greenPipes: an integrated data analysis pipeline for greenCUT&RUN and CUT&RUN genome-localization datasets.

MOTIVATION: To study gene regulation through transcription factors and chromatin modifiers, a variety of genome-wide techniques are used. Recently, CUT&RUN-based technologies have become popular, but a pipeline for the comprehensive analysis of CUT&RUN datasets is currently lacking. Here, we present the "greenPipes" package, which includes fine-tuned parameters specifically for bioinformatic analyses of greenCUT&RUN and CUT&RUN datasets. greenPipes provides additional functionalities for data analysis and data integration with other -omics technologies, which are either not available in other pipelines developed for CUT&RUN datasets or scattered in the literature as individual packages. AVAILABILITY: Source code and a manual of the greenPipes are freely available on GitHub website (https://github.com/snizam001/greenPipes). The test datasets, comprehensive annotation files and other datasets are available at https://osf.io/ruhj9/. SUPPLEMENTARY INFORMATION: The handbook of greenPipes is available online at Bioinformatics as Supplementary text.

Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting transcriptional memory.

Metastasis arises from disseminated tumour cells (DTCs) that are characterized by intrinsic phenotypic plasticity and the capability of seeding to secondary organs. DTCs can remain latent for years before giving rise to symptomatic overt metastasis. In this context, DTCs fluctuate between a quiescent and proliferative state in response to systemic and microenvironmental signals including immune-mediated surveillance. Despite its relevance, how intrinsic mechanisms sustain DTCs plasticity has not been addressed. By interrogating the epigenetic state of metastatic cells, we find that tumour progression is coupled with the activation of oncogenic enhancers that are organized in variable interconnected chromatin domains. This spatial chromatin context leads to the activation of a robust transcriptional response upon repeated exposure to retinoic acid (RA). We show that this adaptive mechanism sustains the quiescence of DTCs through the activation of the master regulator SOX9. Finally, we determine that RA-stimulated transcriptional memory increases the fitness of metastatic cells by supporting the escape of quiescent DTCs from NK-mediated immune surveillance. Overall, these findings highlight the contribution of oncogenic enhancers in establishing transcriptional memories as an adaptive mechanism to reinforce cancer dormancy and immune escape, thus amenable for therapeutic intervention.

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival.

BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.

ATAC and SAGA co-activator complexes utilize co-translational assembly, but their cellular localization properties and functions are distinct.

To understand the function of multisubunit complexes, it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here, we demonstrate that the core modules of ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription co-activator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, a SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histone proteins. In contrast, ATAC complex subunits cannot be detected in the cytoplasm of mammalian cells. However, an endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related co-activators, ATAC and SAGA, assemble using co-translational pathways, but their subcellular localization, cytoplasmic abundance, and functions are distinct.

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1-the largest protein in the complex-as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Alternative mRNA Splicing Controls the Functions of the Histone H3K27 Demethylase UTX/KDM6A.

The UTX/KDM6A histone H3K27 demethylase plays an important role in development and is frequently mutated in cancers such as urothelial cancer. Despite many studies on UTX proteins, variations in mRNA splicing have been overlooked. Using Nanopore sequencing, we present a comprehensive analysis of UTX/KDM6A splicing events in human cell lines and in tissue samples from bladder cancer cases and normal epithelia. We found that the central region of UTX mRNAs encoded by exons 12 to 17 undergoes extensive alternative splicing. Up to half of all stable mRNAs (8-48% in bladder tissues and 18-58% in cell lines) are represented by the UTX canonical isoform lacking exon 14 encoding a nuclear localization sequence, and hence exon 14-containing UTX isoforms exclusively localize to the nucleus, unlike the cytonuclear localization of the canonical isoform. Chromatin association was also higher for exon-14-containing isoforms compared to the canonical UTX. Using quantitative mass spectrometry, we found that all UTX isoforms integrated into the MLL3 and MLL4, PR-DUB and MiDAC complexes. Interestingly, one of the novel UTX isoforms, which lacks exons 14 and 16, fails to interact with PR-DUB and MiDAC complex members. In conclusion, UTX mRNAs undergo extensive alternative splicing, which controls the subcellular localization of UTX and its interactions with other chromatin regulatory complexes.

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1 - the largest protein in the complex - as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

BACKGROUND: Loss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors. RESULTS: Here, we applied and in silico screening approach for 253 disease-related MEN1 missense mutations in order to select a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/MLL2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the MLL1 and MLL2 interactions when compared with JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites. CONCLUSIONS: Our results underline the effects of MEN1 gene mutations in both familial and sporadic tumors of endocrine origin on the interactions of menin with the MLL1 and MLL2 histone H3K4 methyltransferase complexes and with JunD-containing transcription factors. Menin binding pocket mutants R52G, E255K and E359K have differential effects on MLL1/MLL2 and JunD interactions, which translate into differential genomic binding patterns. Our findings encourage future studies addressing the pathophysiological relevance of the separate MLL1/MLL2- and JunD-dependent functions of menin mutants in MEN1 disease model systems.

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism.

X-linked dystonia-parkinsonism (XDP) is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In XDP, the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34'. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in XDP and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34' was further investigated in cellular assays. Subsequently, microexon 34' incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from XDP and control brains tissues. Using cell-based splicing assays, we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34'. In addition, we show that (1) microexon 34'-containing TAF1 mRNAs are detected at similar levels in XDP as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between XDP and controls brains. These results indicate that microexon 34' incorporation into TAF1 mRNA is not affected in XDP brains. Our findings shift the current paradigm of XDP by discounting alternative splicing of TAF1 microexon 34' as the molecular basis for this disease.

greenCUT&RUN: Efficient Genomic Profiling of GFP-Tagged Transcription Factors and Chromatin Regulators.

Genome-wide mapping of transcription factors and chromatin regulators is important to distinguish their direct from indirect effects on gene transcription or chromatin function. Novel approaches for studying their genomic localization under native conditions, such us cleavage under target and release using nuclease (CUT&RUN), offer higher resolution and lower sequencing costs than classical chromatin immunoprecipitation (ChIP) assays, and require fewer cells but they still depend on the availability of high-quality antibodies. Here, we describe detailed and robust protocols for greenCUT&RUN, which is a generic CUT&RUN-based approach for mapping the genome-wide localization of green fluorescent protein (GFP)-tagged factors in intact mammalian cells. The greenCUT&RUN method makes use of a micrococcal nuclease (MNase) coupled to a high affinity nanobody against GFP, which exploits the accessibility of multiple surfaces of the GFP tag, thus eliminating issues of antibody variability and availability. We also provide efficient protocols for the expression and purification of two different GFP nanobodies, which recognize non-overlapping GFP epitopes and can be combined for a further gain in sensitivity and accuracy. Compared to traditional CUT&RUN, genomic localization by greenCUT&RUN reduces handling time and experimental variability. GreenCUT&RUN is a versatile, robust, and universal procedure for surveying the genome-wide localization of GFP-tagged versions of proteins that drive key transcriptional programs and regulate chromatin function. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Standard greenCUT&RUN for GFP-tagged proteins in mammalian cells Alternate Protocol: High-Ca++ /low-salt greenCUT&RUN for GFP-tagged histone proteins in mammalian cells Support Protocol: Expression and purification of GFP nanobody-MNase fusion proteins for greenCUT&RUN.

A Box of Chemistry to Inhibit the MEN1 Tumor Suppressor Gene Promoting Leukemia.

Targeting protein-protein interactions (PPIs) with small-molecule inhibitors has become a hotbed of modern drug development. In this review, we describe a new class of PPI inhibitors that block menin from binding to MLL proteins. Menin is encoded by the MEN1 tumor suppressor, but acts as an essential cofactor for MLL/KMT2A-rearranged leukemias. The most promising menin-MLL inhibitors belong to the thienopyrimidine class and have recently entered phase I/II clinical trials for treating acute leukemias characterized by MLL/KMT2A translocations or NPM1 mutations. As single agents, thienopyrimidine compounds eradicate leukemia in a xenograft models of primary leukemic cells belonging to the MLL-rearranged or NPM1-mutant subtypes. These compounds are well tolerated with few or no side effects, which is remarkable given the tumor-suppressor function of menin. The menin-MLL inhibitors highlight how leukemia patients could benefit from a targeted epigenetic therapy with novel PPI inhibitors obtained by directed chemical evolution.

De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

Epigenetics and transcription regulation during eukaryotic diversification: the saga of TFIID.

The basal transcription factor TFIID is central for RNA polymerase II-dependent transcription. Human TFIID is endowed with chromatin reader and DNA-binding domains and protein interaction surfaces. Fourteen TFIID TATA-binding protein (TBP)-associated factor (TAF) subunits assemble into the holocomplex, which shares subunits with the Spt-Ada-Gcn5-acetyltransferase (SAGA) coactivator. Here, we discuss the structural and functional evolution of TFIID and its divergence from SAGA. Our orthologous tree and domain analyses reveal dynamic gains and losses of epigenetic readers, plant-specific functions of TAF1 and TAF4, the HEAT2-like repeat in TAF2, and, importantly, the pre-LECA origin of TFIID and SAGA. TFIID evolution exemplifies the dynamic plasticity in transcription complexes in the eukaryotic lineage.

Co-translational assembly of mammalian nuclear multisubunit complexes.

Cells dedicate significant energy to build proteins often organized in multiprotein assemblies with tightly regulated stoichiometries. As genes encoding subunits assembling in a multisubunit complex are dispersed in the genome of eukaryotes, it is unclear how these protein complexes assemble. Here, we show that mammalian nuclear transcription complexes (TFIID, TREX-2 and SAGA) composed of a large number of subunits, but lacking precise architectural details are built co-translationally. We demonstrate that dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). The lack of co-translational interaction can lead to degradation of the partner protein. Thus, protein synthesis and complex assembly are linked in building mammalian multisubunit complexes, suggesting that co-translational assembly is a general principle in mammalian cells to avoid non-specific interactions and protein aggregation. These findings will also advance structural biology by defining endogenous co-translational building blocks in the architecture of multisubunit complexes.

Chaperonin CCT checkpoint function in basal transcription factor TFIID assembly.

TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5-TAF6-TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6-TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.

Genomic integrity of ground-state pluripotency.

Pluripotent cells appear to be in a transient state during early development. These cells have the capability to transition into embryonic stem cells (ESCs). It has been reported that mouse pluripotent cells cultivated in chemically defined media sustain the ground state of pluripotency. Because the epigenetic pattern of pluripotent cells reflects their environment, culture under different conditions causes epigenetic changes, which could lead to genomic instability. This study focused on the DNA methylation pattern of repetitive elements (REs) and their activation levels under two ground-state conditions and assessed the genomic integrity of ESCs. We measured the methylation and expression level of REs in different media. The results indicated that although the ground-state conditions show higher REs activity, they did not lead to DNA damage; therefore, the level of genomic instability is lower under the ground-state compared with the conventional condition. Our results indicated that when choosing an optimum condition, different features of the condition must be considered to have epigenetically and genomically stable stem cells.

Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.

Mitotic Chromosomes: Not So Silent After All.

Mitotic chromosome condensation is believed to be incompatible with transcription. Palozola et al. (2017) now report in Science that a transcription program is maintained during mitosis and that transcriptional re-activation at mitotic exit is geared to support cell growth rather than cell identity.