Andrea Piunti

Epigenetic balance of gene expression by Polycomb and COMPASS families.

A balancing act in modifying chromatin Chromatin modifiers add chemical groups to histones, the proteins that package DNA. These modifications are central to cellular development, and mutations in their molecular machinery are linked to a variety of human diseases. Piunti and Shilatifard review the balance between the prototypic chromatin modifiers Polycomb and COMPASS complexes and their role in gene regulation and normal development. Although originally identified as indispensible regulators of fruit fly development, related roles have been identified in other organisms. Furthermore, mutations in human homologs have been implicated in various cancers. As such, these complexes may serve as effective targets for epigenetic therapies. Science, this issue p. 10.1126/science.aad9780 BACKGROUND Multicellular organisms depend on the precise orchestration of gene expression to direct embryonic development and to maintain tissue homeostasis through their life spans. Exactly how such cell type–specific patterns of gene expression are established, maintained, and passed on to the next generation is one of the most fundamental questions of biology. Eukaryotes package their DNA into nucleosomes to form chromatin fibers. Chromatin plays a central role in regulating accessibility to DNA in many different DNA templated processes, including machineries that transcribe DNA into RNA, i.e., transcription. Transcriptional control through sequence-specific DNA binding factors (transcription factors) is well established; however, proteins that change chromatin structure (chromatin modifiers and remodelers) provide an additional layer of regulation and are considered major epigenetic determinants of cell identity and function. Among the numerous chromatin modifiers, the members of the Polycomb group (PcG) and the Trithorax group (TrxG) of proteins, in particular, have been scrutinized genetically and biochemically for decades. However, new and unexpected functions for these complexes are constantly emerging because of the intense interest in the critical role these proteins play in maintaining a balanced state of gene expression. ADVANCES In the classical view, PcG and TrxG proteins regulate the repressed and activated states of gene expression, respectively. Both PcG and TrxG are organized in multiprotein complexes, which include the Polycomb repressive complex 1 and 2 (PRC1 and PRC2, respectively), and the complex of proteins associated with Set1 (COMPASS) family. Polycomb and COMPASS families are well known for their opposing roles in balancing gene expression, a phenomenon initially characterized using classical Drosophila melanogaster genetic approaches at a time when their biochemical functions were still unknown. Later studies demonstrated that Polycomb and COMPASS complexes have enzymatic activities modifying different sites on a common target, the nucleosome. Nucleosomes can be posttranslationally modified in a variety of ways, many of which strongly correlate with different states of gene expression. Through their ability to regulate gene expression, several components of both the Polycomb and COMPASS complexes are involved in a plethora of crucial biological processes ranging from the regulation of embryonic development to widespread involvement in neoplastic pathogenesis. OUTLOOK Recent genome-wide studies have demonstrated that a large number of the components of the Polycomb and COMPASS families are often mutated in different forms of cancer. Some mutations result in gene deletion or early termination, such as loss-of-function (LOF) mutations, whereas gain-of-function (GOF) mutations increase or change their normal activities. Although it cannot be excluded that some of those are passenger rather than driver mutations, they suggest a relevant function of these proteins in tumorigenesis. Moreover, animal models have provided convincing evidence supporting a role for these complexes in tumor progression. However, even after decades of study, how Polycomb and COMPASS control normal or aberrant gene regulatory networks is not fully understood yet. From the perspective of their catalytic activities, the degree to which catalytic versus noncatalytic functions contribute to their roles in development and cancer has just begun to emerge. Concurrently, the possibility that PcG and TrxG enzymatic activities modify non-nucleosome substrates remains a fascinating, although largely unexplored, hypothesis. Ongoing efforts to decipher how mutations affecting members of these complexes disturb transcriptional balances and promote oncogenesis could provide critically needed new strategies for cancer therapeutics. The balanced state of gene expression. The scale symbolizes the transcriptional status of a gene. Each dish contains a nucleosome that is either lysine 4 trimethylated (green light) or lysine 27 tri-methylated (red light) on histone H3 (one tail is depicted for simplicity). These two histone marks strongly correlate, respectively, with transcriptional activation induced by COMPASS and transcriptional repression induced by PcG. [Figure by Mark Miller. Nucleosomes are adapted from a custom model from 3D Molecular Designs] Epigenetic regulation of gene expression in metazoans is central for establishing cellular diversity, and its deregulation can result in pathological conditions. Although transcription factors are essential for implementing gene expression programs, they do not function in isolation and require the recruitment of various chromatin-modifying and -remodeling machineries. A classic example of developmental chromatin regulation is the balanced activities of the Polycomb group (PcG) proteins within the PRC1 and PRC2 complexes, and the Trithorax group (TrxG) proteins within the COMPASS family, which are highly mutated in a large number of human diseases. In this review, we will discuss the latest findings regarding the properties of the PcG and COMPASS families and the insight they provide into the epigenetic control of transcription under physiological and pathological settings.

Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.

Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability

Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.

Detection of Histone H3 mutations in cerebrospinal fluid-derived tumor DNA from children with diffuse midline glioma

Diffuse midline gliomas (including diffuse intrinsic pontine glioma, DIPG) are highly morbid glial neoplasms of the thalamus or brainstem that typically arise in young children and are not surgically resectable. These tumors are characterized by a high rate of histone H3 mutation, resulting in replacement of lysine 27 with methionine (K27M) in genes encoding H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B). Detection of these gain-of-function mutations has clinical utility, as they are associated with distinct tumor biology and clinical outcomes. Given the paucity of tumor tissue available for molecular analysis and relative morbidity of midline tumor biopsy, CSF-derived tumor DNA from patients with diffuse midline glioma may serve as a viable alternative for clinical detection of histone H3 mutation. We demonstrate the feasibility of two strategies to detect H3 mutations in CSF-derived tumor DNA from children with brain tumors (n = 11) via either targeted Sanger sequencing of H3F3A and HIST1H3B, or H3F3A c.83 A > T detection via nested PCR with mutation-specific primers. Of the six CSF specimens from children with diffuse midline glioma in our cohort, tumor DNA sufficient in quantity and quality for analysis was isolated from five (83%), with H3.3K27M detected in four (66.7%). In addition, H3.3G34V was identified in tumor DNA from a patient with supratentorial glioblastoma. Test sensitivity (87.5%) and specificity (100%) was validated via immunohistochemical staining and Sanger sequencing in available matched tumor tissue specimens (n = 8). Our results indicate that histone H3 gene mutation is detectable in CSF-derived tumor DNA from children with brain tumors, including diffuse midline glioma, and suggest the feasibility of “liquid biopsy” in lieu of, or to complement, tissue diagnosis, which may prove valuable for stratification to targeted therapies and monitoring treatment response.

A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation

Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4-RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR-Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.

Abstract A46: Heterotypic nucleosomes and PRC2 role in diffuse intrinsic pontine gliomas

Diffuse intrinsic pontine gliomas (DIPG) harbor a characteristic lysine-to-methionine mutation on histone H3 (H3K27M) that potently inhibits Polycomb Repressive Complex 2 (PRC2) enzymatic activity; however, the precise roles of H3K27M and PRC2 in tumorigenesis remain elusive. Here we perform epigenomic profiling of patient-derived H3K27M mutant DIPG cell lines and demonstrate that H3K27M resides in nucleosomes with H3K27acetylation (H3K27ac) and colocalizes with bromodomain proteins at actively transcribed genes. In contrast, PRC2 is excluded from H3K27M occupied regions but, strikingly, the PRC2 complex is required for maintaining oncogenic potential. Finally, we demonstrate that pharmacologic bromodomain inhibition suppresses tumor growth in vivo. Altogether our results indicate that H3K27M drives accumulation of H3K27ac and point to bromodomain proteins inhibition as a powerful clinical strategy against DIPG. Citation Format: Andrea Piunti. Heterotypic nucleosomes and PRC2 role in diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A46.