Epigenetic effectors read marks written on chromatin to regulate function and fidelity of the genome. Here, we show that this coordinated read-write activity of the epigenetic machinery extends to the cytoskeleton, with PBRM1 in the PBAF chromatin remodeling complex reading microtubule methyl marks written by the SETD2 histone methyltransferase. PBRM1 binds SETD2 methyl marks via BAH domains, recruiting PBAF components to the mitotic spindle. This read-write activity was required for normal mitosis: Loss of SETD2 methylation or pathogenic BAH domain mutations disrupt PBRM1 microtubule binding and PBAF recruitment and cause genomic instability. These data reveal PBRM1 functions beyond chromatin remodeling with domains that allow it to integrate chromatin and cytoskeletal activity via its acetyl-binding BD and methyl-binding BAH domains, respectively. Conserved coordinated activity of the epigenetic machinery on the cytoskeleton opens a previously unknown window into how chromatin remodeler defects can drive disease via both epigenetic and cytoskeletal dysfunction.
Karki M, Jangid RK, Anish R, Seervai RNH, Bertocchio JP, Hotta T, Msaouel P, Jung SY, Grimm SL, Coarfa C, Weissman BE, Ohi R, Verhey KJ, Hodges HC, Burggren W, Dere R, Park IY, Prasad BVV, Rathmell WK, Walker CL, Tripathi DN. A cytoskeletal function for PBRM1 reading methylated microtubules. Sci Adv. 2021 Apr 2;7(14):eabf2866. PMID: 33811077.
Over 60% of supratentorial (ST) ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE driven ZRfus tumors by CUT&RUN, ChIP, ATAC, and RNA sequencing and compared to human ZRfus driven ependymoma. In addition to direct canonical NF-kB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with Plagl family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional co-activators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by Plagl transcription factor proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks.
Arabzade A, Zhao Y, Varadharajan S, Chen HC, Jessa S, Rivas B, Stuckert AJ, Solis M, Kardian A, Tlais D, Golbourn BJ, Stanton AJ, Chan YS, Olson C, Karlin KL, Kong K, Kupp R, Hu B, Injac SG, Ngo M, Wang PR, De Leon LA, Sahm F, Kawauchi D, Pfister SM, Lin CY, Hodges HC, Singh I, Westbrook TF, Chintagumpala MM, Blaney SM, Parsons DW, Pajtler KW, Agnihotri S, Gilbertson RJ, Yi J, Jabado N, Kleinman CL, Bertrand KC, Deneen B, Mack SC. ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma. Cancer Discov. 2021 Mar 19:candisc.1066.2020. PMID: 33741710.
The chromatin remodeler CHD8 is among the most frequently mutated genes in autism spectrum disorder (ASD). CHD8 has a dosage-sensitive role in ASD, but when and how it becomes critical to human social function is unclear. Here, we conducted genomic analyses of heterozygous and homozygous Chd8 mouse embryonic stem cells and differentiated neural progenitors. We identify dosage-sensitive CHD8 transcriptional targets, sites of regulated accessibility, and an unexpected cooperation with SOX transcription factors. Collectively, our findings reveal that CHD8 negatively regulates expression of neuronal genes to maintain pluripotency and also during differentiation. Thus, CHD8 is essential for both the maintenance of pluripotency and neural differentiation, providing mechanistic insight into its function with potential implications for ASD.
Sood S, Weber CM, Hodges HC, Krokhotin A, Shalizi A, Crabtree GR. CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation. Proc Natl Acad Sci U S A. 2020 Sep 8;117(36):22331-22340. PMID: 32839322; PMCID: PMC7486765
Many malignancies display heterogeneous features that support cancer progression. Emerging high-resolution methods provide a view of heterogeneity that recognizes the influence of diverse cell types and cell states of the tumor microenvironment. Here we outline a hierarchical organization of tumor heterogeneity from a genomic perspective, summarize the origins of spatially patterned metabolic features, and review recent developments in single-cell and spatially resolved techniques for genome-wide study of multicellular tissues. We also discuss how integrating these approaches can yield new insights into human cancer and emerging immune therapies. Applying these technologies for the analysis of primary tumors, patient-derived xenografts, and in vitro systems holds great promise for understanding the hierarchical structure and environmental influences that underlie tumor ecosystems. [PDF]
EA Smith & HC Hodges, "The spatial and genomic hierarchy of tumor ecosystems revealed by single-cell technologies" Trends in Cancer 5(7): 411-425 (2019).
Cyclic nucleotide binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain-specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, while the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise fashion, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP binding signals during PKA activation.
Y Hao, J England, L Bellucci, E Paci, HC Hodges, S Taylor, RA Maillard. "Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains" Nature Communications 10(1): 3984, (2019).
Renal medullary carcinoma (RMC) is defined in part by loss of SMARCB1, a subunit of BAF (SWI/SNF) and PBAF complexes. RMC is one of the most aggressive renal cell carcinomas. It predominantly afflicts young adults and adolescents with sickle cell trait and other sickle hemoglobinopathies, and is refractory to targeted and anti-angiogenic therapies used in patients with clear-cell renal cell carcinoma. Platinum-based cytotoxic chemotherapy is the mainstay for RMC treatment. Based on recent advances in the diagnosis, management, and clinical trial development for RMC, a panel of experts met in October 2017 and developed updated consensus recommendations to inform clinicians, researchers, and patients. Because RMC often aggressively recurs while patients are still recovering from nephrectomy, upfront chemotherapy should be considered for the majority of patients, including those with localized disease. After safety and dosing information have been established in adults, phase II and III trials enrolling patients with RMC should allow patients aged 12 years and older to be accrued. Patients with the very rare unclassified renal cell carcinoma with medullary phenotype variant should be included in RMC trials. Medical providers should be aware that RMC can afflict subjects of all races, and not only those of the African descent, and that the presence of sickle cell trait, or of other sickle hemoglobinopathies, can impact drug responses and toxicity.
P Msaouel, AL Hong, EA Mullen, MB Atkins, CL Walker, CH Lee, MA Carden, G Genovese, WM Linehan, P Rao, MJ Merino, H Grodman, JS Dome, CV Fernandez, JI Geller, AB Apolo, NC Daw, HC Hodges, M Moxey-Mims, D Wei, DP Bottaro, M Staehler, JA Karam, WK Rathmell, NM Tannir (2018) "Updated recommendations on the diagnosis, management, and clinical trial eligibility criteria for patients with renal medullary carcinoma" Clinical Genitourinary Cancer 17(1):1-6 (2019).
Chromatin regulation is a critical aspect of nuclear function. Mechanisms crucial for normal nuclear function and epigenetic control include compartmentalization of biochemical reactions by liquid-phase separated condensates and signal-dependent regulation of protein stability. Synthetic control of these phenomena by small molecules provides deep insight into essential activities such as histone modification, BAF (SWI/SNF) and PBAF remodeling, Polycomb repression, enhancer looping by cohesin and CTCF, as well as many other processes that contribute to transcription. As a result, a complete understanding of the spatiotemporal mechanisms that underlie chromatin regulation increasingly requires the use of fast-acting drugs and chemical probes. Here, we provide a comprehensive review of next-generation chemical biology tools to interrogate the chromatin regulatory landscape, including selective PROTAC E3 ubiquitin ligase degraders, degrons, fluorescent ligands, dimerizers, inhibitors, and other drugs. These small molecules provide important insights into the mechanisms that govern gene regulation, DNA repair, development, and diseases like cancer.
Cermakova K, Hodges HC (2018) "Next-Generation Drugs and Probes for Chromatin Biology: From Targeted Protein Degradation to Phase Separation" Molecules 23(8): 1958.
Protein kinases are dynamic molecular switches that sample multiple conformational states. The regulatory subunit of protein kinase A (PKA) harbors two cAMP-binding domains [cyclic nucleotide-binding (CNB) domains] that oscillate between inactive and active conformations dependent on cAMP binding. The cooperative binding of cAMP to the CNB domains activates an allosteric interaction network that enables PKA to progress from the inactive to active conformation, unleashing the activity of the catalytic subunit. Despite its importance in the regulation of many biological processes, the molecular mechanism responsible for the observed cooperativity during the activation of PKA remains unclear. Here, we use optical tweezers to probe the folding cooperativity and energetics of domain communication between the cAMP-binding domains in the apo state and bound to the catalytic subunit. Our study provides direct evidence of a switch in the folding-energy landscape of the two CNB domains from energetically independent in the apo state to highly cooperative and energetically coupled in the presence of the catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decrease the folding cooperativity between them. Taken together, our results provide a thermodynamic foundation for the conformational plasticity that enables protein kinases to adapt and respond to signaling molecules.
England JP, Hao Y, Bai L, Glick V, Hodges HC, Taylor SS and Maillard RA (2018) "Switching of the folding-energy landscape governs the allosteric activation of protein kinase A" Proc Natl Acad Sci USA 115(32): 7478-7485.
Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.
Sharma S*, Cermakova K*, De Rijck J, Demeulemeester J, Fabry M, El Ashkar S, Van Belle S, Lepsik M, Tesina P, Duchoslav V, Novak P, Hubalek M, Srb P, Christ F, Rezacova P, Hodges HC, Debyser Z, Veverka V (2018) "Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation" Proc Natl Acad Sci USA 115(30): 7053-7062.
Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induces pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in disorders with SMARCA4 mutations in a wide variety of tissues.
Hodges HC*, Stanton BZ*, Cermakova K, Chang CY, Miller EL, Kirkland JG, Ku WL, Veverka V, Zhao K, Crabtree GR (2018) "Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers," Nature Structural and Molecular Biology 25(1): 61-72.
The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures.
Miller EL, Hargreaves DC, Kadoch C, Chang CY, Calarco JP, Hodges C, Buenrostro JD, Cui K, Greenleaf WJ, Zhao K, Crabtree GR (2017) "TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin," Nature Structural and Molecular Biology 24(4): 344-352.
Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF-PRC opposition underlying development and disease.
Stanton BZ*, Hodges C*, Calarco JP, Braun SM, Ku WL, Kadoch C, Zhao K, Crabtree GR (2017) "Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin," Nature Genetics 49(2): 282-288.
During the last decade, a host of epigenetic mechanisms were found to contribute to cancer and other human diseases. Several genomic studies have revealed that ~20% of malignancies have alterations of the subunits of polymorphic BAF and PBAF complexes, making them among the most frequently mutated complexes in cancer. Recurrent mutations arise in genes encoding several BAF/PBAF subunits, including ARID1A, ARID2, PBRM1, SMARCA4, and SMARCB1. These subunits share some degree of conservation with subunits from related ATP-dependent chromatin remodeling complexes in model organisms, where a large body of work provides insight into their roles in cancer. Here we review the roles of BAF- and PBAF-like complexes in these organisms, and relate these findings to recent discoveries in cancer epigenomics. We review several roles of BAF and PBAF complexes in cancer, including transcriptional regulation, DNA repair, and regulation of chromatin architecture and topology. More recent results highlight the need for new techniques to study these complexes. [PDF]
Hodges C, Kirkland JK, and Crabtree GR (2016) "The many roles of BAF (mSWI/SNF) and PBAF complexes in cancer," Cold Spring Harbor Perspectives in Medicine 6(8): pii: a026930.
Subunits of mammalian SWI/SNF (mSWI/SNF or BAF) complexes have recently been implicated as tumor suppressors in human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of endogenous mSWI/SNF complexes, which identified several new dedicated, stable subunits not found in yeast SWI/SNF complexes, including BCL7A, BCL7B and BCL7C, BCL11A and BCL11B, BRD9 and SS18. Incorporating these new members, we determined mSWI/SNF subunit mutation frequency in exome and whole-genome sequencing studies of primary human tumors. Notably, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, mutations affecting more than one subunit, defined here as compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer, exhibiting a broad mutation pattern, similar to that of TP53. Thus, proper functioning of polymorphic BAF complexes may constitute a major mechanism of tumor suppression.
Kadoch C*, Hargreaves DC*, Hodges C, Elias L, Ho L, Ranish J, Crabtree GR (2013). "Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy," Nature Genetics 45(6): 592-601.
A central goal of chromatin biology is to reveal how posttranslational histone marks modulate gene expression; however, relatively little is known about the spatial or temporal dynamics of these marks. We previously showed that a dynamic model of histone mark nucleation, propagation, and turnover fits the mean enrichment profiles from 99% of noncentromeric histone H3 lysine 9 trimethylation (H3K9me3) domains in mouse embryonic stem cells without the need for boundary or insulator elements. Here we report the full details of this "inherently bounded" model of histone modification dynamics and describe several dynamic features of the model using H3K9me3 as a paradigm. By analyzing the kinetic and structural constraints that drive formation of inherently bounded domains, we find that such domains are optimized when the rates of marking and turnover are comparable. Additionally, we find that to establish such domains, propagation of the histone marks must occur primarily through local contacts.
Hodges C and Crabtree GR (2012) "Dynamics of inherently bounded histone modification domains" PNAS 109(33): 13296-13301.
Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ∼0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome.
Hathaway NA*, Bell O*, Hodges C, Miller EL, Neel DS, and Crabtree GR. (2012) "Dynamics and memory of heterochromatin in living cells," Cell 149(7): 1447-14460.
Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of yeast RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process through the distribution of transcribed nucleosome positions. Notably, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer and histone-histone dissociation quantitatively explains our observations and unifies them with results obtained from other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression.
Bintu L*, Kopaczynska M*, Hodges C, Lubkowska L, Kashlev M, Bustamante C (2011) "The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes," Nature Structural and Molecular Biology 18(12): 1394-1399.
AAA+ unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.
Maillard RA, Chistol G, Sen M, Righini M, Tan J, Kaiser CM, Hodges C, Martin A, Bustamante C (2011) "ClpX(P) generates mechanical force to unfold and translocate its protein substrates," Cell 145(3): 459-469.
RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pause-free velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtained direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. The interplay between polymerase dynamics and nucleosome fluctuations provides a physical basis for the regulation of eukaryotic transcription.
Hodges C*, Bintu L*, Lubkowska L, Kashlev M, and Bustamante C (2009) "Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II," Science 325(5940): 626-628.
We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation--and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases--one codon-and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.