interest, opening up new avenues for studying genome organization, gene expression regulation, and chromatin dynamics in living cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cloning of genetic constructs Basic Protocol 2: Transient transfection in mammalian cells and live-cell imaging Basic Protocol 3: Generation of SIMBA-expressing stable cell lines Basic Protocol 4: Manipulation of genomic loci using SIMBA.

Simulation of STAT and HP1 interaction by molecular docking.

Heterochromatin Protein 1 (HP1) is a major component of heterochromatin. Multiple proteins have been shown to interact with HP1 with the HP1-binding motif PxVxL/I, thereby affecting heterochromatin stability. The HP1-interacting proteins include the signal transducer and activator of transcription (STAT) protein, which can be regulated by phosphorylation on a tyrosine around amino acid 700 in the carboxyl terminus. Previous research has shown that unphosphorylated STAT (uSTAT) binds to HP1 via a PxVxI HP1-binding motif and maintains the stability of heterochromatin, while phosphorylated STAT (pSTAT) dissociates from HP1, resulting in heterochromatin disruption. To understand the theoretical basis of the biochemical observations, we employed computational modeling to investigate STAT-HP1 binding configurations and the effect of STAT phosphorylation on their interaction. Using STAT3 and HP1α protein structures for molecular docking and thermodynamic calculations, our computations predict that uSTAT homodimers have a higher affinity for HP1 and a lower affinity for DNA than pSTAT homodimers, and that phosphorylation induces a conformational change in STAT, shifting its binding preference from HP1 to DNA. The results of our modeling studies support the idea that phosphorylation drives STAT from HP1-binding to DNA-binding, suggesting a potential role for uSTAT in both maintaining and initiating heterochromatin formation.

Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

Long, noncoding RNAs (lncRNAs) are indispensable for normal cell physiology and, consequently, are tightly regulated in human cells. Yet, unlike mRNA, substantially less is known about the mechanisms for lncRNA degradation. It is important to delineate the regulatory control of lncRNA degradation, particularly for lncRNA telomeric repeat-containing RNA (TERRA), as the TERRA-telomere R-loops dictate cell cycle progression and genomic stability. We now report that the exosome complex component Exosc9 degrades lncRNA TERRA in human mammary epithelial cells. Heterochromatin protein 1 alpha (HP1α) recruits Exosc9 to the telomeres; specifically, the SUMO-modified form of HP1α supports interaction with Exosc9 and, as previously reported, lncRNA TERRA. The telomeric enrichment of Exosc9 is cell cycle-dependent and consistent with the loss of telomeric TERRA in the S/G2 phase. Elevated Exosc9 is frequently observed and drives the growth of endocrine therapy-resistant (ET-R) HR+ breast cancer (BCa) cells. Specifically, the knockdown of Exosc9 inversely impacts telomeric R-loops and the integrity of the chromosome ends of ET-R cells. Consistently, Exosc9 levels dictate DNA damage and the sensitivity of ET-R BCa cells to PARP inhibitors. In this regard, Exosc9 may serve as a promising biomarker for predicting the response to PARP inhibitors as a targeted monotherapy for ET-R HR+ BCa.

Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii.

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A. luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation.

The spatial segregation of pericentromeric heterochromatin (PCH) into distinct, membrane-less nuclear compartments involves the binding of Heterochromatin Protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid phase separation properties in vitro, its mechanistic impact on the structure and dynamics of PCH condensate formation in vivo remains largely unresolved. Here, using a minimal theoretical framework, we systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. We reveal that the specific affinity of HP1 for H3K9me2/3 loci facilitates coacervation in nucleo and promotes the formation of stable PCH condensates at HP1 levels far below the concentration required to observe phase separation in purified protein assays in vitro. These heterotypic HP1-chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent phase separation. The dynamical cross talk between HP1 and the viscoelastic chromatin scaffold also leads to anomalously slow equilibration kinetics, which strongly depend on the genomic distribution of H3K9me2/3 domains and result in the coexistence of multiple long-lived, microphase-separated PCH compartments. The morphology of these complex coacervates is further found to be governed by the dynamic establishment of the underlying H3K9me2/3 landscape, which may drive their increasingly abnormal, aspherical shapes during cell development. These findings compare favorably to 4D microscopy measurements of HP1 condensate formation in live Drosophila embryos and suggest a general quantitative model of PCH formation based on the interplay between HP1-based phase separation and chromatin polymer mechanics.

Fluorescent protein lifetimes report densities and phases of nuclear condensates during embryonic stem-cell differentiation.

Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1α (HP1α) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1α condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.

Cooperative DNA-binding activities of Chp2 are critical for its function in heterochromatin assembly.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein that plays a critical role in heterochromatin assembly. HP1 proteins share a basic structure consisting of an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD) linked by a disordered hinge region. The CD recognizes histone H3 lysine 9 methylation, a hallmark of heterochromatin, while the CSD forms a dimer to recruit other chromosomal proteins. HP1 proteins have been shown to bind DNA or RNA primarily through the hinge region. However, how DNA or RNA binding contributes to their function remains elusive. Here, we focus on Chp2, one of the two HP1 proteins in fission yeast, and investigate how Chp2's DNA-binding ability contributes to its function. Similar to other HP1 proteins, the Chp2 hinge exhibits clear DNA-binding activity. Interestingly, the Chp2 CSD also shows robust DNA-binding activity. Mutational analysis revealed that basic residues in the Chp2 hinge and at the N-terminus of the CSD are essential for DNA binding, and the combined amino acid substitutions of these residues alter Chp2 stability, impair Chp2 heterochromatin localization and lead to a silencing defect. These results demonstrate that the cooperative DNA-binding activities of Chp2 play an important role in heterochromatin assembly in fission yeast.

How to evict HP1 from H3: Using a complex salt bridge.

In an effort to unravel the unknown "binary switch" mechanisms underlying the "histone code" hypothesis of gene silencing and activation, we study the dynamics of Heterochromatin Protein 1 (HP1). We find in the literature that when HP1 is bound to tri-methylated Lysine9 (K9me3) of histone-H3 through an aromatic cage consisting of two tyrosines and one tryptophan, it is evicted upon phosphorylation of Serine10 (S10phos) during mitosis. In this work, the kick-off intermolecular interaction of the eviction process is proposed and described in detail on the basis of quantum mechanical calculations: specifically, an electrostatic interaction competes with the cation-π interaction and draws away K9me3 from the aromatic cage. An arginine, abundant in the histonic environment, can form an intermolecular "complex salt bridge" with S10phos and dislodge HP1. The study attempts to reveal the role of phosphorylation of Ser10 on the H3 tail in atomic detail.

Dominant-negative variants in CBX1 cause a neurodevelopmental disorder.

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.

Tissue-specific chromatin-binding patterns of Caenorhabditis elegans heterochromatin proteins HPL-1 and HPL-2 reveal differential roles in the regulation of gene expression.

Heterochromatin is characterized by an enrichment of repetitive elements and low gene density and is often maintained in a repressed state across cell division and differentiation. The silencing is mainly regulated by repressive histone marks such as H3K9 and H3K27 methylated forms and the heterochromatin protein 1 (HP1) family. Here, we analyzed in a tissue-specific manner the binding profile of the two HP1 homologs in Caenorhabditis elegans, HPL-1 and HPL-2, at the L4 developmental stage. We identified the genome-wide binding profile of intestinal and hypodermal HPL-2 and intestinal HPL-1 and compared them with heterochromatin marks and other features. HPL-2 associated preferentially to the distal arms of autosomes and correlated positively with the methylated forms of H3K9 and H3K27. HPL-1 was also enriched in regions containing H3K9me3 and H3K27me3 but exhibited a more even distribution between autosome arms and centers. HPL-2 showed a differential tissue-specific enrichment for repetitive elements conversely with HPL-1, which exhibited a poor association. Finally, we found a significant intersection of genomic regions bound by the BLMP-1/PRDM1 transcription factor and intestinal HPL-1, suggesting a corepressive role during cell differentiation. Our study uncovers both shared and singular properties of conserved HP1 proteins, providing information about genomic binding preferences in relation to their role as heterochromatic markers.

Canonical and non-canonical functions of STAT in germline stem cell maintenance.

BACKGROUND: Maintenance of the Drosophila male germline stem cells (GSCs) requires activation of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway by niche signals. The precise role of JAK/STAT signaling in GSC maintenance, however, remains incompletely understood. RESULTS: Here, we show that, GSC maintenance requires both canonical and non-canonical JAK/STAT signaling, in which unphosphorylated STAT (uSTAT) maintains heterochromatin stability by binding to heterochromatin protein 1 (HP1). We found that GSC-specific overexpressing STAT, or even the transcriptionally inactive mutant STAT, increases GSC number and partially rescues the GSC-loss mutant phenotype due to reduced JAK activity. Furthermore, we found that both HP1 and STAT are transcriptional targets of the canonical JAK/STAT pathway in GSCs, and that GSCs exhibit higher heterochromatin content. CONCLUSIONS: These results suggest that persistent JAK/STAT activation by niche signals leads to the accumulation of HP1 and uSTAT in GSCs, which promote heterochromatin formation important for maintaining GSC identity. Thus, the maintenance of Drosophila GSCs requires both canonical and non-canonical STAT functions within GSCs for heterochromatin regulation.

The HP1α protein is mandatory to repress the circadian clock and its output genes during the 12 h period of transcriptional repression.

The transcriptional repressions driven by the circadian core clock repressors RevErbα, E4BP4, and CRY1/PER1 involve feedback loops which are mandatory for generating the circadian rhythms. These repressors are known to bind to cognate DNA binding sites, but how their circadian bindings trigger the cascade of events leading to these repressions remain to be elucidated. Through molecular and genetic analyses, we now demonstrate that the chromatin protein HP1α plays a key role in these transcriptional repressions of both the circadian clock (CC) genes and their cognate output genes (CCGs). We show that these CC repressors recruit the HP1α protein downstream from a repressive cascade, and that this recruitment is mandatory for the maintenance of both the CC integrity and the expression of the circadian genes. We further show that the presence of HP1α is critical for both the repressor-induced chromatin compaction and the generation of "transcriptionally repressed biomolecular hydrophobic condensates" and demonstrates that HP1α is mandatory within the CC output genes for both the recruitment of DNA methylating enzymes on the intronic deoxyCpG islands and their subsequent methylation.

Characterization of Hepatoma-Derived Growth Factor-Related Protein 2 Interactions with Heterochromatin.

The expression of genetic information is tightly controlled by chromatin regulatory proteins, including those in the heterochromatin gene repression family. Many of these regulatory proteins work together on the chromatin substrate to precisely regulate gene expression during mammalian development, giving rise to many different tissues in higher organisms from a fixed genomic template. Here we identify and characterize the interactions of two related heterochromatin regulatory proteins, heterochromatin protein 1 alpha (HP1α) and M-phase phosphoprotein 8 (MPP8), with hepatoma-derived growth factor-related protein 2 (HRP2). We find in biochemical experiments that HRP2 copurifies and co-sediments with heterochromatin-associated proteins, including HP1α and MPP8. Using the Chromatin in vivo Assay in multiple cell types, we demonstrate that HP1α-mediated gene repression dynamics are altered by the presence of HRP2. Furthermore, the knockout of HRP2 in MDA-MB-231 cells results in significant changes to chromatin structure and stability, which alter gene expression patterns. Here, we detail a mechanism by which HRP2 contributes to epigenetic transcriptional regulation through engagement with heterochromatin-associated proteins to stabilize the chromatin landscape and influence gene expression.

HP1 proteins regulate nucleolar structure and function by secluding pericentromeric constitutive heterochromatin.

Nucleoli are nuclear compartments regulating ribosome biogenesis and cell growth. In embryonic stem cells (ESCs), nucleoli containing transcriptionally active ribosomal genes are spatially separated from pericentromeric satellite repeat sequences packaged in largely repressed constitutive heterochromatin (PCH). To date, mechanisms underlying such nuclear partitioning and the physiological relevance thereof are unknown. Here we show that repressive chromatin at PCH ensures structural integrity and function of nucleoli during cell cycle progression. Loss of heterochromatin proteins HP1α and HP1ß causes deformation of PCH, with reduced H3K9 trimethylation (H3K9me3) and HP1γ levels, absence of H4K20me3 and upregulated major satellites expression. Spatially, derepressed PCH aberrantly associates with nucleoli accumulating severe morphological defects during S/G2 cell cycle progression. Hp1α/ß deficiency reduces cell proliferation, ribosomal RNA biosynthesis and mobility of Nucleophosmin, a major nucleolar component. Nucleolar integrity and function require HP1α/ß proteins to be recruited to H3K9me3-marked PCH and their ability to dimerize. Correspondingly, ESCs deficient for both Suv39h1/2 H3K9 HMTs display similar nucleolar defects. In contrast, Suv4-20h1/2 mutant ESCs lacking H4K20me3 at PCH do not. Suv39h1/2 and Hp1α/ß deficiency-induced nucleolar defects are reminiscent of those defining human ribosomopathy disorders. Our results reveal a novel role for SUV39H/HP1-marked repressive constitutive heterochromatin in regulating integrity, function and physiology of nucleoli.

Diverse silent chromatin states modulate genome compartmentalization and loop extrusion barriers.

The relationships between chromosomal compartmentalization, chromatin state and function are poorly understood. Here by profiling long-range contact frequencies in HCT116 colon cancer cells, we distinguish three silent chromatin states, comprising two types of heterochromatin and a state enriched for H3K9me2 and H2A.Z that exhibits neutral three-dimensional interaction preferences and which, to our knowledge, has not previously been characterized. We find that heterochromatin marked by H3K9me3, HP1α and HP1ß correlates with strong compartmentalization. We demonstrate that disruption of DNA methyltransferase activity greatly remodels genome compartmentalization whereby domains lose H3K9me3-HP1α/ß binding and acquire the neutrally interacting state while retaining late replication timing. Furthermore, we show that H3K9me3-HP1α/ß heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the silent portion of the genome and establishes connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.

Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells.

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.