Genes & Development

Pioneer transcription factors act at the top of gene-regulatory networks by promoting accessible chromatin at the cis-regulatory regions that drive gene expression. Despite their ability to bind closed chromatin, pioneer factors occupy distinct binding sites in different tissues. The pioneer factor Zelda promotes the undifferentiated fate in both the early Drosophila embryo and in the neural stem cells (neuroblasts) of the larval brain. Tissue-specific binding by Zelda identifies cell-type specific enhancers, which are enriched for different DNA-sequence motifs. We investigated the features that promoted cell-type specific occupancy by testing the role of conserved, structured protein domains in the capacity of Zelda to promote the embryonic and neuroblast cell fates. We unexpectedly identified that the most deeply conserved region in Zelda, the second zinc finger, has opposing functions in the embryo and neuroblasts. We showed that this zinc finger is a previously unrecognized DNA-binding domain that is specifically required for Zelda binding to a G-rich motif in larval neuroblasts. The pioneering function of Zelda depends largely on the C-terminal cluster of zinc fingers that promotes binding in the early embryo, suggesting that pioneer function may depend on how Zelda engages the genome. As opposed to co-factor expression or chromatin environment, our data identify tissue-specific usage of two, widely separated DNA-binding domains as the mechanism controlling tissue-specific binding and activity.

Spo11 initiates meiotic recombination by introducing programmed DNA double-strand breaks. DNA cleavage occurs via a topoisomerase-like mechanism involving hybrid active sites formed at the dimer interface. However, in contrast to its topoisomerase relative (Topo VI), Spo11 does not form a stable dimer, likely to prevent uncontrolled DNA cleavage. Here, we investigated the dimerization of S. cerevisiae Spo11 in complex with its partners Ski8, Rec102, and Rec104. We show that the Spo11 complex dimerizes transiently on DNA, forming unstable dimeric complexes with duplex and branched DNA substrates. Guided by AlphaFold modeling of a pre-cleavage complex, we identified mutations that reduce dimerization. Surprisingly, DSB formation is resilient to mutagenesis of the Spo11 dimer interface, implying that additional factors promote dimerization in vivo. Finally, we found that Rec102 exerts a key DNA-binding function, essential for catalysis, and show that it also participates in dimerization through trans contacts with Ski8. Our work provides new insights into the mechanism of Spo11 dimerization and the role of its partners in initiating meiotic recombination.

Heterochromatin plays a critical role in nuclear organization and the regulation of gene expression by directing 3D genome organization, regulating lineage-specific gene expression, and ensuring the repression of transposable elements and endogenous retroviruses. Functionally and structurally distinct chromatin domains are defined by the so-called histone code, which consists of combinations of post-translational histone modifications deposited by "code writers" and recognized by "code readers." The primary mark of heterochromatin, trimethylation of histone H3 at lysine 9 (H3K9me3), is deposited by histone methyltransferases, such as SetDB1, and serves as a binding platform for readers, most notably HP1 family proteins. Using a reporter system to monitor the dynamics of heterochromatin establishment and maintenance, we demonstrated that transient tethering of HP1 triggers the SetDB1-dependent establishment of stable heterochromatin. This finding indicates the presence of a feedback mechanism wherein the reader of the H3K9me3 mark recruits the writer. We further discovered that the genetic interaction between SetDB1 and HP1 is mirrored by a direct physical interaction. This interaction requires the auto-methylation of two conserved histone mimic motifs located in unstructured regions of SetDB1. HP1 binds these SetDB1 motifs using the same molecular interface it employs to recognize the modified histone tail. Our findings show that SetDB1 auto-methylation is essential for the spreading and stable maintenance of heterochromatin. This includes its roles in processes such as X-chromosome inactivation and the negative feedback regulation of a large gene family encoding KRAB-ZNF transcriptional repressors. Thus, the primary heterochromatin mark is not limited to nucleosomes but is also deployed on the marks writer itself. This fosters a direct physical interaction between the writer and the reader, ensuring key features of heterochromatin: its spreading to establish extended domains and its stable maintenance through cell divisions.

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2{Delta}ex3-4;Trp53-/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

Before the spliceosome engages a pre-mRNA to excise its introns, the catalytic small nuclear RNA (snRNA) U6 is inactive because of base pairing with U4 snRNA; thus, spliceosome activation requires unwinding of base paired U4/U6, composed of stem I and stem II. The Ski2-like ATPase and RNA helicase Brr2p facilitates U4/U6 unwinding and the ultimately irreversible release of U4; however, the molecular mechanism behind Brr2p-mediated U4/U6 unwinding and the roles of the snRNAs in unwinding remains incompletely understood. To investigate the mechanism in vivo in budding yeast, we screened an unwinding deficient, cold-sensitive brr2 mutant, associated with retinitis pigmentosa in humans, for genetic interactions with mutations in U4 snRNA. Destabilizing U4 mutations in either stem I or stem II suppressed the brr2 mutant, providing functional evidence that Brr2p disrupts both stems in vivo. Further, destabilizing mutations in the intervening 5 stem loop of U4 also suppressed the brr2 mutant, and in vitro Brr2p displaced Prp31p from this stem loop, implicating Brr2p in disruption of this structure, too. Unexpectedly and counterintuitively, many destabilizing mutations in U4/U6 stem I exacerbated the brr2 mutant. These mutations disrupted an intramolecular stem loop (U4-ISL1) in U4 that is mutually exclusive with U4/U6 stem I. We found that U4-ISL1 is required for splicing in vivo and for U4/U6 unwinding in vitro. Altogether, these results implicate Brr2p in disrupting all U4 secondary structures upstream of its initial U4 binding site and implicate an important role for U4 in antagonizing U4/U6 reannealing during Brr2p-mediated U4/U6 unwinding.

The mechanisms that control the dynamic composition of RNAPII elongation complexes govern major transitions in the transcription cycle yet are poorly understood. Here, we show that the transcription elongation factor Spt5 determines elongation complex composition to promote productive elongation and the transition to termination. Using an unbiased genetic screen and genomic approaches in Saccharomyces cerevisiae, we provide evidence that dephosphorylation of the Spt5 C-terminal repeat domain (CTR) by Glc7/PP1 is required to dislodge the Paf1 complex (Paf1C) from RNAPII near the cleavage and polyadenylation site (CPS). Mutations in Paf1C or the Spt5 CTR that dissociate Paf1C from RNAPII bypass the requirement for two critical regulators of Glc7 in the cleavage and polyadenylation factor that promote Glc7 enrichment at the 3 ends of genes. Depletion of Glc7 causes aberrant retention of Paf1C past the CPS and a dramatic increase in readthrough transcription, which is fully suppressed by Paf1C mutations. Our results demonstrate that Paf1C retention antagonizes transcription termination and that Glc7-mediated restructuring of the RNAPII elongation complex to evict Paf1C at the CPS is a critical step in the transition from elongation to termination.

During early transcription, RNA polymerase II (RNAPII) undergoes a series of structural transitions controlled by cyclin-dependent kinases. Whether protein ubiquitylation and proteasomal degradation affect the fate of RNAPII close to promoters is less well understood. Here we show that the deubiquitylating enzyme USP11 and its heterodimeric partner USP7 form a trimeric complex with TCEAL1, a member of the poorly understood TCEAL (TCEA/TFIIS-like) protein family. TCEAL1 shares sequence homology with the RNAPII interaction domain of the TCEA/TFIIS elongation factor, which controls the fate of backtracked RNAPII. TCEAL1 stabilizes complexes of USP11 with USP7 and with RNAPII. TCEAL1 is recruited to core promoters when transcription elongation is blocked and globally enhances the chromatin association of RNAPII during early transcription. Mechanistically, the USP11/USP7/TCEAL1 complex competes with TFIIS for binding to core promoters and protects RPB8, an essential subunit of RNAPII, from degradation, likely preventing excessive TFIIS-mediated transcript cleavage and RNAPII disassembly. In neuroblastoma and other tumors, TCEAL1-dependent genes define a TGF beta-dependent gene expression program that is characteristic for mesenchymal and invasive tumor cell types, suggesting that the USP11/USP7/TCEAL1 trimer stabilizes RNAPII during early transcription to support a critical oncogenic gene expression program (190 words).

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the ON/OFF state of targets like the Hox genes. Trithorax encodes a methyltransferase specific to histone H3 lysine-4 (H3K4). However, there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo. Here, we demonstrate two key roles for replication-dependent histone H3.2K4 in target control. We find that H3.2K4 antagonizes Polycomb group catalytic activity and that it is required for proper target gene activation. We conclude that H3.2K4 directly regulates expression of Polycomb targets.

HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-MODY) and hypomorphic HNF1A variants confer type 2 diabetes risk, but a lack of experimental systems has limited our understanding of how the transcription factor HNF1 regulates adult human islet function. Here, we combined human islet genetics, RNA sequencing, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) chromatin mapping, patch-clamp electrophysiology and transplantation-based assays to elucidate HNF1-regulated mechanisms in mature pancreatic and {beta} cells. shRNA-mediated suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion both in vitro and after transplantation into immunocompromised mice, recapitulating phenotypes observed in HNF1A-MODY patients. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATP-transporters and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional de-repression through HNF1. CUT&RUN mapping of HNF1 DNA-binding sites in primary human islets verified that a subset of HNF1-regulated genes were direct targets. These data provide unprecedented mechanistic links between HNF1A loss and diabetic phenotypes in mature human and {beta} cells.

The restrictor, ZC3H4/WDR82, is the major termination factor for antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 co-purifies with PP1 phosphatase and PP1 phosphatase nuclear targeting subunit, PNUTS, which binds directly to the WDR82 subunit of restrictor. AlphaFold predicts a quaternary complex, PPWZ, in which PP1-associated PNUTS and ZC3H4 both contact WDR82. To investigate the role of protein dephosphorylation in PPWZ activity, we expressed a substrate trap comprising inactive PP1H66K linked to the PNUTS C-terminus. PP1H66K-PNUTS binds pol II large subunit and nuclear exosome components. PP1H66K-PNUTS, but not PP1WT-PNUTS, functions as a dominant-negative inhibitor of antisense termination and CTD Ser5 dephosphorylation. Both these activities require the PNUTS WDR82 binding domain that interacts with restrictor. We show that CTD Ser5 hyperphosphorylation is associated with higher processivity and reduced pausing that would counteract termination, and propose that Ser5 dephosphorylation by PPWZ is coupled to termination. In summary, we identify the PP1 phosphatase activity of the PPWZ complex as essential for terminator function and propose that this heterotetramer is the physiologically relevant form of restrictor.

Abstract/SummaryThe eukaryotic genome is organized to enable the precise regulation of gene expression required for development. This organization is established during early development when the embryo transitions from a fertilized germ cell to the totipotent zygote. To understand the factors and processes that drive genomic organization, we focused on the pioneer factor GAGA factor (GAF) that is required for early embryonic development in Drosophila. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. We show that this non-uniform distribution is driven by binding to the highly abundant GA-satellite repeats. At GA-repeats, GAF is necessary to form heterochromatin and silence transcription. Thus, GAF is required to establish both active and silent regions. We propose that foci formation enables GAF to have opposing transcriptional roles within a single nucleus. Our data support a model in which modulation of the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains that are essential for the robust regulation of gene expression.

The gastrointestinal (GI) tract consists of highly specialized organs from the proximal esophagus to the distal colon, each with unique functions. Rare congenital malformations of the GI tract, including organ atresia, agenesis or mis-patterning are linked to gene mutations although the molecular basis of these malformations has been poorly studied due to lack of model systems to study human development. We identified a patient with compound heterozygous mutations in the transcription factor RFX6 with pancreatic agenesis as previously described. In addition, the patient had duodenal mal-rotation and atresia suggesting that establishment of the proximal small intestine was impaired in these patients. To identify the molecular basis of the intestinal malformation we generated induced pluripotent stem cell lines from this patient, and derived human intestinal organoid (HIOs) to identify how mutations in RFX6 impact intestinal patterning and function. We identified that the duodenal identity of HIOs and patient tissues had adopted a more distal small intestinal signature, including expression of SATB2, normally expressed in the ileum and colon. CRISPR-mediated correction of RFX6 restored duodenal identity, including expression of PDX1, which is required for duodenal development. Using transcriptomic approaches in HIOs and Xenopus embryos we identified that PDX1 is a downstream transcriptional target of RFX6 and that PDX1 expression in a RFX6 mutant background was sufficient to rescue duodenal identity. However, RFX6 had a PDX1-independent role in regulating expression of components of WNT, HH, and BMP signaling pathways that are critical for establishing early regional identity in the GI tract. In summary, we have identified that RFX6 is one of the most upstream regulators early intestinal patterning in vertebrates and that it acts by regulating key transcriptional and signaling pathways.

Upon DNA replication stress, cells utilize the post-replication repair pathway to repair single-stranded DNA and maintain genome integrity. Post-replication repair is divided into two branches: error-prone translesion synthesis, signaled by PCNA mono-ubiquitination, and error-free template switching, signaled by PCNA poly-ubiquitination. In Saccharomyces cerevisiae, Rad5 is involved in both branches of repair during DNA replication stress. When the PCNA poly-ubiquitination function of Rad5 is disrupted, Rad5 recruits translesion synthesis polymerases to stalled replication forks, resulting in mutagenic repair. Details of how mutagenic repair is carried out, as well as the relationship between Rad5-mediated mutagenic repair and the canonical PCNA-mediated mutagenic repair, remain to be understood. We find that Rad5-mediated mutagenic repair requires the translesion synthesis polymerase {zeta} but does not require other yeast translesion polymerase activities. Furthermore, we show that Rad5-mediated mutagenic repair is independent of PCNA binding by Rev1 and so is separable from canonical mutagenic repair. In the absence of error-free template switching, both modes of mutagenic repair contribute additively to replication stress response in a replication timing-independent manner. Cellular contexts where error-free template switching is compromised are not simply laboratory phenomena, as we find that a natural variant in RAD5 is defective in PCNA poly-ubiquitination and therefore defective in error-free repair, resulting in Rad5- and PCNA-mediated mutagenic repair. Our results highlight the importance of Rad5 in regulating spontaneous mutagenesis and genetic diversity in S. cerevisiae through different modes of post-replication repair.

tRNA genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae. tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.

Epigenetic dysregulation is widespread in cancer. However, the specific epigenetic regulators and the processes they control to drive cancer phenotypes are poorly understood. Here, we employed a novel, scalable and high-throughput in vivo method to perform iterative functional screens of over 250 epigenetic regulatory genes within autochthonous oncogenic KRAS-driven lung tumors. We identified multiple novel epigenetic tumor suppressor and tumor dependency genes. We show that a specific HBO1 complex and the MLL1 complex are among the most impactful tumor suppressive epigenetic regulators in lung. The histone modifications generated by the HBO1 complex are frequently absent or reduced in human lung adenocarcinomas. The HBO1 and MLL1 complexes regulate chromatin accessibility of shared genomic regions, lineage fidelity and the expression of canonical tumor suppressor genes. The HBO1 and MLL1 complexes are epistatic during lung tumorigenesis, and their functional correlation is conserved in human cancer cell lines. Together, these results demonstrate the value of quantitative methods to generate a phenotypic roadmap of epigenetic regulatory genes in tumorigenesis in vivo.

We have formed new short telomeres in Schizosaccharomyces pombe using an inducible nuclease that cuts near telomere repeats in cells that lack, cannot recruit or cannot fully activate telomerase. Sequencing these new telomeres showed that cells can divide at least 4 times with [~]30 bp of non-telomeric sequence at the chromosome end in cells lacking telomerase, which contrasts with current models for the roles of terminal single-stranded telomere repeats and the telomere proteins in telomere protection and replication. Cells that cannot recruit or activate telomerase had similar results, with additional rearrangements or telomere repeat addition, respectively.

The nucleosome acidic patch is a hub of coordinated engagement by proteins that regulate genomic function. Here we report that S. cerevisiae Dot5 contains an arginine-rich HMGN-like motif that mediates nucleosome acidic patch binding and is required for the cell growth, DNA repair and heterochromatin defects exhibited when the protein is overexpressed. The heterologous expression of camelid single chain antibodies to the nucleosome acidic patch confers a similar range of phenotypes, with the most severe observed when an arginine-anchor mode of binding analogous to many endogenous factors is employed. This highlights a delicate balance between nucleosome acidic patch interactors critical for normal cellular functions and dysregulated in disease.

The distinct contributions of replication-dependent and replication-independent histones to development and genome function remain unclear. In this study, we investigate how the distinct protein identities of the histone H3.2 and H3.3 subtypes contribute to development and gene regulation in Drosophila. Comparing animals in which the replication-independent H3.3 genes were mutated to produce the replication-dependent H3.2 protein with those carrying deletions of the replication-independent H3.3 genes revealed that replication-independent H3.3 is essential for fertility, adult locomotor behavior, and normal longevity. However, development to adulthood does not depend on which replication-independent H3 subtype is expressed from the H3.3 loci. Moreover, replication-independent H3.3 is not required to establish or maintain global patterns of chromatin accessibility or gene expression in the adult brain. Surprisingly, we find that expression of H3.2 from the replication-dependent HisC locus is essential in post-replicative cells in the absence of replication-independent H3.3, and we uncover a critical role for the HIRA histone chaperone complex in preserving genome function when replication-independent H3.3 is deleted. We conclude that an available pool of H3 is more critical than the specific identity of H3 in the pool.

The CMG helicase is the metastable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, indicating that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2{Delta} is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.

Biomolecular condensates, such as germ granules, organize RNAi pathways critical for fertility and genome regulation. Yet, the protein composition and functional contributions of these condensates remain poorly defined. Here, we applied TurboID proximity labeling to the Caenorhabditis elegans germ granule protein SIMR-1, integrating mass spectrometry with genetic screening, CRISPR-based tagging, and small RNA sequencing. This systematic approach identified several previously uncharacterized germ granule proteins that contribute to fertility, germline immortality, exogenous RNAi, and transgenerational inheritance. Small RNA sequencing of 21 mutants revealed broad and class-specific defects in siRNA and miRNA biogenesis, with distinct factors associated with defects in WAGO-class 22G-RNAs, CSR-class 22G-RNAs, or histone-directed small RNAs. Among these, we identified PINT-1, a highly disordered protein that directly interacts with and is recruited to germ granules by the PIWI Argonaute PRG-1. PINT-1 is required for piRNA-dependent and -independent secondary siRNA biogenesis and germline development. Comparative genomics revealed that PINT-1 has co-evolved with PRG-1 across nematodes, with a conserved structured N-terminus and a rapidly diverging repeat-rich intrinsically disordered region. Together, our findings expand the germ granule proteome and reveal how distinct condensate components contribute to specialized functions within the small RNA pathways, while highlighting an evolutionarily co-adapted PIWI interactor critical for siRNA biogenesis.